Dr. Maecker received a BS in Microbiology from Purdue University and a PhD in Cancer Biology from Stanford University. He did postdoctoral work with Ronald and Shoshana Levy at Stanford, and was an Assistant Professor of Biology at Loyola University Chicago, as well as a Senior Scientist at BD Biosciences, San Jose, CA. He is currently a Professor of Microbiology and Immunology, and Director of the Human Immune Monitoring Center, at Stanford University.

Administrative Appointments

  • Director, Human Immune Monitoring Center (2009 - Present)
  • Member, Institute for Immunity, Transplantation, and Infection (2009 - Present)
  • Member, Immunology Interdisciplinary Degree Program (2012 - Present)
  • Director, Service Centers and Enabling Technologies, Stanford University School of Medicine (2014 - Present)
  • Member, Stanford Diabetes Research Center (2017 - Present)

Boards, Advisory Committees, Professional Organizations

  • Member, Scientific Advisory Board, Cytek (2017 - Present)
  • Member, SITC Biomarker Task Force (2014 - Present)
  • Member, Stanford Cancer Institute Scientific Review Committee (2011 - Present)
  • Co-chair, FOCIS Human Immunophenotyping Consortium (HIPC) (2009 - Present)

Professional Education

  • PhD, Stanford University, Cancer Biology (1988)
  • BS, Purdue University, Microbiology (1984)

Community and International Work

  • Human Immunophenotyping Consortium


    Standardization of human immune monitoring

    Partnering Organization(s)




    Ongoing Project


    Opportunities for Student Involvement



  • Vernon Maino, Holden Maecker, Douglas Petry. "United States Patent 2007/109161 Detection of HIV-infected cell subsets", Becton, Dickinson, and Company, Jan 1, 2007
  • Shoshana Levy, Rosemarie Dekruyff, Dale Umetsu, Holden Maecker. "United States Patent 2001/0044418 Treatment of allergies", Stanford University, Jan 1, 2001

Research & Scholarship

Current Research and Scholarly Interests

A major theme in our group is to define metrics of immune competence in various settings, including cancer immunotherapy, organ transplantation, allergy, and chronic viral infection. We use CyTOF mass cytometry, often in combination with other technologies, to broadly survey immune features at the cellular level, then examine links between features or groups of features and clinical outcome. A long-term goal is to create an assay of global immune competence that could predict risk for various immune-related outcomes in both healthy individuals and in disease.


2020-21 Courses

Stanford Advisees

  • Doctoral Dissertation Reader (AC)
    Bryan Xie


All Publications

  • Aging and CMV discordance are associated with increased immune diversity between monozygotic twins. Immunity & ageing : I & A Yan, Z., Maecker, H. T., Brodin, P., Nygaard, U. C., Lyu, S. C., Davis, M. M., Nadeau, K. C., Andorf, S. 2021; 18 (1): 5


    BACKGROUND: Broadly, much of variance in immune system phenotype has been linked to the influence of non-heritable factors rather than genetics. In particular, two non-heritable factors: aging and human cytolomegavirus (CMV) infection, have been known to account for significant inter-individual immune variance. However, many specific relationships between them and immune composition remain unclear, especially between individuals over narrower age ranges. Further exploration of these relationships may be useful for informing personalized intervention development.RESULTS: To address this need, we evaluated 41 different cell type frequencies by mass cytometry and identified their relationships with aging and CMV seropositivity. Analyses were done using 60 healthy individuals, including 23 monozygotic twin pairs, categorized into young (12-31years) and middle-aged (42-59years). Aging and CMV discordance were associated with increased immune diversity between monozygotic twins overall, and particularly strongly in various T cell populations. Notably, we identified 17 and 11 cell subset frequencies as relatively influenced and uninfluenced by non-heritable factors, respectively, with results that largely matched those from studies on older-aged cohorts. Next, CD4+ T cell frequency was shown to diverge with age in twins, but with lower slope than in demographically similar non-twins, suggesting that much inter-individual variance in this cell type can be attributed to interactions between genetic and environmental factors. Several cell frequencies previously associated with memory inflation, such as CD27- CD8+ T cells and CD161+ CD4+ T cells, were positively correlated with CMV seropositivity, supporting findings that CMV infection may incur rapid aging of the immune system.CONCLUSIONS: Our study confirms previous findings that aging, even within a relatively small age range and by mid-adulthood, and CMV seropositivity, both contribute significantly to inter-individual immune diversity. Notably, we identify several key immune cell subsets that vary considerably with aging, as well as others associated with memory inflation which correlate with CMV seropositivity.

    View details for DOI 10.1186/s12979-021-00216-1

    View details for PubMedID 33461563

  • Network for biomarker immunoprofiling for cancer immunotherapy: Cancer Immune Monitoring and Analysis Centers and Cancer Immunologic Data Commons (CIMAC-CIDC). Clinical cancer research : an official journal of the American Association for Cancer Research Chen, H. X., Song, M., Maecker, H. T., Gnjatic, S., Patton, D., Lee, J. J., Adam, S. J., Moravec, R., Liu, X. S., Cerami, E., Lindsay, J., Hodi, F. S., Wu, C., Wistuba, I. I., Al-Atrash, G., Bernatchez, C., Bendall, S. C., Hewitt, S. M., Sharon, E., Streicher, H., Enos, R. A., Bowman, M. D., Tatard-Leitman, V. M., Sanchez-Espiridion, B., Ranasinghe, S., Pichavant, M., Del Valle, D. M., Yu, J., Janssens, S., Peterson-Klaus, J., Rowe, C., Bongers, G., Jenq, R. R., Chang, C., Abrams, J. S., Mooney, M., Doroshow, J. H., Harris, L. N., Thurin, M. 2021


    Immunoprofiling to identify biomarkers and integration with clinical trials outcome are critical to improve immunotherapy approaches for cancer patients. However, the translational potential of individual studies is often limited by small sample size of trials and the complexity of immuno-oncology biomarkers. Variability in assays further limits comparison and interpretation of data across studies and laboratories. To enable a systematic approach to biomarker identification and correlation with clinical outcome across trials, the Cancer Immune Monitoring and Analysis Centers and Cancer Immunologic Data Commons (CIMAC-CIDC) Network was established through support of the Cancer MoonshotSM Initiative of the National Cancer Institute and the Partnership for Accelerating Cancer Therapies (PACT) with industry partners via the Foundation for the National Institutes of Health. The CIMAC-CIDC Network is composed of four academic centers (CIMACs) with multidisciplinary expertise in the field of cancer immunotherapy that provide validated and harmonized assays for immune profiling. A data coordinating center (CIDC) provides the computational expertise and resources for biomarker data storage and analysis platforms for correlation with clinical data. This overview highlights strategies for assay harmonization to enable cross-trial and cross-site data analysis and describes key elements for establishing a network to enhance immuno-oncology biomarker development. These include an operational infrastructure; validation and harmonization of core immunoprofiling assays; platforms for data ingestion and integration; and access to specimens from clinical trials. Published in the same volume are reports of harmonization for core analyses: whole exome sequencing, RNA sequencing, cytometry by time of flight, and immunohistochemistry/immunofluorescence.

    View details for DOI 10.1158/1078-0432.CCR-20-3241

    View details for PubMedID 33419780

  • Acute Chelation Therapy-Associated Changes in Urine Gadolinium, Self-reported Flare Severity, and Serum Cytokines in Gadolinium Deposition Disease. Investigative radiology Maecker, H. T., Siebert, J. C., Rosenberg-Hasson, Y., Koran, L. M., Ramalho, M., Semelka, R. C. 2021


    The aim of this study was to determine the following in patients who have undergone magnetic resonance imaging with gadolinium-based contrast agents (GBCAs) and meet the proposed diagnostic criteria for gadolinium deposition disease (GDD): (1) the effectiveness of chelation therapy (CT) with intravenous Ca-diethylenetriaminepentaacetic acid in removing retained gadolinium (Gd) and factors affecting the amount removed; (2) the frequency of CT-induced Flare, that is, GDD diagnostic symptom worsening, and factors affecting Flare intensity; (3) whether, as reported in a separate cohort, GDD patients' serum cytokine levels differ significantly from those in healthy normal controls and change significantly in response to CT; and (4) whether urine Gd, Flare reaction, and serum cytokine findings in GDD patients are mimicked in non-ill patients described as having gadolinium storage condition (GSC).Twenty-one GDD subjects and 3 GSC subjects underwent CT. Patients provided pre-CT and post-CT 24-hour urine samples for Gd content determination along with pre-CT and 24-hour post-CT serum samples for cytokine analysis. Patients rated potential Flare 24 hours after CT. Pre-CT and post-CT 24-hour urine Gd analyses and Luminex serum cytokine assays were performed blind to patients' GDD and GSC status and all other data except age and sex. Serum cytokine levels in a healthy normal control group of age- and sex-matched subjects drawn from Stanford influenza vaccination studies were measured once, contemporaneously with those of GDD and GSC patients, using the same Luminex assay.Urine Gd amounts increased post-CT by 4 times or more after 87% of the 30 CT sessions. The most important factors appeared to be the time since the last GBCA dose and the cumulative dose received. Urine Gd amounts for GDD and GSC patients fell in the same ranges. All GDD patients, and no GSC patient, reported a Flare 24 hours post-CT. Linear regression found that Flare intensity was significantly predicted by a model including pre- and post-CT Gd amounts and the number of GBCA-enhanced magnetic resonance imaging. Post-CT, multiple cytokines showed strong positive relationships with GDD patients' Flare intensity in multivariable models. The pre-CT serum levels of 12 cytokines were significantly different in GDD patients compared with healthy flu vaccine controls. The small number of GSC patients precluded analogous statistical testing. Post-CT, GDD patients' serum levels of 20 cytokines were significantly decreased, and 2 cytokines significantly increased. These cytokines did not exhibit the same change pattern in the 3 GSC patients. The small number of GSC patients precluded statistical comparisons of GSC to GDD patients' results.In this preliminary study, 24-hour urine Gd content increased markedly and similarly in GDD and GSC patients after Ca-diethylenetriaminepentaacetic acid CT. Post-CT Flare reaction developed only in GDD patients. The current study is the second finding significantly different serum cytokine levels in GDD patients compared with healthy normal controls. These differences and the difference between GDD and GSC patients' Flare and cytokine responses to CT suggest some inflammatory, immunologic, or other physiological differences in patients with GDD. Further research into the treatment and physiological underpinnings of GDD is warranted.

    View details for DOI 10.1097/RLI.0000000000000752

    View details for PubMedID 33449576

  • Mass Cytometry Defines Virus-Specific CD4+ T Cells in Influenza Vaccination. ImmunoHorizons Subrahmanyam, P. B., Holmes, T. H., Lin, D., Su, L. F., Obermoser, G., Banchereau, J., Pascual, V., Garcia-Sastre, A., Albrecht, R. A., Palucka, K., Davis, M. M., Maecker, H. T. 2020; 4 (12): 774–88


    The antiviral response to influenza virus is complex and multifaceted, involving many immune cell subsets. There is an urgent need to understand the role of CD4+ T cells, which orchestrate an effective antiviral response, to improve vaccine design strategies. In this study, we analyzed PBMCs from human participants immunized with influenza vaccine, using high-dimensional single-cell proteomic immune profiling by mass cytometry. Data were analyzed using a novel clustering algorithm, denoised ragged pruning, to define possible influenza virus-specific clusters of CD4+ T cells. Denoised ragged pruning identified six clusters of cells. Among these, one cluster (Cluster 3) was found to increase in abundance following stimulation with influenza virus peptide ex vivo. A separate cluster (Cluster 4) was found to expand in abundance between days 0 and 7 postvaccination, indicating that it is vaccine responsive. We examined the expression profiles of all six clusters to characterize their lineage, functionality, and possible role in the response to influenza vaccine. Clusters 3 and 4 consisted of effector memory cells, with high CD154 expression. Cluster 3 expressed cytokines like IL-2, IFN-gamma, and TNF-alpha, whereas Cluster 4 expressed IL-17. Interestingly, some participants had low abundance of Clusters 3 and 4, whereas others had higher abundance of one of these clusters compared with the other. Taken together, we present an approach for identifying novel influenza virus-reactive CD4+ T cell subsets, a method that could help advance understanding of the immune response to influenza, predict responsiveness to vaccines, and aid in better vaccine design.

    View details for DOI 10.4049/immunohorizons.1900097

    View details for PubMedID 33310880

  • Vi-Vaccinations Induce Heterogeneous Plasma Cell Responses That Associate With Protection From Typhoid Fever FRONTIERS IN IMMUNOLOGY Cross, D. L., Verheul, M. K., Leipold, M. D., Obermoser, G., Jin, C., Jones, E., Starr, J. S., Mohorianu, I., Blohmke, C. J., Maecker, H. T., Napolitani, G., Hill, J., Pollard, A. J. 2020; 11
  • SITC cancer immunotherapy resource document: a compass in the land of biomarker discovery. Journal for immunotherapy of cancer Hu-Lieskovan, S., Bhaumik, S., Dhodapkar, K., Grivel, J. J., Gupta, S., Hanks, B. A., Janetzki, S., Kleen, T. O., Koguchi, Y., Lund, A. W., Maccalli, C., Mahnke, Y. D., Novosiadly, R. D., Selvan, S. R., Sims, T., Zhao, Y., Maecker, H. T. 2020; 8 (2)


    Since the publication of the Society for Immunotherapy of Cancer's (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune escape mechanisms which can develop in each patient. Therefore, the development of robust biomarkers for each immunotherapy strategy, enabling rational patient selection and the design of precise combination therapies, is key for the continued success and improvement of immunotherapy. In this document, we summarize and update established biomarkers, guidelines, and regulatory considerations for clinical immune biomarker development, discuss well-known and novel technologies for biomarker discovery and validation, and provide tools and resources that can be used by the biomarker research community to facilitate the continued development of immuno-oncology and aid in the goal of durable responses in all patients.

    View details for DOI 10.1136/jitc-2020-000705

    View details for PubMedID 33268350

  • Study Protocol for Teen Inflammation Glutamate Emotion Research (TIGER) FRONTIERS IN HUMAN NEUROSCIENCE Walker, J. C., Teresi, G. I., Weisenburger, R. L., Segarra, J. R., Ojha, A., Kulla, A., Sisk, L., Gu, M., Spielman, D. M., Rosenberg-Hasson, Y., Maecker, H. T., Singh, M. K., Gotlib, I. H., Ho, T. C. 2020; 14
  • Immune biomarkers link air pollution exposure to blood pressure in adolescents. Environmental health : a global access science source Prunicki, M., Cauwenberghs, N., Ataam, J. A., Movassagh, H., Kim, J. B., Kuznetsova, T., Wu, J. C., Maecker, H., Haddad, F., Nadeau, K. 2020; 19 (1): 108


    BACKGROUND: Childhood exposure to air pollution contributes to cardiovascular disease in adulthood. Immune and oxidative stress disturbances might mediate the effects of air pollution on the cardiovascular system, but the underlying mechanisms are poorly understood in adolescents. Therefore, we aimed to identify immune biomarkers linking air pollution exposure and blood pressure levels in adolescents.METHODS: We randomly recruited 100 adolescents (mean age, 16years) from Fresno, California. Using central-site data, spatial-temporal modeling, and distance weighting exposures to the participant's home, we estimated average pollutant levels [particulate matter (PM), polyaromatic hydrocarbons (PAH), ozone (O3), carbon monoxide (CO) and nitrogen oxides (NOx)]. We collected blood samples and vital signs on health visits. Using proteomic platforms, we quantitated markers of inflammation, oxidative stress, coagulation, and endothelial function. Immune cellular characterization was performed via mass cytometry (CyTOF). We investigated associations between pollutant levels, cytokines, immune cell types, and blood pressure (BP) using partial least squares (PLS) and linear regression, while adjusting for important confounders.RESULTS: Using PLS, biomarkers explaining most of the variance in air pollution exposure included markers of oxidative stress (GDF-15 and myeloperoxidase), acute inflammation (C-reactive protein), hemostasis (ADAMTS, D-dimer) and immune cell types such as monocytes. Most of these biomarkers were independently associated with the air pollution levels in fully adjusted regression models. In CyTOF analyses, monocytes were enriched in participants with the highest versus the lowest PM2.5 exposure. In both PLS and linear regression, diastolic BP was independently associated with PM2.5, NO, NO2, CO and PAH456 pollution levels (P≤0.009). Moreover, monocyte levels were independently related to both air pollution and diastolic BP levels (P≤0.010). In in vitro cell assays, plasma of participants with high PM2.5 exposure induced endothelial dysfunction as evaluated by eNOS and ICAM-1 expression and tube formation.CONCLUSIONS: For the first time in adolescents, we found that ambient air pollution levels were associated with oxidative stress, acute inflammation, altered hemostasis, endothelial dysfunction, monocyte enrichment and diastolic blood pressure. Our findings provide new insights on pollution-related immunological and cardiovascular disturbances and advocate preventative measures of air pollution exposure.

    View details for DOI 10.1186/s12940-020-00662-2

    View details for PubMedID 33066786

  • Autoimmunity to Hypocretin/Orexin and Molecular Mimicry to Flu in Type 1 Narcolepsy Luo, G., Ambati, A., Lin, L., Partinen, M., Ji, X., Maecker, H., Mignot, E. WILEY. 2020: S53
  • An initial investigation of serum cytokine levels in patients with gadolinium retention. Radiologia brasileira Maecker, H. T., Wang, W., Rosenberg-Hasson, Y., Semelka, R. C., Hickey, J., Koran, L. M. 2020; 53 (5): 306–13


    Objective: To determine whether individuals with proposed gadolinium deposition disease (GDD) have elevated serum levels of pro-inflammatory and pro-fibrotic cytokines, and whether specific cytokines are correlated with certain symptoms.Materials and Methods: Twenty-four participants recruited between May 2016 and June 2017 met GDD diagnostic criteria. The 64 control subjects provided serum samples before prophylactic flu vaccination. Serum cytokine levels were obtained with Luminex serum cytokine assay using eBiosciences/Affymetrix human 62-plex kits. Wilcoxon rank-sum tests were performed to assess the difference between the median fluorescence intensity values for the participants and the control group. Generalized linear models were built to evaluate the association between each cytokine of interest and selected participant symptoms.Results: Serum levels of 14 cytokines, including nine pro-inflammatory cytokines, were statistically significantly elevated compared to controls (p ≤ 0.05). Hypotheses regarding pro-fibrotic cytokines and cytokine links to specific symptoms' intensity were not confirmed.Conclusion: The statistically significantly elevated cytokines may be markers of susceptibility to GDD or agents of symptom induction. These findings suggest that individuals developing symptoms characteristic of GDD after a contrast-assisted magnetic resonance imaging should be studied to investigate whether gadolinium retention and elevated cytokines may be related to their symptoms.

    View details for DOI 10.1590/0100-3984.2019.0075

    View details for PubMedID 33071374

  • Systems biological assessment of immunity to mild versus severe COVID-19 infection in humans. Science (New York, N.Y.) Arunachalam, P. S., Wimmers, F., Mok, C. K., Perera, R. A., Scott, M., Hagan, T., Sigal, N., Feng, Y., Bristow, L., Tak-Yin Tsang, O., Wagh, D., Coller, J., Pellegrini, K. L., Kazmin, D., Alaaeddine, G., Leung, W. S., Chan, J. M., Chik, T. S., Choi, C. Y., Huerta, C., Paine McCullough, M., Lv, H., Anderson, E., Edupuganti, S., Upadhyay, A. A., Bosinger, S. E., Maecker, H. T., Khatri, P., Rouphael, N., Peiris, M., Pulendran, B. 2020


    COVID-19 represents a global crisis, yet major knowledge gaps remain about human immunity to SARS-CoV-2. We analyzed immune responses in 76 COVID-19 patients and 69 healthy individuals from Hong Kong and Atlanta. In PBMCs of COVID-19 patients, there was reduced expression of HLA-DR and pro-inflammatory cytokines by myeloid cells, and impaired mTOR-signaling and IFN-alpha production by plasmacytoid DCs. In contrast, there were enhanced plasma levels of inflammatory mediators, including EN-RAGE, TNFSF14, and oncostatin-M, which correlated with disease severity and increased bacterial products in human plasma. Single-cell transcriptomics revealed no type-I IFN, reduced HLA-DR in myeloid cells of severe patients, and transient expression of IFN-stimulated genes. This was consistent with bulk PBMC transcriptomics, and transient, low plasma IFN-alpha levels during infection. These results reveal mechanisms and potential therapeutic targets for COVID-19.

    View details for DOI 10.1126/science.abc6261

    View details for PubMedID 32788292

  • Transcriptional changes in peanut-specific CD4+ T cells over the course of oral immunotherapy. Clinical immunology (Orlando, Fla.) Wang, W., Lyu, S., Ji, X., Gupta, S., Manohar, M., Dhondalay, G. K., Chinthrajah, S., Andorf, S., Boyd, S. D., Tibshirani, R., Galli, S. J., Nadeau, K. C., Maecker, H. T. 2020: 108568


    Oral immunotherapy (OIT) can successfully desensitize allergic individuals to offending foods such as peanut. Our recent clinical trial (NCT02103270) of peanut OIT allowed us to monitor peanut-specific CD4+ T cells, using MHC-peptide Dextramers, over the course of OIT. We used a single-cell targeted RNAseq assay to analyze these cells at 0, 12, 24, 52, and 104 weeks of OIT. We found a transient increase in TGFbeta-producing cells at 52 weeks in those with successful desensitization, which lasted until 117 weeks. We also performed clustering and identified 5 major clusters of Dextramer+ cells, which we tracked over time. One of these clusters appeared to be anergic, while another was consistent with recently described TFH13 cells. The other 3 clusters appeared to be Th2 cells by their coordinated production of IL-4 and IL-13, but they varied in their expression of STAT signaling proteins and other markers. A cluster with high expression of STAT family members also showed a possible transient increase at week 24 in those with successful desensitization. Single cell TCRalphabeta repertoire sequences were too diverse to track clones over time. Together with increased TGFbeta production, these changes may be mechanistic predictors of successful OIT that should be further investigated.

    View details for DOI 10.1016/j.clim.2020.108568

    View details for PubMedID 32783912

  • Cytokine profile in plasma of severe COVID-19 does not differ from ARDS and sepsis. JCI insight Wilson, J. G., Simpson, L. J., Ferreira, A., Rustagi, A., Roque, J. A., Asuni, A., Ranganath, T., Grant, P. M., Subramanian, A. K., Rosenberg-Hasson, Y., Maecker, H., Holmes, S., Levitt, J. E., Blish, C., Rogers, A. J. 2020


    BACKGROUND: Elevated levels of inflammatory cytokines have been associated with poor outcomes among COVID-19 patients. It is unknown, however, how these levels compare to those observed in critically ill patients with ARDS or sepsis due to other causes.METHODS: We used a luminex assay to determine expression of 76 cytokines from plasma of hospitalized COVID-19 patients and banked plasma samples from ARDS and sepsis patients. Our analysis focused on detecting statistical differences in levels of 6 cytokines associated with cytokine storm (IL-1b, IL-1RA, IL-6, IL-8, IL-18, and TNFalpha) between patients with moderate COVID-19, severe COVID-19, and ARDS or sepsis.RESULTS: 15 hospitalized COVID-19 patients, 9 of whom were critically ill, were compared to critically ill patients with ARDS (n = 12) or sepsis (n = 16). There were no statistically significant differences in baseline levels of IL-1b, IL-1RA, IL-6, IL-8, IL-18, and TNFalpha between patients with COVID-19 and critically ill controls with ARDS or sepsis.CONCLUSIONS: Levels of inflammatory cytokines were not higher in severe COVID-19 patients than in moderate COVID-19 or critically ill patients with ARDS or sepsis in this small cohort. Broad use of immunosuppressive therapies in ARDS has failed in numerous Phase 3 studies; use of these therapies in unselected patients with COVID-19 may be unwarranted.FUNDING: A.J.R.: Stanford ICU Biobank NHLBI K23 HL125663. C.A.B.: Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Diseases #1016687; NIH/NIAID U19AI057229-16 (PI MM Davis); Stanford Maternal Child Health Research Institute; Chan Zuckerberg Biohub.

    View details for DOI 10.1172/jci.insight.140289

    View details for PubMedID 32706339

  • MYC functions as a switch for natural killer cell-mediated immune surveillance of lymphoid malignancies. Nature communications Swaminathan, S., Hansen, A. S., Heftdal, L. D., Dhanasekaran, R., Deutzmann, A., Fernandez, W. D., Liefwalker, D. F., Horton, C., Mosley, A., Liebersbach, M., Maecker, H. T., Felsher, D. W. 2020; 11 (1): 2860


    The MYC oncogene drives T- and B- lymphoid malignancies, including Burkitt's lymphoma (BL) and Acute Lymphoblastic Leukemia (ALL). Here, we demonstrate a systemic reduction in natural killer (NK) cell numbers in SRalpha-tTA/Tet-O-MYCON mice bearing MYC-driven T-lymphomas. Residual mNK cells in spleens of MYCON T-lymphoma-bearing mice exhibit perturbations in the terminal NK effector differentiation pathway. Lymphoma-intrinsic MYC arrests NK maturation by transcriptionally repressing STAT1/2 and secretion of Type I Interferons (IFNs). Treating T-lymphoma-bearing mice with Type I IFN improves survival by rescuing NK cell maturation. Adoptive transfer of mature NK cells is sufficient to delay both T-lymphoma growth and recurrence post MYC inactivation. In MYC-driven BL patients, low expression of both STAT1 and STAT2 correlates significantly with the absence of activated NK cells and predicts unfavorable clinical outcomes. Our studies thus provide a rationale for developing NK cell-based therapies to effectively treat MYC-driven lymphomas in the future.

    View details for DOI 10.1038/s41467-020-16447-7

    View details for PubMedID 32503978

  • A Novel Utility to Correct for Plate/Batch/Lot and Nonspecific Binding Artifacts in Luminex Data. Journal of immunology (Baltimore, Md. : 1950) Maecker, H. T., Rosenberg-Hasson, Y., Kolstad, K. D., Steen, V. D., Chung, L. S. 2020


    Cytokines and other secreted soluble proteins are routinely assayed as fluorescence intensities on the Luminex (Luminex, Austin, TX) platform. As with any immunoassay, a portion of the measured Ab binding can be nonspecific. Use of spiked-in microbead controls (e.g., AssayChex Process, Control Panel; Radix Biosolutions, Georgetown, TX) can determine the level of nonspecific binding on a per specimen basis. A statistical approach for correction of this assay's nonspecific binding artifact was first described in earlier work. The current paper describes a novel utility written in the R language (, that refines correction for nonspecific binding in three important ways: 1) via local polynomial regression, the utility allows for curvature in relationships between soluble protein median fluorescence intensities and nonspecific binding median fluorescence intensities; 2) to stabilize correction, the fit of the nonlinear regression function is obtained via repeated cross-validation; and 3) the utility addresses possible bias due to technical error in measured nonspecific binding. The utility first logarithm transforms and then removes plate/batch/lot artifacts from median fluorescence intensities prior to correction for nonspecific binding, even when plates/batches/lots are unbalanced with respect to experimental factors of interest. Continuous (e.g., age) and categorical (e.g., diagnosis) covariates are accommodated in plate/batch/lot artifact correction. We present application of the utility to a panel of 62 cytokines in a sample of human patients diagnosed with systemic sclerosis and to an experiment that examined multiple lots of a human 51-cytokine panel. The R script for our new utility is publicly available for download from the web.

    View details for DOI 10.4049/jimmunol.2000017

    View details for PubMedID 32376648

  • JAK-STAT Activity in Peripheral Blood Cells and Kidney Tissue in IgA Nephropathy. Clinical journal of the American Society of Nephrology : CJASN Tao, J., Mariani, L., Eddy, S., Maecker, H., Kambham, N., Mehta, K., Hartman, J., Wang, W., Kretzler, M., Lafayette, R. A. 2020


    BACKGROUND AND OBJECTIVES: IgA nephropathy is the most common primary glomerular disease in the world. Marked by mesangial inflammation and proliferation, it generally leads to progressive kidney fibrosis. As the Janus kinase signal transducer and activator of transcription pathway has been implicated as an important mediator of diabetic kidney disease and FSGS, detailed investigation of this pathway in IgA nephropathy was undertaken to establish the basis for targeting this pathway across glomerular diseases.DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Well characterized patients with IgA nephropathy and controls were studied, allowing us to compare 77 patients with biopsy-proven IgA nephropathy with 45 healthy subjects. STAT phosphorylation was assessed in peripheral blood monocytes (PBMCs) by phosphoflow before and after cytokine stimulation. Kidney Janus kinase signal transducer and activator of transcription activity was studied by immunofluorescence and by transcriptomic studies. An STAT1 activity score was established using downstream transcriptional targets of pSTAT1 and associated with disease and clinical outcomes.RESULTS: We found PBMCs to have upregulated pSTAT production at baseline in patients with IgA nephropathy with a limited reserve to respond to cytokine stimulation compared with controls. Increased staining in glomerular mesangium and endothelium was seen for Jak-2 and pSTAT1 and in the tubulointerstitial for JAK2, pSTAT1, and pSTAT3. Activation of the Janus kinase signal transducer and activator of transcription pathway was further supported by increased pSTAT1 and pSTAT3 scores in glomerular and tubulointerstitial sections of the kidney (glomerular activation Z scores: 7.1 and 4.5, respectively; P values: <0.001 and <0.001, respectively). Clinically, phosphoflow results associated with proteinuria and kidney function, and STAT1 activation associated with proteinuria but was not associated with progression.CONCLUSIONS: Janus kinase signal transducer and activator of transcription signaling was activated in patients with IgA nephropathy compared with controls. There were altered responses in peripheral immune cells and increased message and activated proteins in the kidney. These changes variably related to proteinuria and kidney function.

    View details for DOI 10.2215/CJN.11010919

    View details for PubMedID 32354727

  • A Prospective, Phase 1 Trial of Nivolumab, Ipilimumab, and Radiotherapy in Patients with Advanced Melanoma. Clinical cancer research : an official journal of the American Association for Cancer Research Postow, M. A., Knox, S. J., Goldman, D. A., Elhanati, Y., Mavinkurve, V., Wong, P., Halpenny, D. F., Reddy, S. A., Vado, K. P., McCabe, D., Ramirez, K. A., Macri, M., Schwarzenberger, P., Ricciardi, T., Ryan, A., Venhaus, R. R., Momtaz, P., Shoushtari, A. N., Callahan, M. K., Chapman, P. B., Wolchok, J. D., Subrahmanyam, P. B., Maecker, H. T., Panageas, K. S., Barker, C. A. 2020


    PURPOSE: Preclinical data suggests radiotherapy is beneficial in combination with immune checkpoint blockade. Clinical trials have explored radiotherapy with single agent immune checkpoint blockade, but no trials have reported radiotherapy with the combination of nivolumab and ipilimumab.EXPERIMENTAL DESIGN: We conducted a phase 1 study of patients with stage IV melanoma receiving nivolumab and ipilimumab with two different dose-fractionation schemes of radiotherapy. Patients had at least one melanoma metastasis that would benefit from palliative radiotherapy and one metastasis that would not be irradiated. Nivolumab 1mg/kg + ipilimumab 3mg/kg and extracranial radiotherapy with a dose of 30 Gy in 10 fractions was administered in Cohort A, and then 27 Gy in 3 fractions was administered in Cohort B. The primary outcome was safety.RESULTS: Twenty patients were treated (10 in each cohort). The rates of treatment related grade 3-4 adverse events in Cohort A and Cohort B were 40% and 30%, respectively. There were no grade ≥3 adverse events attributed to radiation. Patients responded to treatment outside of the irradiated volume (Cohort A 5/10; Cohort B 1/9). No evaluable patients had progression of irradiated metastases. Immunologic changes were seen in the peripheral blood with increases in T cell receptor diversity in some responding patients.CONCLUSIONS: Radiotherapy with nivolumab and ipilimumab was safe compared to historical data of nivolumab and ipilimumab alone. Immunologic effects were observed in the peripheral blood. Randomized studies are ongoing to assess whether RT increases the efficacy of nivolumab and ipilimumab.

    View details for DOI 10.1158/1078-0432.CCR-19-3936

    View details for PubMedID 32205463

  • Impaired Immune Health in Survivors of Diffuse Large B-Cell Lymphoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology Shree, T., Li, Q., Glaser, S. L., Brunson, A., Maecker, H. T., Haile, R. W., Levy, R., Keegan, T. H. 2020: JCO1901937


    PURPOSE: Therapeutic advances for diffuse large B-cell lymphoma (DLBCL) have led to an increasing number of survivors. Both DLBCL and its treatments perturb the immune system, yet little is known about immune health during extended survivorship.METHODS: In this retrospective cohort study, we compared 21,690 survivors of DLBCL from the California Cancer Registry (CCR) to survivors of breast, prostate, head and neck, and melanoma cancers. We linked their CCR records to a statewide database documenting hospital, emergency room, and ambulatory surgery visits and investigated the incidence of autoimmune conditions, immune deficiencies, and infections 1-10 years after cancer diagnosis.RESULTS: We found elevated incidence rate ratios (IRRs) for many immune-related conditions in survivors of DLBCL compared with other cancer survivors, including significantly and consistently elevated IRRs for viral and fungal pneumonias (up to 10.8-fold), meningitis (up to 5.3-fold), as well as humoral deficiency (up to 17.6-fold) and autoimmune cytopenias (up to 12-fold). IRRs for most conditions remained high even in the late survivorship period (5-10 years after cancer diagnosis). The elevated risks could not be explained by exposure to chemotherapy, stem-cell transplantation, or rituximab, except for IRRs for humoral deficiency, which were consistently higher after the incorporation of rituximab into DLBCL treatments.CONCLUSION: To our knowledge, this is the largest cohort study with extended follow-up to demonstrate impaired immune health in survivors of DLBCL. The observed persistent, elevated risks for autoimmune diseases, immune deficiencies, and infectious conditions may reflect persistent immune dysregulation caused by lymphoma or treatment and may lead to excess morbidity and mortality during survivorship. Improved understanding of these risks could meaningfully improve long-term care of patients with DLBCL.

    View details for DOI 10.1200/JCO.19.01937

    View details for PubMedID 32083991

  • Sustained outcomes in oral immunotherapy for peanut allergy (POISED study): a large, randomised, double-blind, placebo-controlled, phase 2 study Chinthrajah, S., Purington, N., Andorf, S., Long, A., O'Laughlin, K., Lyu, S., Manohar, M., Boyd, S., Tibshirani, R., Maecker, H., Mukai, K., Tsai, M., Desai, M., Galli, S., Nadeau, K. MOSBY-ELSEVIER. 2020: AB181
  • High-Parametric Evaluation of Human Invariant Natural Killer T Cells to Delineate Heterogeneity in Allo- and Autoimmunity. Blood Erkers, T., Xie, B., Kenyon, L. J., Smith, B., Rieck, M., Jensen, K. P., Ji, X., Basina, M., Strober, S., Negrin, R. S., Maecker, H. T., Meyer, E. 2020


    Human invariant natural killer T cells (iNKTs) are a rare innate-like lymphocyte population that recognize glycolipids presented on CD1d. Studies in mice have shown that these cells are heterogenous and capable of enacting diverse functions, and the composition of iNKT subsets can alter disease outcomes. In contrast, far less is known about how heterogeneity in human iNKTs relates to disease. To address this, we use a high-dimensional, data-driven approach to devise a framework to parse human iNKT heterogeneity. Our data revealed novel and previously described iNKT phenotypes with distinct functions. In particular, we found two phenotypes of interest: 1) a population with Th1 function that was increased with iNKT activation characterized by HLA-II+CD161- expression, and 2) a population with enhanced cytotoxic function characterized by CD4-CD94+ expression. These populations, respectively, correlate with acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation and with new onset type 1 diabetes. Our study identifies human iNKT phenotypes associated with human disease that could aid in the development of biomarkers or therapeutics targeting iNKTs.

    View details for DOI 10.1182/blood.2019001903

    View details for PubMedID 31935280

  • Immunologic effects of forest fire exposure show increases in IL-1β and CRP. Allergy Prunicki, M. M., Dant, C. C., Cao, S., Maecker, H., Haddad, F., Kim, J. B., Snyder, M., Wu, J., Nadeau, K. 2020

    View details for DOI 10.1111/all.14251

    View details for PubMedID 32112439

  • Autoantibody-positive healthy individuals with lower lupus risk display a unique immune endotype. The Journal of allergy and clinical immunology Slight-Webb, S., Smith, M., Bylinska, A., Macwana, S., Guthridge, C., Lu, R., Merrill, J. T., Chakravarty, E., Arriens, C., Munroe, M. E., Maecker, H. T., Utz, P. J., Guthridge, J. M., James, J. A. 2020


    Autoimmune diseases comprise a spectrum of illnesses and are on the rise worldwide. Although anti-nuclear antibodies (ANA) are detected in many autoimmune diseases, up to 20% of healthy women are ANA+ and most will never develop clinical symptoms. Further, disease transition is higher among ANA+ African Americans compared to European Americans.To determine the immune features that might define and prevent transition to clinical autoimmunity in ANA+ healthy individuals.We comprehensively phenotype immune profiles of African Americans and European Americans who are ANA- healthy, ANA+ healthy, or have systemic lupus erythematosus (SLE) using single cell mass cytometry, next-generation RNA sequencing, multiplex cytokine profiling, and phospho-signaling analyses.We found that SLE patients of both races displayed T cell expansion and elevated expression of Type I and II interferon pathways compared to both ANA- and ANA+ healthy individuals. We discovered a unique immune signature that suggests a suppressive immune phenotype and reduced CD11C+ autoimmunity-associated B cells in healthy ANA+ European Americans that is absent in their SLE or even healthy ANA- counterparts, or among African American cohorts. In contrast, ANA+ healthy African Americans exhibited elevated expression of T cell activation markers and higher plasma levels of IL-6 compared to healthy ANA+ European Americans.We propose that this novel immune signature identified in ANA+ healthy European Americans protects them from T cell expansion, heightened activation of interferon pathways, and disease transition.

    View details for DOI 10.1016/j.jaci.2020.04.047

    View details for PubMedID 32446964

  • High-Parameter Immune Profiling with CyTOF. Methods in molecular biology (Clifton, N.J.) Sahaf, B., Rahman, A., Maecker, H. T., Bendall, S. C. 2020; 2055: 351–68


    Mass cytometry, or CyTOF, is a useful technology for high-parameter single-cell phenotyping, especially from suspension cells such as blood or PBMC. It is particularly appealing to monitor the systemic immune changes that could accompany cancer immunotherapy. Here we present a reference panel for identification of all major immune cell populations, with flexibility for addition of trial-specific markers. We also describe best-practice measures for minimizing and tracking batch variability. These include: sample barcoding, use of spiked-in reference cells, and lyophilization of the antibody cocktail. Our protocol assumes the use of cryopreserved PBMC, both for convenience of batching samples and for maximum comparability across patients and time points. Finally, we show an option for automated analysis using the Astrolabe platform (Astrolabe Diagnostics, Inc.).

    View details for DOI 10.1007/978-1-4939-9773-2_16

    View details for PubMedID 31502160

  • The Application of Cytokine Expression Assays to Differentiate Active From Previously Treated Syphilis. The Journal of infectious diseases Kojima, N., Siebert, J. C., Maecker, H., Rosenberg-Hasson, Y., Leon, S. R., Vargas, S. K., Konda, K. A., Caceres, C. F., Klausner, J. D. 2020; 222 (4): 690–94


    To investigate the role of serum cytokine assays to distinguish between active from treated syphilis among serofast patients, we recruited individuals into a prospective cohort study. Participants underwent routine syphilis screening. We selected specimens from a majority cohort of serofast participants with treated and active syphilis. We analyzed specimens with a 62-cytokine multiplex bead-based enzyme-linked immunosorbent assay. Cytokines, brain-derived neurotrophic factor and tumor necrosis factor β, were most predictive. We built a decision tree that was 82.4% accurate, 100% (95% confidence interval, 82%-100%) sensitive, and 45% (18%-75%) specific. Our decision tree differentiated between serum specimens from serofast participants with treated syphilis versus active syphilis.

    View details for DOI 10.1093/infdis/jiaa127

    View details for PubMedID 32189000

  • Cardiovascular Complications in Patients with COVID-19: Consequences of Viral Toxicities and Host Immune Response Curr Cardiol Rep Zhu, H., Rhee, J., Cheng, P., Waliany, S., Chang, A., Witteles, R. M., Maecker, H., Davis, M. M., Nguyen, P. K., Wu, S. M. 2020; 22 (5)
  • The FluPRINT dataset, a multidimensional analysis of the influenza vaccine imprint on the immune system. Scientific data Tomic, A., Tomic, I., Dekker, C. L., Maecker, H. T., Davis, M. M. 2019; 6 (1): 214


    Machine learning has the potential to identify novel biological factors underlying successful antibody responses to influenza vaccines. The first attempts have revealed a high level of complexity in establishing influenza immunity, and many different cellular and molecular components are involved. Of note is that the previously identified correlates of protection fail to account for the majority of individual responses across different age groups and influenza seasons. Challenges remain from the small sample sizes in most studies and from often limited data sets, such as transcriptomic data. Here we report the creation of a unified database, FluPRINT, to enable large-scale studies exploring the cellular and molecular underpinnings of successful antibody responses to influenza vaccines. Over 3,000 parameters were considered, including serological responses to influenza strains, serum cytokines, cell phenotypes, and cytokine stimulations. FluPRINT, facilitates the application of machine learning algorithms for data mining. The data are publicly available and represent a resource to uncover new markers and mechanisms that are important for influenza vaccine immunogenicity.

    View details for DOI 10.1038/s41597-019-0213-4

    View details for PubMedID 31636302

  • Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). European journal of immunology Cossarizza, A., Chang, H., Radbruch, A., Acs, A., Adam, D., Adam-Klages, S., Agace, W. W., Aghaeepour, N., Akdis, M., Allez, M., Almeida, L. N., Alvisi, G., Anderson, G., Andra, I., Annunziato, F., Anselmo, A., Bacher, P., Baldari, C. T., Bari, S., Barnaba, V., Barros-Martins, J., Battistini, L., Bauer, W., Baumgart, S., Baumgarth, N., Baumjohann, D., Baying, B., Bebawy, M., Becher, B., Beisker, W., Benes, V., Beyaert, R., Blanco, A., Boardman, D. A., Bogdan, C., Borger, J. G., Borsellino, G., Boulais, P. E., Bradford, J. A., Brenner, D., Brinkman, R. R., Brooks, A. E., Busch, D. H., Buscher, M., Bushnell, T. P., Calzetti, F., Cameron, G., Cammarata, I., Cao, X., Cardell, S. L., Casola, S., Cassatella, M. A., Cavani, A., Celada, A., Chatenoud, L., Chattopadhyay, P. K., Chow, S., Christakou, E., Cicin-Sain, L., Clerici, M., Colombo, F. S., Cook, L., Cooke, A., Cooper, A. M., Corbett, A. J., Cosma, A., Cosmi, L., Coulie, P. G., Cumano, A., Cvetkovic, L., Dang, V. D., Dang-Heine, C., Davey, M. S., Davies, D., De Biasi, S., Del Zotto, G., Dela Cruz, G. V., Delacher, M., Della Bella, S., Dellabona, P., Deniz, G., Dessing, M., Di Santo, J. P., Diefenbach, A., Dieli, F., Dolf, A., Dorner, T., Dress, R. J., Dudziak, D., Dustin, M., Dutertre, C., Ebner, F., Eckle, S. B., Edinger, M., Eede, P., Ehrhardt, G. R., Eich, M., Engel, P., Engelhardt, B., Erdei, A., Esser, C., Everts, B., Evrard, M., Falk, C. S., Fehniger, T. A., Felipo-Benavent, M., Ferry, H., Feuerer, M., Filby, A., Filkor, K., Fillatreau, S., Follo, M., Forster, I., Foster, J., Foulds, G. A., Frehse, B., Frenette, P. S., Frischbutter, S., Fritzsche, W., Galbraith, D. W., Gangaev, A., Garbi, N., Gaudilliere, B., Gazzinelli, R. T., Geginat, J., Gerner, W., Gherardin, N. A., Ghoreschi, K., Gibellini, L., Ginhoux, F., Goda, K., Godfrey, D. I., Goettlinger, C., Gonzalez-Navajas, J. M., Goodyear, C. S., Gori, A., Grogan, J. L., Grummitt, D., Grutzkau, A., Haftmann, C., Hahn, J., Hammad, H., Hammerling, G., Hansmann, L., Hansson, G., Harpur, C. M., Hartmann, S., Hauser, A., Hauser, A. E., Haviland, D. L., Hedley, D., Hernandez, D. C., Herrera, G., Herrmann, M., Hess, C., Hofer, T., Hoffmann, P., Hogquist, K., Holland, T., Hollt, T., Holmdahl, R., Hombrink, P., Houston, J. P., Hoyer, B. F., Huang, B., Huang, F., Huber, J. E., Huehn, J., Hundemer, M., Hunter, C. A., Hwang, W. Y., Iannone, A., Ingelfinger, F., Ivison, S. M., Jack, H., Jani, P. K., Javega, B., Jonjic, S., Kaiser, T., Kalina, T., Kamradt, T., Kaufmann, S. H., Keller, B., Ketelaars, S. L., Khalilnezhad, A., Khan, S., Kisielow, J., Klenerman, P., Knopf, J., Koay, H., Kobow, K., Kolls, J. K., Kong, W. T., Kopf, M., Korn, T., Kriegsmann, K., Kristyanto, H., Kroneis, T., Krueger, A., Kuhne, J., Kukat, C., Kunkel, D., Kunze-Schumacher, H., Kurosaki, T., Kurts, C., Kvistborg, P., Kwok, I., Landry, J., Lantz, O., Lanuti, P., LaRosa, F., Lehuen, A., LeibundGut-Landmann, S., Leipold, M. D., Leung, L. Y., Levings, M. K., Lino, A. C., Liotta, F., Litwin, V., Liu, Y., Ljunggren, H., Lohoff, M., Lombardi, G., Lopez, L., Lopez-Botet, M., Lovett-Racke, A. E., Lubberts, E., Luche, H., Ludewig, B., Lugli, E., Lunemann, S., Maecker, H. T., Maggi, L., Maguire, O., Mair, F., Mair, K. H., Mantovani, A., Manz, R. A., Marshall, A. J., Martinez-Romero, A., Martrus, G., Marventano, I., Maslinski, W., Matarese, G., Mattioli, A. V., Maueroder, C., Mazzoni, A., McCluskey, J., McGrath, M., McGuire, H. M., McInnes, I. B., Mei, H. E., Melchers, F., Melzer, S., Mielenz, D., Miller, S. D., Mills, K. H., Minderman, H., Mjosberg, J., Moore, J., Moran, B., Moretta, L., Mosmann, T. R., Muller, S., Multhoff, G., Munoz, L. E., Munz, C., Nakayama, T., Nasi, M., Neumann, K., Ng, L. G., Niedobitek, A., Nourshargh, S., Nunez, G., O'Connor, J., Ochel, A., Oja, A., Ordonez, D., Orfao, A., Orlowski-Oliver, E., Ouyang, W., Oxenius, A., Palankar, R., Panse, I., Pattanapanyasat, K., Paulsen, M., Pavlinic, D., Penter, L., Peterson, P., Peth, C., Petriz, J., Piancone, F., Pickl, W. F., Piconese, S., Pinti, M., Pockley, A. G., Podolska, M. J., Poon, Z., Pracht, K., Prinz, I., Pucillo, C. E., Quataert, S. A., Quatrini, L., Quinn, K. M., Radbruch, H., Radstake, T. R., Rahmig, S., Rahn, H., Rajwa, B., Ravichandran, G., Raz, Y., Rebhahn, J. A., Recktenwald, D., Reimer, D., Reis E Sousa, C., Remmerswaal, E. B., Richter, L., Rico, L. G., Riddell, A., Rieger, A. M., Robinson, J. P., Romagnani, C., Rubartelli, A., Ruland, J., Saalmuller, A., Saeys, Y., Saito, T., Sakaguchi, S., Sala-de-Oyanguren, F., Samstag, Y., Sanderson, S., Sandrock, I., Santoni, A., Sanz, R. B., Saresella, M., Sautes-Fridman, C., Sawitzki, B., Schadt, L., Scheffold, A., Scherer, H. U., Schiemann, M., Schildberg, F. A., Schimisky, E., Schlitzer, A., Schlosser, J., Schmid, S., Schmitt, S., Schober, K., Schraivogel, D., Schuh, W., Schuler, T., Schulte, R., Schulz, A. R., Schulz, S. R., Scotta, C., Scott-Algara, D., Sester, D. P., Shankey, T. V., Silva-Santos, B., Simon, A. K., Sitnik, K. M., Sozzani, S., Speiser, D. E., Spidlen, J., Stahlberg, A., Stall, A. M., Stanley, N., Stark, R., Stehle, C., Steinmetz, T., Stockinger, H., Takahama, Y., Takeda, K., Tan, L., Tarnok, A., Tiegs, G., Toldi, G., Tornack, J., Traggiai, E., Trebak, M., Tree, T. I., Trotter, J., Trowsdale, J., Tsoumakidou, M., Ulrich, H., Urbanczyk, S., van de Veen, W., van den Broek, M., van der Pol, E., Van Gassen, S., Van Isterdael, G., van Lier, R. A., Veldhoen, M., Vento-Asturias, S., Vieira, P., Voehringer, D., Volk, H., von Borstel, A., von Volkmann, K., Waisman, A., Walker, R. V., Wallace, P. K., Wang, S. A., Wang, X. M., Ward, M. D., Ward-Hartstonge, K. A., Warnatz, K., Warnes, G., Warth, S., Waskow, C., Watson, J. V., Watzl, C., Wegener, L., Weisenburger, T., Wiedemann, A., Wienands, J., Wilharm, A., Wilkinson, R. J., Willimsky, G., Wing, J. B., Winkelmann, R., Winkler, T. H., Wirz, O. F., Wong, A., Wurst, P., Yang, J. H., Yang, J., Yazdanbakhsh, M., Yu, L., Yue, A., Zhang, H., Zhao, Y., Ziegler, S. M., Zielinski, C., Zimmermann, J., Zychlinsky, A. 2019; 49 (10): 1457–1973


    These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

    View details for DOI 10.1002/eji.201970107

    View details for PubMedID 31633216

  • Reversal of epigenetic aging and immunosenescent trends in humans. Aging cell Fahy, G. M., Brooke, R. T., Watson, J. P., Good, Z., Vasanawala, S. S., Maecker, H., Leipold, M. D., Lin, D. T., Kobor, M. S., Horvath, S. 2019: e13028


    Epigenetic "clocks" can now surpass chronological age in accuracy for estimating biological age. Here, we use four such age estimators to show that epigenetic aging can be reversed in humans. Using a protocol intended to regenerate the thymus, we observed protective immunological changes, improved risk indices for many age-related diseases, and a mean epigenetic age approximately 1.5years less than baseline after 1year of treatment (-2.5-year change compared to no treatment at the end of the study). The rate of epigenetic aging reversal relative to chronological age accelerated from -1.6year/year from 0-9month to -6.5year/year from 9-12month. The GrimAge predictor of human morbidity and mortality showed a 2-year decrease in epigenetic vs. chronological age that persisted six months after discontinuing treatment. This is to our knowledge the first report of an increase, based on an epigenetic age estimator, in predicted human lifespan by means of a currently accessible aging intervention.

    View details for DOI 10.1111/acel.13028

    View details for PubMedID 31496122

  • Comprehensive Immune Monitoring of Clinical Trials to Advance Human Immunotherapy. Cell reports Hartmann, F. J., Babdor, J., Gherardini, P. F., Amir, E. D., Jones, K., Sahaf, B., Marquez, D. M., Krutzik, P., O'Donnell, E., Sigal, N., Maecker, H. T., Meyer, E., Spitzer, M. H., Bendall, S. C. 2019; 28 (3): 819


    The success of immunotherapy has led to a myriad of clinical trials accompanied by efforts to gain mechanistic insight and identify predictive signatures for personalization. However, many immune monitoring technologies face investigator bias, missing unanticipated cellular responses in limited clinical material. We present here a mass cytometry (CyTOF) workflow for standardized, systems-level biomarker discovery in immunotherapy trials. To broadly enumerate immune cell identity and activity, we established and extensively assessed a reference panel of 33 antibodies to cover major cell subsets, simultaneously quantifying activation and immune checkpoint molecules in a single assay. This assay enumerates ≥98% of peripheral immune cells with ≥4 positively identifying antigens. Robustness and reproducibility are demonstrated on multiple samples types, across two research centers and by orthogonal measurements. Using automated analysis, we identify stratifying immune signatures in bone marrow transplantation-associated graft-versus-host disease. Together, this validated workflow ensures comprehensive immunophenotypic analysis and data comparability and will accelerate biomarker discovery.

    View details for DOI 10.1016/j.celrep.2019.06.049

    View details for PubMedID 31315057

  • Diminished B-Cell Response After Repeat Influenza Vaccination JOURNAL OF INFECTIOUS DISEASES Sanyal, M., Holmes, T. H., Maecker, H. T., Albrecht, R. A., Dekker, C. L., He, X., Greenberg, H. B. 2019; 219 (10): 1586–95
  • Vaccine-Induced Memory CD8(+) T Cells Provide Clinical Benefit in HER2 Expressing Breast Cancer: A Mouse to Human Translational Study CLINICAL CANCER RESEARCH Crosby, E. J., Gwin, W. R., Blackwell, K., Marcom, P., Chang, S., Maecker, H. T., Broadwater, G., Hyslop, T. M., Kim, S., Rogatko, A., Lubkov, V., Snyder, J. C., Osada, T., Hobeika, A. C., Morse, M. A., Lyerly, H., Hartman, Z. C. 2019; 25 (9): 2725–36
  • Getting the Most from Your High-Dimensional Cytometry Data. Immunity Olsen, L. R., Pedersen, C. B., Leipold, M. D., Maecker, H. T. 2019; 50 (3): 535–36

    View details for PubMedID 30893579

  • A clinically meaningful metric of immune age derived from high-dimensional longitudinal monitoring. Nature medicine Alpert, A., Pickman, Y., Leipold, M., Rosenberg-Hasson, Y., Ji, X., Gaujoux, R., Rabani, H., Starosvetsky, E., Kveler, K., Schaffert, S., Furman, D., Caspi, O., Rosenschein, U., Khatri, P., Dekker, C. L., Maecker, H. T., Davis, M. M., Shen-Orr, S. S. 2019


    Immune responses generally decline with age. However, the dynamics of this process at the individual level have not been characterized, hindering quantification of an individual's immune age. Here, we use multiple 'omics' technologies to capture population- and individual-level changes in the human immune system of 135 healthy adult individuals of different ages sampled longitudinally over a nine-year period. We observed high inter-individual variability in the rates of change of cellular frequencies that was dictated by their baseline values, allowing identification of steady-state levels toward which a cell subset converged and the ordered convergence of multiple cell subsets toward an older adult homeostasis. These data form a high-dimensional trajectory of immune aging (IMM-AGE) that describes a person's immune status better than chronological age. We show that the IMM-AGE score predicted all-cause mortality beyond well-established risk factors in the Framingham Heart Study, establishing its potential use in clinics for identification of patients at risk.

    View details for PubMedID 30842675

  • Penalized Supervised Star Plots: Example Application in Influenza-Specific CD4+T Cells VIRAL IMMUNOLOGY Holmes, T. H., Subrahmanyam, P. B., Wang, W., Maecker, H. T. 2019; 32 (2): 102–9
  • The anatomy of single cell mass cytometry data CYTOMETRY PART A Olsen, L. R., Leipold, M. D., Pedersen, C. B., Maecker, H. 2019; 95A (2): 156–72
  • Mycophenolate mofetil reduces STAT3 phosphorylation in systemic lupus erythematosus patients. JCI insight Slight-Webb, S., Guthridge, J. M., Chakravarty, E. F., Chen, H., Lu, R., Macwana, S., Bean, K., Maecker, H. T., Utz, P. J., James, J. A. 2019; 4 (2)


    Systemic lupus erythematosus (SLE) is a highly variable autoimmune disease that can involve severe organ-threatening symptoms, such as lupus nephritis. Certain drugs, such as mycophenolate mofetil (MMF), are effective at reducing morbidity associated with nephritis; however, the immune pathways associated with disease suppression are poorly defined. Here, we provide evidence that MMF inhibits phosphorylation of STAT3 and other associated immune pathways. Using mass cytometry and bead-based or ELISA assays, the systemic phenotype of SLE patients not taking (MMF-) or taking (MMF+) MMF were studied. MMF+ SLE patients had significant reductions in total numbers of transitional B cells, plasmablasts, and T cells, specifically CD4+ Th17-type and CD4+ Treg-type cells, compared with MMF- patients. Plasma soluble mediators were decreased in MMF+ patients including chemokines (MIG/CXCL9 and SDF-1alpha/CXCL12) and growth factors (VEGF-A and PDGF-BB). Soluble mediators and cell subsets grouped by functional properties revealed significant modifications associated with STAT3 and B cell pathways. Further, healthy PBMCs treated with IL-6 revealed a reduction in p-STAT3 following the addition of mycophenolic acid (the active metabolite of MMF). In conclusion, the inhibition of STAT3 phosphorylation by MMF may explain the effectiveness of this treatment in SLE patients, since increased levels of p-STAT3 are associated with disease pathology.

    View details for PubMedID 30674728

  • Sustained outcomes in oral immunotherapy for peanut allergy (POISED study): a large, randomised, double-blind, placebo-controlled, phase 2 study. Lancet (London, England) Chinthrajah, R. S., Purington, N., Andorf, S., Long, A., O'Laughlin, K. L., Lyu, S. C., Manohar, M., Boyd, S. D., Tibshirani, R., Maecker, H., Plaut, M., Mukai, K., Tsai, M., Desai, M., Galli, S. J., Nadeau, K. C. 2019


    Dietary avoidance is recommended for peanut allergies. We evaluated the sustained effects of peanut allergy oral immunotherapy (OIT) in a randomised long-term study in adults and children.In this randomised, double-blind, placebo-controlled, phase 2 study, we enrolled participants at the Sean N Parker Center for Allergy and Asthma Research at Stanford University (Stanford, CA, USA) with peanut allergy aged 7-55 years with a positive result from a double-blind, placebo-controlled, food challenge (DBPCFC; ≤500 mg of peanut protein), a positive skin-prick test (SPT) result (≥5 mm wheal diameter above the negative control), and peanut-specific immunoglobulin (Ig)E concentration of more than 4 kU/L. Participants were randomly assigned (2·4:1·4:1) in a two-by-two block design via a computerised system to be built up and maintained on 4000 mg peanut protein through to week 104 then discontinued on peanut (peanut-0 group), to be built up and maintained on 4000 mg peanut protein through to week 104 then to ingest 300 mg peanut protein daily (peanut-300 group) for 52 weeks, or to receive oat flour (placebo group). DBPCFCs to 4000 mg peanut protein were done at baseline and weeks 104, 117, 130, 143, and 156. The pharmacist assigned treatment on the basis of a randomised computer list. Peanut or placebo (oat) flour was administered orally and participants and the study team were masked throughout by use of oat flour that was similar in look and feel to the peanut flour and nose clips, as tolerated, to mask taste. The statistician was also masked. The primary endpoint was the proportion of participants who passed DBPCFCs to a cumulative dose of 4000 mg at both 104 and 117 weeks. The primary efficacy analysis was done in the intention-to-treat population. Safety was assessed in the intention-to-treat population. This trial is registered at, NCT02103270.Between April 15, 2014, and March 2, 2016, of 152 individuals assessed, we enrolled 120 participants, who were randomly assigned to the peanut-0 (n=60), peanut-300 (n=35), and placebo groups (n=25). 21 (35%) of peanut-0 group participants and one (4%) placebo group participant passed the 4000 mg challenge at both 104 and 117 weeks (odds ratio [OR] 12·7, 95% CI 1·8-554·8; p=0·0024). Over the entire study, the most common adverse events were mild gastrointestinal symptoms, which were seen in 90 of 120 patients (50/60 in the peanut-0 group, 29/35 in the peanut-300 group, and 11/25 in the placebo group) and skin disorders, which were seen in 50/120 patients (26/60 in the peanut-0 group, 15/35 in the peanut-300 group, and 9/25 in the placebo group). Adverse events decreased over time in all groups. Two participants in the peanut groups had serious adverse events during the 3-year study. In the peanut-0 group, in which eight (13%) of 60 participants passed DBPCFCs at week 156, higher baseline peanut-specific IgG4 to IgE ratio and lower Ara h 2 IgE and basophil activation responses were associated with sustained unresponsiveness. No treatment-related deaths occurred.Our study suggests that peanut OIT could desensitise individuals with peanut allergy to 4000 mg peanut protein but discontinuation, or even reduction to 300 mg daily, could increase the likelihood of regaining clinical reactivity to peanut. Since baseline blood tests correlated with week 117 treatment outcomes, this study might aid in optimal patient selection for this therapy.National Institute of Allergy and Infectious Diseases.

    View details for DOI 10.1016/S0140-6736(19)31793-3

    View details for PubMedID 31522849

  • Pembrolizumab in Relapsed and Refractory Mycosis Fungoides and Sézary Syndrome: A Multicenter Phase II Study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology Khodadoust, M. S., Rook, A. H., Porcu, P., Foss, F., Moskowitz, A. J., Shustov, A., Shanbhag, S., Sokol, L., Fling, S. P., Ramchurren, N., Pierce, R., Davis, A., Shine, R., Li, S., Fong, S., Kim, J., Yang, Y., Blumenschein, W. M., Yearley, J. H., Das, B., Patidar, R., Datta, V., Cantu, E., McCutcheon, J. N., Karlovich, C., Williams, P. M., Subrahmanyam, P. B., Maecker, H. T., Horwitz, S. M., Sharon, E., Kohrt, H. E., Cheever, M. A., Kim, Y. H. 2019: JCO1901056


    To assess the efficacy of pembrolizumab in patients with advanced relapsed or refractory mycosis fungoides (MF) or Sézary syndrome (SS).CITN-10 is a single-arm, multicenter phase II trial of 24 patients with advanced MF or SS. Patients were treated with pembrolizumab 2 mg/kg every 3 weeks for up to 24 months. The primary end point was overall response rate by consensus global response criteria.Patients had advanced-stage disease (23 of 24 with stage IIB to IV MF/SS) and were heavily pretreated with a median of four prior systemic therapies. The overall response rate was 38% with two complete responses and seven partial responses. Of the nine responding patients, six had 90% or more improvement in skin disease by modified Severity Weighted Assessment Tool, and eight had ongoing responses at last follow-up. The median duration of response was not reached, with a median response follow-up time of 58 weeks. Immune-related adverse events led to treatment discontinuation in four patients. A transient worsening of erythroderma and pruritus occurred in 53% of patients with SS. This cutaneous flare reaction did not result in treatment discontinuation for any patient. The flare reaction correlated with high PD-1 expression on Sézary cells but did not associate with subsequent clinical responses or lack of response. Treatment responses did not correlate with expression of PD-L1, total mutation burden, or an interferon-γ gene expression signature.Pembrolizumab demonstrated significant antitumor activity with durable responses and a favorable safety profile in patients with advanced MF/SS.

    View details for DOI 10.1200/JCO.19.01056

    View details for PubMedID 31532724

  • SIMON, an Automated Machine Learning System, Reveals Immune Signatures of Influenza Vaccine Responses. Journal of immunology (Baltimore, Md. : 1950) Tomic, A., Tomic, I., Rosenberg-Hasson, Y., Dekker, C. L., Maecker, H. T., Davis, M. M. 2019


    Machine learning holds considerable promise for understanding complex biological processes such as vaccine responses. Capturing interindividual variability is essential to increase the statistical power necessary for building more accurate predictive models. However, available approaches have difficulty coping with incomplete datasets which is often the case when combining studies. Additionally, there are hundreds of algorithms available and no simple way to find the optimal one. In this study, we developed Sequential Iterative Modeling "OverNight" (SIMON), an automated machine learning system that compares results from 128 different algorithms and is particularly suitable for datasets containing many missing values. We applied SIMON to data from five clinical studies of seasonal influenza vaccination. The results reveal previously unrecognized CD4+ and CD8+ T cell subsets strongly associated with a robust Ab response to influenza Ags. These results demonstrate that SIMON can greatly speed up the choice of analysis modalities. Hence, it is a highly useful approach for data-driven hypothesis generation from disparate clinical datasets. Our strategy could be used to gain biological insight from ever-expanding heterogeneous datasets that are publicly available.

    View details for DOI 10.4049/jimmunol.1900033

    View details for PubMedID 31201239

  • Value of Neutrophil to Lymphocyte Ratio and Its Trajectory in Patients Hospitalized With Acute Heart Failure and Preserved Ejection Fraction. The American journal of cardiology Boralkar, K. A., Kobayashi, Y., Amsallem, M., Ataam, J. A., Moneghetti, K. J., Cauwenberghs, N., Horne, B. D., Knowlton, K. U., Maecker, H., Kuznetsova, T., Heidenreich, P. A., Haddad, F. 2019


    The neutrophil to lymphocyte ratio (NLR) has been proposed as a simple and routinely obtained marker of inflammation. This study sought to determine whether the NLR on admission as well as NLR trajectory would be complementary to the Get with the Guidelines Heart Failure (GWTG-HF) risk score in patients hospitalized with acute heart failure with preserved ejection fraction (HFpEF).Using the Stanford Translational Research Database, we identified 443 patients between January 2002 and December 2013 hospitalized with acute HFpEF and with complete data of NLR both on admission and at discharge. The primary endpoint was all-cause mortality. Mean age was 77 ± 16 years, 58% were female, with a high prevalence of diabetes mellitus (35.4%), coronary artery disease (58.2%), systemic hypertension (96.6%) and history of atrial fibrillation (57.5%). Over a median follow-up of 2.2 years, 121 (27.3%) patients died. The median NLR on admission was 6.5 (IQR 3.6 - 11.1); a majority of patients decreased their NLR during the course of hospitalization. On multivariable Cox modeling, both NLR on admission (HR 1.18 95% CI (1.00 - .38), p = 0.04) and absolute NLR trajectory (HR 1.26 95% CI (1.10 - 1.45), p = 0.001) were shown to be incremental to GWTG-HF risk score (p < 0.05) for outcome prediction. Adding the NLR or absolute NLR trajectory to the GWTG-HF risk score significantly improved the area under the operator-receiver curve and the reclassification up to 3 years after admission.This simple, readily available marker of inflammation may be useful when stratifying the risk of patients hospitalized with HFpEF.

    View details for DOI 10.1016/j.amjcard.2019.10.020

    View details for PubMedID 31753313

  • Guidelines for Gating Flow Cytometry Data for Immunological Assays. Methods in molecular biology (Clifton, N.J.) Staats, J., Divekar, A., McCoy, J. P., Maecker, H. T. 2019; 2032: 81–104


    "Gating" refers to the selection of successive subpopulations of cells for analysis in flow cytometry. It is usually performed manually, based on expert knowledge of cell characteristics. However, there can be considerable disagreement in how gates should be applied, even between individuals experienced in the field. While clinical software often automates gating, and some guidelines do exist (especially for clinical assays), there are no comprehensive guidelines across the various types of immunological assays performed using flow cytometry. Here we attempt to provide such guidelines, focused on the most general and pervasive types of gates, why they are important, and what recommendations can be made regarding their use. We do so through the display of example data, collected by academic, government, and industry representatives. These guidelines should be of value to both novice and experienced flow cytometrists analyzing a wide variety of immunological assays.

    View details for DOI 10.1007/978-1-4939-9650-6_5

    View details for PubMedID 31522414

  • Autoimmunity to hypocretin and molecular mimicry to flu in type 1 narcolepsy. Proceedings of the National Academy of Sciences of the United States of America Luo, G., Ambati, A., Lin, L., Bonvalet, M., Partinen, M., Ji, X., Maecker, H. T., Mignot, E. J. 2018


    Type 1 narcolepsy (T1N) is caused by hypocretin/orexin (HCRT) neuronal loss. Association with the HLA DQB1*06:02/DQA1*01:02 (98% vs. 25%) heterodimer (DQ0602), T cell receptors (TCR) and other immune loci suggest autoimmunity but autoantigens are unknown. Onset is seasonal and associated with influenza A, notably pandemic 2009 H1N1 (pH1N1) infection and vaccination (Pandemrix). Peptides derived from HCRT and influenza A, including pH1N1, were screened for DQ0602 binding and presence of cognate DQ0602 tetramer-peptide-specific CD4+ T cells tested in 35 T1N cases and 22 DQ0602 controls. Higher reactivity to influenza pHA273-287 (pH1N1 specific), PR8 (H1N1 pre-2009 and H2N2)-specific NP17-31 and C-amidated but not native version of HCRT54-66 and HCRT86-97 (HCRTNH2) were observed in T1N. Single-cell TCR sequencing revealed sharing of CDR3beta TRBV4-2-CASSQETQGRNYGYTF in HCRTNH2 and pHA273-287-tetramers, suggesting molecular mimicry. This public CDR3beta uses TRBV4-2, a segment modulated by T1N-associated SNP rs1008599, suggesting causality. TCR-alpha/beta CDR3 motifs of HCRT54-66-NH2 and HCRT86-97-NH2 tetramers were extensively shared: notably public CDR3alpha, TRAV2-CAVETDSWGKLQF-TRAJ24, that uses TRAJ24, a chain modulated by T1N-associated SNPs rs1154155 and rs1483979. TCR-alpha/beta CDR3 sequences found in pHA273-287, NP17-31, and HCRTNH2 tetramer-positive CD4+ cells were also retrieved in single INF-gamma-secreting CD4+ sorted cells stimulated with Pandemrix, independently confirming these results. Our results provide evidence for autoimmunity and molecular mimicry with flu antigens modulated by genetic components in the pathophysiology of T1N.

    View details for PubMedID 30541895

  • The anatomy of single cell mass cytometry data. Cytometry. Part A : the journal of the International Society for Analytical Cytology Olsen, L. R., Leipold, M. D., Pedersen, C. B., Maecker, H. T. 2018


    Mass cytometry enables the measurement of up to 50 features on single cell. This has catalyzed a shift toward multidimensional data analysis methods, rather than the manual gating strategies as traditionally for in flow cytometry data. This shift means that data scientists are involved in the analysis process to an increasing degree. As the data is analyzed in a more unbiased fashion, where noisy or uninformative observations are not easily excluded, a deeper knowledge of the origin, noise, and modalities of the data is therefore needed to embark on useful data analysis. In this primer, we introduce the idiosyncrasies of mass cytometry data with a focus on the technical properties of how data generated with the CyTOF system, and the characteristics of protein expression in the cells of the hematopoietic continuum, specifically targeted toward data scientists. We also provide a comprehensive online repository of scripts, tutorials, and example data.

    View details for PubMedID 30277658

  • Interleukin 4 is inactivated via selective disulfide-bond reduction by extracellular thioredoxin. Proceedings of the National Academy of Sciences of the United States of America Plugis, N. M., Weng, N., Zhao, Q., Palanski, B. A., Maecker, H. T., Habtezion, A., Khosla, C. 2018


    Thioredoxin 1 (TRX), an essential intracellular redox regulator, is also secreted by mammalian cells. Recently, we showed that TRX activates extracellular transglutaminase 2 via reduction of an allosteric disulfide bond. In an effort to identify other extracellular substrates of TRX, macrophages derived from THP-1 cells were treated with NP161, a small-molecule inhibitor of secreted TRX. NP161 enhanced cytokine outputs of alternatively activated macrophages, suggesting that extracellular TRX regulated the activity of interleukin 4 (IL-4) and/or interleukin 13 (IL-13). To test this hypothesis, the C35S mutant of human TRX was shown to form a mixed disulfide bond with recombinant IL-4 but not IL-13. Kinetic analysis revealed a kcat/KM value of 8.1 muM-1min-1 for TRX-mediated recognition of IL-4, which established this cytokine as the most selective partner of extracellular TRX to date. Mass spectrometry identified the C46-C99 bond of IL-4 as the target of TRX, consistent with the essential role of this disulfide bond in IL-4 activity. To demonstrate the physiological relevance of our biochemical findings, recombinant TRX was shown to attenuate IL-4-dependent proliferation of cultured TF-1 erythroleukemia cells and also to inhibit the progression of chronic pancreatitis in an IL-4-driven mouse model of this disease. By establishing that IL-4 is posttranslationally regulated by TRX-promoted reduction of a disulfide bond, our findings highlight a novel regulatory mechanism of the type 2 immune response that is specific to IL-4 over IL-13.

    View details for PubMedID 30104382

  • JAK-STAT signaling is activated in the kidney and peripheral blood cells of patients with focal segmental glomerulosclerosis. Kidney international Tao, J., Mariani, L., Eddy, S., Maecker, H., Kambham, N., Mehta, K., Hartman, J., Wang, W., Kretzler, M., Lafayette, R. A. 2018


    Focal segmental glomerular sclerosis (FSGS) is a devastating disease with limited treatment options and poor prognosis. Activated JAK-STAT signaling has been implicated in other kidney diseases. Since new technologies allow us to better evaluate changes in systemic and renal JAK-STAT activity as it relates to kidney function, we examined this in 106 patients with biopsy-proven FSGS compared to 47 healthy control individuals. Peripheral immune function was assessed in peripheral blood mononuclear cells by phosphoflow studies before and after cytokine stimulation. Kidney JAK-STAT activity was measured by immunofluorescence and by transcriptomics. A STAT1 activity score was calculated by evaluating message status of downstream targets of pSTAT1. Peripheral blood mononuclear cells were found to be upregulated in terms of pSTAT production at baseline in FSGS and to have limited reserve to respond to various cytokines. Increased staining for components of the JAK-STAT system in FSGS by microscopy was found. Furthermore, we found transcriptomic evidence for activation of JAK-STAT that increased pSTAT 1 and pSTAT 3 in glomerular and tubulointerstitial sections of the kidney. Some of these changes were associated with the likelihood of remission of proteinuria and progression of disease. JAK-STAT signaling is altered in patients with FSGS as compared to healthy controls with activated peripheral immune cells, increased message in the kidney and increased activated proteins in the kidney. Thus, our findings support immune activation in this disease and point to the JAK-STAT pathway as a potential target for treatment of FSGS.

    View details for PubMedID 30093081

  • Baseline immune profile by CyTOF can predict response to an investigational adjuvanted vaccine in elderly adults. Journal of translational medicine Lingblom, C. M., Kowli, S., Swaminathan, N., Maecker, H. T., Lambert, S. L. 2018; 16 (1): 153


    BACKGROUND: Mass cytometry, or CyTOF (Cytometry by Time-of-Flight), permits the simultaneous detection of over 40 phenotypic and functional immune markers in individual cells without the issues of spectral overlap seen in traditional flow cytometry.METHODS: In this study, we applied CyTOF to comprehensively characterize the circulating immune cell populations in elderly individuals both before and after administration of an investigational adjuvanted protein vaccine against respiratory syncytial virus (RSV) in a Phase 1a trial. Antigen-specific T cell responses to RSV by IFNgamma ELISPOT had been observed in most but not all recipients in the highest dose cohort in this trial. Here, CyTOF was used to characterize the cellular response profile of ELISPOT responders and non-responders in this vaccine dose cohort.RESULTS: Both CD4+ and CD8+ T cell antigen-specific IFNgamma responses were observed. Principal components analysis revealed baseline differences between responders and non-responders, including differences in activated (HLA-DR+) CD4+ and CD8+ T cells, which were higher in non-responders versus responders. Using viSNE to analyze RSV-responsive CD4+ and CD8+ T cells, we also found increased expression of HLA-DR, CCR7, CD127 and CD69 in non-responders versus responders.CONCLUSIONS: High parameter CyTOF can help profile immune components associated with differential vaccine responsiveness.

    View details for PubMedID 29866115

  • Comparison of CyTOF assays across sites: Results of a six-center pilot study JOURNAL OF IMMUNOLOGICAL METHODS Leipold, M. D., Obermoser, G., Fenwick, C., Kleinstuber, K., Rashidi, N., McNevin, J. P., Nau, A. N., Wagar, L. E., Rozot, V., Davis, M. M., DeRosa, S., Pantaleo, G., Scriba, T. J., Walker, B. D., Olsen, L. R., Maecker, H. T. 2018; 453: 37–43


    For more than five years, high-dimensional mass cytometry has been employed to study immunology. However, these studies have typically been performed in one laboratory on one or few instruments. We present the results of a six-center study using healthy control human peripheral blood mononuclear cells (PBMCs) and commercially available reagents to test the intra-site and inter-site variation of mass cytometers and operators. We used prestained controls generated by the primary center as a reference to compare against samples stained at each individual center. Data were analyzed at the primary center, including investigating the effects of two normalization methods. All six sites performed similarly, with CVs for both Frequency of Parent and median signal intensity (MSI) values<30%. Increased background was seen when using the premixed antibody cocktail aliquots at each site, suggesting that cocktails are best made fresh. Both normalization methods tested performed adequately for normalizing MSI values between centers. Clustering algorithms revealed slight differences between the prestained and the sites-stained samples, due mostly to the increased background of a few antibodies. Therefore, we believe that multicenter mass cytometry assays are feasible.

    View details for PubMedID 29174717

    View details for PubMedCentralID PMC5805584

  • Food allergy and omics. The Journal of allergy and clinical immunology Dhondalay, G. K., Rael, E., Acharya, S., Zhang, W., Sampath, V., Galli, S. J., Tibshirani, R., Boyd, S. D., Maecker, H., Nadeau, K. C., Andorf, S. 2018; 141 (1): 20–29


    Food allergy (FA) prevalence has been increasing over the last few decades and is now a global health concern. Current diagnostic methods for FA result in a high number of false-positive results, and the standard of care is either allergen avoidance or use of epinephrine on accidental exposure, although currently with no other approved treatments. The increasing prevalence of FA, lack of robust biomarkers, and inadequate treatments warrants further research into the mechanism underlying food allergies. Recent technological advances have made it possible to move beyond traditional biological techniques to more sophisticated high-throughput approaches. These technologies have created the burgeoning field of omics sciences, which permit a more systematic investigation of biological problems. Omics sciences, such as genomics, epigenomics, transcriptomics, proteomics, metabolomics, microbiomics, and exposomics, have enabled the construction of regulatory networks and biological pathway models. Parallel advances in bioinformatics and computational techniques have enabled the integration, analysis, and interpretation of these exponentially growing data sets and opens the possibility of personalized or precision medicine for FA.

    View details for PubMedID 29307411

  • Multiparameter Intracellular Cytokine Staining FLOW CYTOMETRY PROTOCOLS, 4TH EDITION Lovelace, P., Maecker, H. T., Hawley, T. S., Hawley, R. G. 2018; 1678: 151–66


    Intracellular cytokine staining is a popular method for visualizing cellular responses, most often T-cell responses to antigenic or mitogenic stimulation. It can be coupled with staining for other functional markers, such as upregulation of CD107 or CD154, as well as phenotypic markers that define specific cellular subsets, e.g., effector and memory T-cell compartments, NK cells, or monocytes. Recent advances in multicolor flow cytometry instrumentation and software have allowed the routine combination of 12 or more markers, creating some technical and analytical challenges along the way, and exposing a need for standardization in the field. Here, we will review best practices for antibody panel design and procedural variables for multicolor intracellular cytokine staining, and present an optimized protocol with variations designed for use with specific markers and sample types.

    View details for PubMedID 29071680

  • Mass Cytometry Assays for Antigen-Specific T Cells Using CyTOF FLOW CYTOMETRY PROTOCOLS, 4TH EDITION Lin, D., Maecker, H. T., Hawley, T. S., Hawley, R. G. 2018; 1678: 37–47


    T Cells specific for a single antigen tend to be rare, even after expansion of memory cells. They are commonly detected by in vitro stimulation with peptides or protein, followed by staining for intracellular cytokines. In this protocol, we use CyTOF® mass cytometry to collect single-cell data on a large number of cytokines/chemokines, as well as cell-surface proteins that characterize T cells and other immune cells. We also include a method for magnetic bead enrichment of antigen-stimulated T cells, based on their expression of CD154 and CD69. Coupling magnetic enrichment with highly multi-parameter mass cytometry, this method enables the ability to dissect the frequency, phenotype, and function of antigen-specific T cells in greater detail than previously possible. Rare cell subsets can be examined, while minimizing run times on the CyTOF.

    View details for PubMedID 29071674

    View details for PubMedCentralID PMC5798871

  • Distinct predictive biomarker candidates for response to anti-CTLA-4 and anti-PD-1 immunotherapy in melanoma patients. Journal for immunotherapy of cancer Subrahmanyam, P. B., Dong, Z., Gusenleitner, D., Giobbie-Hurder, A., Severgnini, M., Zhou, J., Manos, M., Eastman, L. M., Maecker, H. T., Hodi, F. S. 2018; 6 (1): 18


    While immune checkpoint blockade has greatly improved clinical outcomes in diseases such as melanoma, there remains a need for predictive biomarkers to determine who will likely benefit most from which therapy. To date, most biomarkers of response have been identified in the tumors themselves. Biomarkers that could be assessed from peripheral blood would be even more desirable, because of ease of access and reproducibility of sampling.We used mass cytometry (CyTOF) to comprehensively profile peripheral blood of melanoma patients, in order to find predictive biomarkers of response to anti-CTLA-4 or anti-PD-1 therapy. Using a panel of ~ 40 surface and intracellular markers, we performed in-depth phenotypic and functional immune profiling to identify potential predictive biomarker candidates.Immune profiling of baseline peripheral blood samples using CyTOF revealed that anti-CTLA-4 and anti-PD-1 therapies have distinct sets of candidate biomarkers. The distribution of CD4+ and CD8+ memory/non-memory cells and other memory subsets was different between responders and non-responders to anti-CTLA-4 therapy. In anti-PD-1 (but not anti-CTLA-4) treated patients, we discovered differences in CD69 and MIP-1β expressing NK cells between responders and non-responders. Finally, multivariate analysis was used to develop a model for the prediction of response.Our results indicate that anti-CTLA-4 and anti-PD-1 have distinct predictive biomarker candidates. CD4+ and CD8+ memory T cell subsets play an important role in response to anti-CTLA-4, and are potential biomarker candidates. For anti-PD-1 therapy, NK cell subsets (but not memory T cell subsets) correlated with clinical response to therapy. These functionally active NK cell subsets likely play a critical role in the anti-tumor response triggered by anti-PD-1.

    View details for PubMedID 29510697

    View details for PubMedCentralID PMC5840795

  • Differences in multiple immune parameters between Indian and U.S. infants. PloS one Rathore, D. K., Holmes, T. H., Nadeau, K. C., Mittal, P., Batra, A., Rosenberg-Hasson, Y., Sopory, S., Gupta, R., Chellani, H. K., Aggarwal, K. C., Bal, V., Natchu, U. C., Bhatnagar, S., Tavassoli, M., Lyell, D. J., Rath, S., Wadhwa, N., Maecker, H. T. 2018; 13 (11): e0207297


    To compare immune phenotypes across two geographic and ethnic communities, we examined umbilical cord blood by flow cytometry and Luminex in parallel cohorts of 53 newborns from New Delhi, India, and 46 newborns from Stanford, California. We found that frequencies of a B cell subset suggested to be B-1-like, and serum IgM concentration were both significantly higher in the Stanford cohort, independent of differences in maternal age. While serum IgA levels were also significantly higher in the Stanford cohort, IgG1, IgG2, and IgG4 were significantly higher in the New Delhi samples. We found that neutrophils, plasmacytoid dendritic cells, CD8+ T cells, and total T cells were higher in the U.S. cohort, while dendritic cells, patrolling monocytes (CD14dimCD16+), natural killer cells, CD4+ T cells, and naive B cells were higher in the India cohort. Within the India cohort, we also identified cell types whose frequency was positively or negatively predictive of occurrence of infection(s) in the first six months of life. Monocytes, total T cells, and memory CD4+ T cells were most prominent in having an inverse relationship with infection. We suggest that these data provide impetus for follow-up studies linking phenotypic differences to environmental versus genetic factors, and to infection outcomes.

    View details for PubMedID 30444901

  • Baseline Gastrointestinal Eosinophilia Is Common in Oral Immunotherapy Subjects With IgE-Mediated Peanut Allergy. Frontiers in immunology Wright, B. L., Fernandez-Becker, N. Q., Kambham, N., Purington, N., Tupa, D., Zhang, W., Rank, M. A., Kita, H., Shim, K. P., Bunning, B. J., Doyle, A. D., Jacobsen, E. A., Boyd, S. D., Tsai, M., Maecker, H., Manohar, M., Galli, S. J., Nadeau, K. C., Chinthrajah, R. S. 2018; 9: 2624


    Rationale: Oral immunotherapy (OIT) is an emerging treatment for food allergy. While desensitization is achieved in most subjects, many experience gastrointestinal symptoms and few develop eosinophilic gastrointestinal disease. It is unclear whether these subjects have subclinical gastrointestinal eosinophilia (GE) at baseline. We aimed to evaluate the presence of GE in subjects with food allergy before peanut OIT. Methods: We performed baseline esophagogastroduodenoscopies on 21 adults before undergoing peanut OIT. Subjects completed a detailed gastrointestinal symptom questionnaire. Endoscopic findings were assessed using the Eosinophilic Esophagitis (EoE) Endoscopic Reference Score (EREFS) and biopsies were obtained from the esophagus, gastric antrum, and duodenum. Esophageal biopsies were evaluated using the EoE Histologic Scoring System. Immunohistochemical staining for eosinophil peroxidase (EPX) was also performed. Hematoxylin and eosin and EPX stains of each biopsy were assessed for eosinophil density and EPX/mm2 was quantified using automated image analysis. Results: All subjects were asymptomatic. Pre-existing esophageal eosinophilia (>5 eosinophils per high-power field [eos/hpf]) was present in five participants (24%), three (14%) of whom had >15 eos/hpf associated with mild endoscopic findings (edema, linear furrowing, or rings; median EREFS = 0, IQR 0-0.25). Some subjects also demonstrated basal cell hyperplasia, dilated intercellular spaces, and lamina propria fibrosis. Increased eosinophils were noted in the gastric antrum (>12 eos/hpf) or duodenum (>26 eos/hpf) in 9 subjects (43%). EPX/mm2 correlated strongly with eosinophil counts (r = 0.71, p < 0.0001). Conclusions: Pre-existing GE is common in adults with IgE-mediated peanut allergy. Eosinophilic inflammation (EI) in these subjects may be accompanied by mild endoscopic and histologic findings. Longitudinal data collection during OIT is ongoing.

    View details for PubMedID 30524424

  • Value of Circulating Cytokine Profiling During Submaximal Exercise Testing in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Scientific reports Moneghetti, K. J., Skhiri, M., Contrepois, K., Kobayashi, Y., Maecker, H., Davis, M., Snyder, M., Haddad, F., Montoya, J. G. 2018; 8 (1): 2779


    Myalgic Encephalomyelitis or Chronic Fatigue Syndrome (ME/CFS) is a heterogeneous syndrome in which patients often experience severe fatigue and malaise following exertion. Immune and cardiovascular dysfunction have been postulated to play a role in the pathophysiology. We therefore, examined whether cytokine profiling or cardiovascular testing following exercise would differentiate patients with ME/CFS. Twenty-four ME/CFS patients were matched to 24 sedentary controls and underwent cardiovascular and circulating immune profiling. Cardiovascular analysis included echocardiography, cardiopulmonary exercise and endothelial function testing. Cytokine and growth factor profiles were analyzed using a 51-plex Luminex bead kit at baseline and 18 hours following exercise. Cardiac structure and exercise capacity were similar between groups. Sparse partial least square discriminant analyses of cytokine profiles 18 hours post exercise offered the most reliable discrimination between ME/CFS and controls (κ = 0.62(0.34,0.84)). The most discriminatory cytokines post exercise were CD40L, platelet activator inhibitor, interleukin 1-β, interferon-α and CXCL1. In conclusion, cytokine profiling following exercise may help differentiate patients with ME/CFS from sedentary controls.

    View details for PubMedID 29426834

  • Lupus Nephritis in Isolation or Accompanied By Extra-Renal Manifestations: Early Lessons from the Accelerating Medicines Partnership James, J. A., Petri, M., Putterman, C., Diamond, B., Wofsy, D., Lee, C., Fine, D., Broder, A. R., Clancy, R. M., Izmirly, P. M., Belmont, M., Bornkamp, N., Davidson, A., Tosta, P., Kalunian, K. C., Park, M., Dall'Era, M., Furie, R., Massarotti, E., Hernandez, G. T., Payan-Schober, F., Connery, S. M., Kamen, D. L., Lee, I., Pendergraft, W., Anolik, J. H., Shah, U., Raychaudhuri, S., Lee, Y. C., Guthridge, J. M., Holers, V., Utz, P. J., Pichavant, M., Gupta, R., Maecker, H. T., Weisman, M., Buyon, J. P. WILEY. 2017
  • Guidelines for the use of flow cytometry and cell sorting in immunological studies EUROPEAN JOURNAL OF IMMUNOLOGY Cossarizza, A., Chang, H., Radbruch, A., Akdis, M., Andrae, I., Annunziato, F., Bacher, P., Barnaba, V., Battistini, L., Bauer, W. M., Baumgart, S., Becher, B., Beisker, W., Berek, C., Blanco, A., Borsellino, G., Boulais, P. E., Brinkman, R. R., Buescher, M., Busch, D. H., Bushnell, T. P., Cao, X., Cavani, A., Chattopadhyay, P. K., Cheng, Q., Chow, S., Clerici, M., Cooke, A., Cosma, A., Cosmi, L., Cumano, A., Dang, V., Davies, D., De Biasi, S., Del Zotto, G., Della Bella, S., Dellabona, P., Deniz, G., Dessing, M., Diefenbach, A., Di Santo, J., Dieli, F., Dolf, A., Donnenberg, V. S., Doerner, T., Ehrhardt, G. A., Endl, E., Engel, P., Engelhardt, B., Esser, C., Everts, B., Dreher, A., Falk, C. S., Fehniger, T. A., Filby, A., Fillatreau, S., Follo, M., Foerster, I., Foster, J., Foulds, G. A., Frenette, P. S., Galbraith, D., Garbi, N., Dolores Garcia-Godoy, M., Geginat, J., Ghoreschi, K., Gibellini, L., Goettlinger, C., Goodyear, C. S., Gori, A., Grogan, J., Gross, M., Gruetzkau, A., Grummitt, D., Hahn, J., Hammer, Q., Hauser, A. E., Haviland, D. L., Hedley, D., Herrera, G., Herrmann, M., Hiepe, F., Holland, T., Hombrink, P., Houston, J. P., Hoyer, B. F., Huang, B., Hunter, C. A., Iannone, A., Jaeck, H., Javega, B., Jonjic, S., Juelke, K., Jung, S., Kaiser, T., Kalina, T., Keller, B., Khan, S., Kienhoefer, D., Kroneis, T., Kunkel, D., Kurts, C., Kvistborg, P., Lannigan, J., Lantz, O., Larbi, A., LeibundGut-Landmann, S., Leipold, M. D., Levings, M. K., Litwin, V., Liu, Y., Lohoff, M., Lombardi, G., Lopez, L., Lovett-Racke, A., Lubberts, E., Ludewig, B., Lugli, E., Maecker, H. T., Martrus, G., Matarese, G., Maueroeder, C., McGrath, M., McInnes, I., Mei, H. E., Melchers, F., Melzer, S., Mielenz, D., Mills, K., Mirrer, D., Mjosberg, J., Moore, J., Moran, B., Moretta, A., Moretta, L., Mosmann, T. R., Mueller, S., Muller, W., Munz, C., Multhoff, G., Munoz, L., Murphy, K. M., Nakayama, T., Nasi, M., Neudoerfl, C., Nolan, J., Nourshargh, S., O'Connor, J., Ouyang, W., Oxenius, A., Palankar, R., Panse, I., Peterson, P., Peth, C., Petriz, J., Philips, D., Pickl, W., Piconese, S., Pinti, M., Pockley, A., Podolska, M., Pucillo, C., Quataert, S. A., Radstake, T. J., Rajwa, B., Rebhahn, J. A., Recktenwald, D., Remmerswaal, E. M., Rezvani, K., Rico, L. G., Robinson, J., Romagnani, C., Rubartelli, A., Ruckert, B., Ruland, J., Sakaguchi, S., Sala-de-Oyanguren, F., Samstag, Y., Sanderson, S., Sawitzki, B., Scheffold, A., Schiemann, M., Schildberg, F., Schimisky, E., Schmid, S. A., Schmitt, S., Schober, K., Schueler, T., Schulz, A., Schumacher, T., Scotta, C., Shankey, T., Shemer, A., Simon, A., Spidlen, J., Stall, A. M., Stark, R., Stehle, C., Stein, M., Steinmetz, T., Stockinger, H., Takahama, Y., Tarnok, A., Tian, Z., Toldi, G., Tornack, J., Traggiai, E., Trotter, J., Ulrich, H., van der Braber, M., van Lier, R. W., Veldhoen, M., Vento-Asturias, S., Vieira, P., Voehringer, D., Volk, H., von Volkmann, K., Waisman, A., Walker, R., Ward, M. D., Warnatz, K., Warth, S., Watson, J. V., Watzl, C., Wegener, L., Wiedemann, A., Wienands, J., Willimsky, G., Wing, J., Wurst, P., Yu, L., Yue, A., Zhang, Q., Zhao, Y., Ziegler, S., Zimmermann, J. 2017; 47 (10): 1584–1797

    View details for PubMedID 29023707

  • Continuous immunotypes describe human immune variation and predict diverse responses PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kaczorowski, K. J., Shekhar, K., Nkulikiyimfura, D., Dekker, C. L., Maecker, H., Davis, M. M., Chakraborty, A. K., Brodin, P. 2017; 114 (30): E6097–E6106


    The immune system consists of many specialized cell populations that communicate with each other to achieve systemic immune responses. Our analyses of various measured immune cell population frequencies in healthy humans and their responses to diverse stimuli show that human immune variation is continuous in nature, rather than characterized by discrete groups of similar individuals. We show that the same three key combinations of immune cell population frequencies can define an individual's immunotype and predict a diverse set of functional responses to cytokine stimulation. We find that, even though interindividual variations in specific cell population frequencies can be large, unrelated individuals of younger age have more homogeneous immunotypes than older individuals. Across age groups, cytomegalovirus seropositive individuals displayed immunotypes characteristic of older individuals. The conceptual framework for defining immunotypes suggested by our results could guide the development of better therapies that appropriately modulate collective immunotypes, rather than individual immune components.

    View details for PubMedID 28696306

  • Cord blood T cell subpopulations and associations with maternal cadmium and arsenic exposures PLOS ONE Nygaard, U. C., Li, Z., Palys, T., Jackson, B., Subbiah, M., Malipatlolla, M., Sampath, V., Maecker, H., Karagas, M. R., Nadeau, K. C. 2017; 12 (6): e0179606


    Arsenic and cadmium are environmental pollutants, and although the evidence for adverse immune effects after prenatal arsenic and cadmium exposures is increasing, little is known about the underlying immunological mechanisms.We investigated the relationship between prenatal arsenic and cadmium exposures and a variety of T cell subpopulations measured in cord blood for 63 participants in the New Hampshire Birth Cohort Study. Post-partum toenail concentrations of arsenic and cadmium were used as an estimate of maternal exposure during pregnancy. The characteristics of cord blood proportions of T lymphocytes and subpopulations (expression of markers for Th1, Th2, Th17, Th1Th17, induced and natural regulatory T cells and NKTs) are presented.In regression analyses, maternal arsenic exposure levels were inversely associated with cord blood T helper memory cells (-21%, 95% CI: -36%, -3%) and the association was found to be stronger in females. They were also inversely associated with activated T helper memory cells, particularly in males (-26%, 95% CI: -43%, -3%). Similarly, inverse associations were observed between cadmium exposure levels and activated T helper memory cells (-16%, 95% CI: -30%, -1%) and also for T helper memory cells in females (-20%, 95% CI: -35%, -3%).The results suggest that prenatal exposures to relatively low levels of arsenic and cadmium may contribute to altered distribution of T cell populations at birth. These changes in theory, could have contributed to the previously reported immunosuppressive effects observed later in infancy/childhood.

    View details for PubMedID 28662050

  • Antigen Availability Shapes T Cell Differentiation and Function during Tuberculosis CELL HOST & MICROBE Moguche, A. O., Musvosvi, M., Penn-Nicholson, A., Plumlee, C. R., Mearns, H., Geldenhuys, H., Smit, E., Abrahams, D., Rozot, V., Dintwe, O., Hoff, S. T., Kromann, I., Ruhwald, M., Bang, P., Larson, R. P., Shafiani, S., Ma, S., Sherman, D. R., Sette, A., Arlehamn, C., McKinney, D. M., Maecker, H., Hanekom, W. A., Hatherill, M., Andersen, P., Scriba, T. J., Urdahl, K. B. 2017; 21 (6): 695-+


    CD4 T cells are critical for protective immunity against Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB). Yet to date, TB vaccine candidates that boost antigen-specific CD4 T cells have conferred little or no protection. Here we examined CD4 T cell responses to two leading TB vaccine antigens, ESAT-6 and Ag85B, in Mtb-infected mice and in vaccinated humans with and without underlying Mtb infection. In both species, Mtb infection drove ESAT-6-specific T cells to be more differentiated than Ag85B-specific T cells. The ability of each T cell population to control Mtb in the lungs of mice was restricted for opposite reasons: Ag85B-specific T cells were limited by reduced antigen expression during persistent infection, whereas ESAT-6-specific T cells became functionally exhausted due to chronic antigenic stimulation. Our findings suggest that different vaccination strategies will be required to optimize protection mediated by T cells recognizing antigens expressed at distinct stages of Mtb infection.

    View details for PubMedID 28618268

    View details for PubMedCentralID PMC5533182

  • Immune Checkpoint Function of CD85j in CD8 T Cell Differentiation and Aging FRONTIERS IN IMMUNOLOGY Gustafson, C. E., Qi, Q., Hutter-Saunders, J., Gupta, S., Jadhav, R., Newell, E., Maecker, H., Weyand, C. M., Goronzy, J. J. 2017; 8: 692


    Aging is associated with an increased susceptibility to infection and a failure to control latent viruses thought to be driven, at least in part, by alterations in CD8 T cell function. The aging T cell repertoire is characterized by an accumulation of effector CD8 T cells, many of which express the negative regulatory receptor CD85j. To define the biological significance of CD85j expression on CD8 T cells and to address the question whether presence of CD85j in older individuals is beneficial or detrimental for immune function, we examined the specific attributes of CD8 T cells expressing CD85j as well as the functional role of CD85j in antigen-specific CD8 T cell responses during immune aging. Here, we show that CD85j is mainly expressed by terminally differentiated effector (TEMRAs) CD8 T cells, which increase with age, in cytomegalovirus (CMV) infection and in males. CD85j+ CMV-specific cells demonstrate clonal expansion. However, TCR diversity is similar between CD85j+ and CD85j- compartments, suggesting that CD85j does not directly impact the repertoire of antigen-specific cells. Further phenotypic and functional analyses revealed that CD85j identifies a specific subset of CMV-responsive CD8 T cells that coexpress a marker of senescence (CD57) but retain polyfunctional cytokine production and expression of cytotoxic mediators. Blocking CD85j binding enhanced proliferation of CMV-specific CD8 T cells upon antigen stimulation but did not alter polyfunctional cytokine production. Taken together, these data demonstrate that CD85j characterizes a population of "senescent," but not exhausted antigen-specific effector CD8 T cells and indicates that CD85j is an important checkpoint regulator controlling expansion of virus-specific T cells during aging. Inhibition of CD85j activity may be a mechanism to promote stronger CD8 T cell effector responses during immune aging.

    View details for PubMedID 28659925

  • Reconstitution of immune cell populations in multiple sclerosis patients after autologous stem cell transplantation. Clinical and experimental immunology Karnell, F. G., LIN, D., Motley, S., Duhen, T., Lim, N., Campbell, D. J., Turka, L. A., Maecker, H. T., Harris, K. M. 2017


    Multiple sclerosis is an inflammatory T cell-mediated autoimmune disease. In a Phase II clinical trial, high-dose immunosuppressive therapy combined with autologous CD34(+) haematopoietic stem cell transplant resulted in 69·2% of subjects remaining disease-free without evidence of relapse, loss of neurological function or new magnetic resonance imaging (MRI) lesions to year 5 post-treatment. A combination of CyTOF mass cytometry and multi-parameter flow cytometry was used to explore the reconstitution kinetics of immune cell subsets in the periphery post-haematopoietic cell transplant (HSCT) and the impact of treatment on the phenotype of circulating T cells in this study population. Repopulation of immune cell subsets progressed similarly for all patients studied 2 years post-therapy, regardless of clinical outcome. At month 2, monocytes and natural killer (NK) cells were proportionally more abundant, while CD4 T cells and B cells were reduced, relative to baseline. In contrast to the changes observed at earlier time-points in the T cell compartment, B cells were proportionally more abundant and expansion in the proportion of naive B cells was observed 1 and 2 years post-therapy. Within the T cell compartment, the proportion of effector memory and late effector subsets of CD4 and CD8 T cells was increased, together with transient increases in proportions of CD45RA-regulatory T cells (Tregs ) and T helper type 1 (Th1 cells) and a decrease in Th17·1 cells. While none of the treatment effects studied correlated with clinical outcome, patients who remained healthy throughout the 5-year study had significantly higher absolute numbers of memory CD4 and CD8 T cells in the periphery prior to stem cell transplantation.

    View details for DOI 10.1111/cei.12985

    View details for PubMedID 28498568

  • Systems approach to uncover signaling networks in primary immunodeficiency diseases. journal of allergy and clinical immunology Choi, J., Fernandez, R., Maecker, H. T., Butte, M. J. 2017

    View details for DOI 10.1016/j.jaci.2017.03.025

    View details for PubMedID 28412396

  • Immunotherapy biomarkers 2016: overcoming the barriers JOURNAL FOR IMMUNOTHERAPY OF CANCER Gulley, J. L., Berzofsky, J. A., Butler, M. O., Cesano, A., Fox, B. A., Gnjatic, S., Janetzki, S., Kalavar, S., Karanikas, V., Khleif, S. N., Kirsch, I., Lee, P. P., Maccalli, C., Maecker, H., Schlom, J., Seliger, B., Siebert, J., Stroncek, D. F., Thurin, M., Yuan, J., Butterfield, L. H. 2017; 5: 29


    This report summarizes the symposium, 'Immunotherapy Biomarkers 2016: Overcoming the Barriers', which was held on April 1, 2016 at the National Institutes of Health in Bethesda, Maryland. The symposium, cosponsored by the Society for Immunotherapy of Cancer (SITC) and the National Cancer Institute (NCI), focused on emerging immunotherapy biomarkers, new technologies, current hurdles to further progress, and recommendations for advancing the field of biomarker development.

    View details for PubMedID 28653584

  • Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY Mukai, K., Gaudenzio, N., Gupta, S., Vivanco, N., Bendall, S. C., Maecker, H. T., Chinthrajah, R. S., Tsai, M., Nadeau, K. C., Galli, S. J. 2017; 139 (3): 889-?


    Basophil activation tests (BATs) have promise for research and for clinical monitoring of patients with allergies. However, BAT protocols vary in blood anticoagulant used and temperature and time of storage before testing, complicating comparisons of results from various studies.We attempted to establish a BAT protocol that would permit analysis of blood within 24 hours of obtaining the sample.Blood from 46 healthy donors and 120 patients with peanut allergy was collected into EDTA or heparin tubes, and samples were stored at 4°C or room temperature for 4 or 24 hours before performing BATs.Stimulation with anti-IgE or IL-3 resulted in strong upregulation of basophil CD203c in samples collected in EDTA or heparin, stored at 4°C, and analyzed 24 hours after sample collection. However, a CD63(hi) population of basophils was not observed in any conditions in EDTA-treated samples unless exogenous calcium/magnesium was added at the time of anti-IgE stimulation. By contrast, blood samples collected in heparin tubes were adequate for quantification of upregulation of basophil CD203c and identification of a population of CD63(hi) basophils, irrespective of whether the specimens were analyzed by means of conventional flow cytometry or cytometry by time-of-flight mass spectrometry, and such tests could be performed after blood was stored for 24 hours at 4°C.BATs to measure upregulation of basophil CD203c and induction of a CD63(hi) basophil population can be conducted with blood obtained in heparin tubes and stored at 4°C for 24 hours.

    View details for DOI 10.1016/j.jaci.2016.04.060

    View details for Web of Science ID 000397295800022

    View details for PubMedCentralID PMC5237629

  • Opening the Door on the CMV Immune Response in Aging. journal of infectious diseases Maecker, H. T. 2017

    View details for DOI 10.1093/infdis/jix081

    View details for PubMedID 28199686

  • Wild immunology assessed by multidimensional mass cytometry. Cytometry. Part A : the journal of the International Society for Analytical Cytology Japp, A. S., Hoffmann, K., Schlickeiser, S., Glauben, R., Nikolaou, C., Maecker, H. T., Braun, J., Matzmohr, N., Sawitzki, B., Siegmund, B., Radbruch, A., Volk, H., Frentsch, M., Kunkel, D., Thiel, A. 2017; 91 (1): 85-95


    A great part of our knowledge on mammalian immunology has been established in laboratory settings. The use of inbred mouse strains enabled controlled studies of immune cell and molecule functions in defined settings. These studies were usually performed in specific-pathogen free (SPF) environments providing standardized conditions. In contrast, mammalians including humans living in their natural habitat are continuously facing pathogen encounters throughout their life. The influences of environmental conditions on the signatures of the immune system and on experimental outcomes are yet not well defined. Thus, the transferability of results obtained in current experimental systems to the physiological human situation has always been a matter of debate. Studies elucidating the diversity of "wild immunology" imprintings in detail and comparing it with those of "clean" lab mice are sparse. Here, we applied multidimensional mass cytometry to dissect phenotypic and functional differences between distinct groups of laboratory and pet shop mice as a source for "wild mice". For this purpose, we developed a 31-antibody panel for murine leukocyte subsets identification and a 35-antibody panel assessing various cytokines. Established murine leukocyte populations were easily identified and diverse immune signatures indicative of numerous pathogen encounters were classified particularly in pet shop mice and to a lesser extent in quarantine and non-SPF mice as compared to SPF mice. In addition, unsupervised analysis identified distinct clusters that associated strongly with the degree of pathogenic priming, including increased frequencies of activated NK cells and antigen-experienced B- and T-cell subsets. Our study unravels the complexity of immune signatures altered under physiological pathogen challenges and highlights the importance of carefully adapting laboratory settings for immunological studies in mice, including drug and therapy testing. © 2016 International Society for Advancement of Cytometry.

    View details for DOI 10.1002/cyto.a.22906

    View details for PubMedID 27403624

  • Autoantibody profiling on a plasmonic nano-gold chip for the early detection of hypertensive heart disease. Proceedings of the National Academy of Sciences of the United States of America Li, X., Kuznetsova, T., Cauwenberghs, N., Wheeler, M., Maecker, H., Wu, J. C., Haddad, F., Dai, H. 2017; 114 (27): 7089–94


    The role of autoimmunity in cardiovascular (CV) diseases has been increasingly recognized. Autoimmunity is most commonly examined by the levels of circulating autoantibodies in clinical practices. Measurement of autoantibodies remains, however, challenging because of the deficiency of reproducible, sensitive, and standardized assays. The lack of multiplexed assays also limits the potential to identify a CV-specific autoantibody profile. To overcome these challenges, we developed a nanotechnology-based plasmonic gold chip for autoantibody profiling. This approach allowed simultaneous detection of 10 CV autoantibodies targeting the structural myocardial proteins, the neurohormonal regulatory proteins, the vascular proteins, and the proteins associated with apoptosis and coagulation. Autoantibodies were measured in four groups of participants across the continuum of hypertensive heart diseases. We observed higher levels of all 10 CV autoantibodies in hypertensive subjects (n= 77) compared with healthy participants (n= 30), and the autoantibodies investigated were related to each other, forming a highly linked network. In addition, we established that autoantibodies to troponin I, annexin-A5, and beta 1-adrenegic receptor best discriminated hypertensive subjects with adverse left ventricular (LV) remodeling or dysfunction (n= 49) from hypertensive subjects with normal LV structure and function (n= 28). By further linking these three significant CV autoantibodies to the innate and growth factors, we revealed a positive but weak association between autoantibodies to troponin I and proinflammatory cytokine IL-18. Overall, we demonstrated that this platform can be used to evaluate autoantibody profiles in hypertensive subjects at risk for heart failure.

    View details for PubMedID 28630342

  • Cytokine signature associated with disease severity in chronic fatigue syndrome patients. Proceedings of the National Academy of Sciences of the United States of America Montoya, J. G., Holmes, T. H., Anderson, J. N., Maecker, H. T., Rosenberg-Hasson, Y., Valencia, I. J., Chu, L., Younger, J. W., Tato, C. M., Davis, M. M. 2017; 114 (34): E7150–E7158


    Although some signs of inflammation have been reported previously in patients with myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS), the data are limited and contradictory. High-throughput methods now allow us to interrogate the human immune system for multiple markers of inflammation at a scale that was not previously possible. To determine whether a signature of serum cytokines could be associated with ME/CFS and correlated with disease severity and fatigue duration, cytokines of 192 ME/CFS patients and 392 healthy controls were measured using a 51-multiplex array on a Luminex system. Each cytokine's preprocessed data were regressed on ME/CFS severity plus covariates for age, sex, race, and an assay property of newly discovered importance: nonspecific binding. On average, TGF-β was elevated (P = 0.0052) and resistin was lower (P = 0.0052) in patients compared with controls. Seventeen cytokines had a statistically significant upward linear trend that correlated with ME/CFS severity: CCL11 (Eotaxin-1), CXCL1 (GROα), CXCL10 (IP-10), IFN-γ, IL-4, IL-5, IL-7, IL-12p70, IL-13, IL-17F, leptin, G-CSF, GM-CSF, LIF, NGF, SCF, and TGF-α. Of the 17 cytokines that correlated with severity, 13 are proinflammatory, likely contributing to many of the symptoms experienced by patients and establishing a strong immune system component of the disease. Only CXCL9 (MIG) inversely correlated with fatigue duration.

    View details for PubMedID 28760971

  • CyTOF Measurement of Immunocompetence Across Major Immune Cell Types. Current protocols in cytometry Subrahmanyam, P. B., Maecker, H. T. 2017; 82: 9.54.1–9.54.12


    The central role of the immune system is becoming appreciated in a wide variety of diseases. Cancer immunotherapy is one area that has yielded much recent success, although not all patients benefit equally. At the same time, recent studies have highlighted the heterogeneity of the human immune system. Despite this heterogeneity, we do not routinely measure immune competence in clinical practice, and there are no consensus assays of healthy immune function. Using mass cytometry (CyTOF), we can simultaneously detect ∼40 markers to identify various cell subsets and determine their function by the expression of cytokines, cytotoxicity, and activation markers. This can help assess 'immunocompetence' and facilitate better implementation of immunotherapies, both in specific disease settings and perhaps eventually as a prognostic tool in healthy subjects. Here we introduce the concepts behind this assay and provide a protocol that we have successfully implemented to identify possible predictive biomarkers of immunotherapy outcome. © 2017 by John Wiley & Sons, Inc.

    View details for PubMedID 28967988

    View details for PubMedCentralID PMC5678938

  • Characterizing CD137 Upregulation on NK cells in Patients Receiving Monoclonal Antibody Therapy. Annals of oncology MAKKOUK, A., Sundaram, V., Chester, C., Chang, S., Colevas, A. D., Sunwoo, J. B., Maecker, H., Desai, M., Kohrt, H. E. 2016


    In the era of personalized cancer medicine, identifying techniques for effectively matching patients to efficacious treatments is a critical step in the treatment process. The advent of anti-cancer immunotherapies necessitates novel approaches to biomarker identification beyond traditional genomic profiling. One promising approach is incorporation of nomograms into treatment decisions. Nomograms are prediction tools, based on statistical modeling, designed to predict treatment outcomes. As a first step toward developing a nomogram, we conducted analyses to predict CD137 expression of natural killer cells after monoclonal antibody (mAb) treatment.Patient, tumor and immune characteristics were collected from 199 patients with breast cancer (N = 62), head/neck cancers (N = 46) and non-Hodgkin's lymphoma (NHL) (N = 91), who were receiving mAb therapy as part of clinical trials. The difference in CD137 expression before and after mAb therapy was assessed by flow cytometry. To evaluate those who respond to mAb therapy via increased CD137 expression, we applied classification and regression trees (CART), multivariable lasso regression tools and Random Forest.The CD137 expression was significantly different for each cancer type [mean (SD): Breast: 6.6 (6.5); Head/Neck: 11.0 (7.0); NHL: 7.5 (7.1), P < 0.0001]. For breast cancer and NHL, FcR polymorphism and baseline CD137 expression were significant predictors of increased CD137 expression; for head/neck cancer, FcR polymorphism and age were significant predictors of increased expression.Our preliminary results suggest that FcR polymorphism, pre-treatment CD137 expression and age are significant predictors of CD137 upregulation in patients. This study demonstrates that the development of a nomogram for therapy response is feasible. Further work validating our models in an independent cohort will provide the next steps in developing a nomogram that may be used to individualize this therapeutic approach for patients (NCT01114256).

    View details for PubMedID 27831501

  • Cytokine profiles in patients with toxoplasmic lymphadenitis in the setting of pregnancy. Cytokine Pomares, C., Holmes, T. H., Estran, R., Press, C. J., Ramirez, R., Talucod, J., Maecker, H., Rosenberg-Hasson, Y., Montoya, J. G. 2016; 90: 14-20


    Majority of Toxoplasma gondii infections are benign and asymptomatic; however, some patients experience toxoplasmic lymphadenitis (TL). Factors associated as to whether infection will be symptomatic or not are unknown.Dye test titers of patients with acute toxoplasmosis (pregnant and not pregnant) with TL (TL+) were compared with those in patients with asymptomatic acute infection (TL-). Additionally, mean levels of 62 serum cytokines were compared between TL+ and TL- pregnant women and between TL+ pregnant and non-pregnant women.During acute infection, mean dye test titer was higher in TL+ than in TL- patients (p=0.021). In addition, out of 62 cytokines, CXCL9andCXCL10 levels were higher (p<0.05) and resistin mean levels were lower (p<0.05) in pregnant women with TL+ compared to TL-. Among patients with TL+, levels of VCAM1andCCL2 were lower (p<0.05) in pregnant women than in non-pregnant women.Here we report differences in dye test titers in patients with acute infection. Cytokine responses vary according to the presence of TL+ and to the pregnancy status. Factors underlying these differences are presently unknown and require further studies to define individual and combined roles of cytokines in TL+.

    View details for DOI 10.1016/j.cyto.2016.09.021

    View details for PubMedID 27744174

  • Defective T Memory Cell Differentiation after Varicella Zoster Vaccination in Older Individuals. PLoS pathogens Qi, Q., Cavanagh, M. M., Le Saux, S., Wagar, L. E., Mackey, S., Hu, J., Maecker, H., Swan, G. E., Davis, M. M., Dekker, C. L., Tian, L., Weyand, C. M., Goronzy, J. J. 2016; 12 (10)


    Vaccination with attenuated live varicella zoster virus (VZV) can prevent zoster reactivation, but protection is incomplete especially in an older population. To decipher the molecular mechanisms underlying variable vaccine responses, T- and B-cell responses to VZV vaccination were examined in individuals of different ages including identical twin pairs. Contrary to the induction of VZV-specific antibodies, antigen-specific T cell responses were significantly influenced by inherited factors. Diminished generation of long-lived memory T cells in older individuals was mainly caused by increased T cell loss after the peak response while the expansion of antigen-specific T cells was not affected by age. Gene expression in activated CD4 T cells at the time of the peak response identified gene modules related to cell cycle regulation and DNA repair that correlated with the contraction phase of the T cell response and consequently the generation of long-lived memory cells. These data identify cell cycle regulatory mechanisms as targets to reduce T cell attrition in a vaccine response and to improve the generation of antigen-specific T cell memory, in particular in an older population.

    View details for DOI 10.1371/journal.ppat.1005892

    View details for PubMedID 27764254

    View details for PubMedCentralID PMC5072604

  • Immune Profiles to Predict Response to Desensitization Therapy in Highly HLA-Sensitized Kidney Transplant Candidates PLOS ONE Yabu, J. M., Siebert, J. C., Maecker, H. T. 2016; 11 (4)


    Kidney transplantation is the most effective treatment for end-stage kidney disease. Sensitization, the formation of human leukocyte antigen (HLA) antibodies, remains a major barrier to successful kidney transplantation. Despite the implementation of desensitization strategies, many candidates fail to respond. Current progress is hindered by the lack of biomarkers to predict response and to guide therapy. Our objective was to determine whether differences in immune and gene profiles may help identify which candidates will respond to desensitization therapy.Single-cell mass cytometry by time-of-flight (CyTOF) phenotyping, gene arrays, and phosphoepitope flow cytometry were performed in a study of 20 highly sensitized kidney transplant candidates undergoing desensitization therapy. Responders to desensitization therapy were defined as 5% or greater decrease in cumulative calculated panel reactive antibody (cPRA) levels, and non-responders had 0% decrease in cPRA. Using a decision tree analysis, we found that a combination of transitional B cell and regulatory T cell (Treg) frequencies at baseline before initiation of desensitization therapy could distinguish responders from non-responders. Using a support vector machine (SVM) and longitudinal data, TRAF3IP3 transcripts and HLA-DR-CD38+CD4+ T cells could also distinguish responders from non-responders. Combining all assays in a multivariate analysis and elastic net regression model with 72 analytes, we identified seven that were highly interrelated and eleven that predicted response to desensitization therapy.Measuring baseline and longitudinal immune and gene profiles could provide a useful strategy to distinguish responders from non-responders to desensitization therapy. This study presents the integration of novel translational studies including CyTOF immunophenotyping in a multivariate analysis model that has potential applications to predict response to desensitization, select candidates, and personalize medicine to ultimately improve overall outcomes in highly sensitized kidney transplant candidates.

    View details for DOI 10.1371/journal.pone.0153355

    View details for Web of Science ID 000374131700042

    View details for PubMedID 27078882

    View details for PubMedCentralID PMC4831845

  • Successful immunotherapy induces previously unidentified allergen-specific CD4+ T-cell subsets. Proceedings of the National Academy of Sciences of the United States of America Ryan, J. F., Hovde, R., Glanville, J., Lyu, S., Ji, X., Gupta, S., Tibshirani, R. J., Jay, D. C., Boyd, S. D., Chinthrajah, R. S., Davis, M. M., Galli, S. J., Maecker, H. T., Nadeau, K. C. 2016; 113 (9): E1286-95


    Allergen immunotherapy can desensitize even subjects with potentially lethal allergies, but the changes induced in T cells that underpin successful immunotherapy remain poorly understood. In a cohort of peanut-allergic participants, we used allergen-specific T-cell sorting and single-cell gene expression to trace the transcriptional "roadmap" of individual CD4+ T cells throughout immunotherapy. We found that successful immunotherapy induces allergen-specific CD4+ T cells to expand and shift toward an "anergic" Th2 T-cell phenotype largely absent in both pretreatment participants and healthy controls. These findings show that sustained success, even after immunotherapy is withdrawn, is associated with the induction, expansion, and maintenance of immunotherapy-specific memory and naive T-cell phenotypes as early as 3 mo into immunotherapy. These results suggest an approach for immune monitoring participants undergoing immunotherapy to predict the success of future treatment and could have implications for immunotherapy targets in other diseases like cancer, autoimmune disease, and transplantation.

    View details for DOI 10.1073/pnas.1520180113

    View details for PubMedID 26811452

  • Platinum-conjugated antibodies for application in mass cytometry. Cytometry. Part A : the journal of the International Society for Analytical Cytology Mei, H. E., Leipold, M. D., Maecker, H. T. 2016; 89 (3): 292-300


    Mass cytometry has overcome limitations of fluorescent single cell cytometry by allowing for the measurement of up to currently ∼40 different parameters on a single cell level. However, the cellular proteome comprises many more potential analytes, and current mass cytometry instrumentation allows for theoretically up to 121 different mass detection channels. The labeling of specific probes with appropriate metal ions is a significant hurdle for exploiting more of mass cytometry's analytical capacity. To this end, we here describe the labeling of antibody with natural abundance or isotopically purified platinum as formulated in cisplatin and circumventing the use of chelator-loaded polymers. We confirm the utility of cisplatin-antibody-conjugates for surface, intracellular, and phosphoepitope-specific immunophenotyping, as well as for application in cell surface CD45-based barcoding. Cisplatin-labeling of antibody increases the analytical capacity of the CyTOF(®) platform by two channels based on available reagents, and has the potential to add a total of six channels for detection of specific probes, thus helping to better extend the analytical mass range of mass cytometers. © 2015 International Society for Advancement of Cytometry.

    View details for DOI 10.1002/cyto.a.22778

    View details for PubMedID 26355391

  • Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium SCIENTIFIC REPORTS Finak, G., Langweiler, M., Jaimes, M., Malek, M., Taghiyar, J., Korin, Y., Raddassi, K., Devine, L., Obermoser, G., Pekalski, M. L., Pontikos, N., Diaz, A., Heck, S., Villanova, F., Terrazzini, N., Kern, F., Qian, Y., Stanton, R., Wang, K., Brandes, A., Ramey, J., Aghaeepour, N., Mosmann, T., Scheuermann, R. H., Reed, E., Palucka, K., pascual, V., Blomberg, B. B., Nestle, F., Nussenblatt, R. B., Brinkman, R. R., Gottardo, R., Maecker, H., McCoy, J. P. 2016; 6


    Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.

    View details for DOI 10.1038/srep20686

    View details for PubMedID 26861911

  • Immunodynamics: a cancer immunotherapy trials network review of immune monitoring in immuno-oncology clinical trials. Journal for immunotherapy of cancer Kohrt, H. E., Tumeh, P. C., Benson, D., Bhardwaj, N., Brody, J., Formenti, S., Fox, B. A., Galon, J., June, C. H., Kalos, M., Kirsch, I., Kleen, T., Kroemer, G., Lanier, L., Levy, R., Lyerly, H. K., Maecker, H., Marabelle, A., Melenhorst, J., Miller, J., Melero, I., Odunsi, K., Palucka, K., Peoples, G., Ribas, A., Robins, H., Robinson, W., Serafini, T., Sondel, P., Vivier, E., Weber, J., Wolchok, J., Zitvogel, L., Disis, M. L., Cheever, M. A. 2016; 4: 15-?


    The efficacy of PD-1/PD-L1 targeted therapies in addition to anti-CTLA-4 solidifies immunotherapy as a modality to add to the anticancer arsenal. Despite raising the bar of clinical efficacy, immunologically targeted agents raise new challenges to conventional drug development paradigms by highlighting the limited relevance of assessing standard pharmacokinetics (PK) and pharmacodynamics (PD). Specifically, systemic and intratumoral immune effects have not consistently correlated with standard relationships between systemic dose, toxicity, and efficacy for cytotoxic therapies. Hence, PK and PD paradigms remain inadequate to guide the selection of doses and schedules, both starting and recommended Phase 2 for immunotherapies. The promise of harnessing the immune response against cancer must also be considered in light of unique and potentially serious toxicities. Refining immune endpoints to better inform clinical trial design represents a high priority challenge. The Cancer Immunotherapy Trials Network investigators review the immunodynamic effects of specific classes of immunotherapeutic agents to focus immune assessment modalities and sites, both systemic and importantly intratumoral, which are critical to the success of the rapidly growing field of immuno-oncology.

    View details for DOI 10.1186/s40425-016-0118-0

    View details for PubMedID 26981245

  • Similarities in the Markers of Inflammation Between Men With Syphilis and Women With Increased Risk of HIV Acquisition. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Kojima, N., Bristow, C. C., Maecker, H., Rosenberg-Hasson, Y., Leon, S. R., Vargas, S. K., Konda, K. A., Caceres, C. F., Klausner, J. D. 2016; 62 (2): 265–66

    View details for PubMedID 26394671

  • Novel technologies and emerging biomarkers for personalized cancer immunotherapy. Journal for immunotherapy of cancer Yuan, J., Hegde, P. S., Clynes, R., Foukas, P. G., Harari, A., Kleen, T. O., Kvistborg, P., Maccalli, C., Maecker, H. T., Page, D. B., Robins, H., Song, W., Stack, E. C., Wang, E., Whiteside, T. L., Zhao, Y., Zwierzina, H., Butterfield, L. H., Fox, B. A. 2016; 4: 3-?


    The culmination of over a century's work to understand the role of the immune system in tumor control has led to the recent advances in cancer immunotherapies that have resulted in durable clinical responses in patients with a variety of malignancies. Cancer immunotherapies are rapidly changing traditional treatment paradigms and expanding the therapeutic landscape for cancer patients. However, despite the current success of these therapies, not all patients respond to immunotherapy and even those that do often experience toxicities. Thus, there is a growing need to identify predictive and prognostic biomarkers that enhance our understanding of the mechanisms underlying the complex interactions between the immune system and cancer. Therefore, the Society for Immunotherapy of Cancer (SITC) reconvened an Immune Biomarkers Task Force to review state of the art technologies, identify current hurdlers, and make recommendations for the field. As a product of this task force, Working Group 2 (WG2), consisting of international experts from academia and industry, assembled to identify and discuss promising technologies for biomarker discovery and validation. Thus, this WG2 consensus paper will focus on the current status of emerging biomarkers for immune checkpoint blockade therapy and discuss novel technologies as well as high dimensional data analysis platforms that will be pivotal for future biomarker research. In addition, this paper will include a brief overview of the current challenges with recommendations for future biomarker discovery.

    View details for DOI 10.1186/s40425-016-0107-3

    View details for PubMedID 26788324

    View details for PubMedCentralID PMC4717548

  • Computationally efficient multidimensional analysis of complex flow cytometry data using second order polynomial histograms CYTOMETRY PART A Zaunders, J., Jing, J., Leipold, M., Maecker, H., Kelleher, A. D., Koch, I. 2016; 89A (1): 44-58
  • Cytokines profile in hypertensive patients with left ventricular remodeling and dysfunction. Journal of the American Society of Hypertension Kuznetsova, T., Haddad, F., Knez, J., Rosenberg-Hasson, Y., Sung, J., Cauwenberghs, N., Thijs, L., Karakikes, I., Maecker, H., Mahaffey, K. W., Wu, J. C., Staessen, J. A. 2015; 9 (12): 975-984 e3


    There is strong evidence that inflammatory mediators play a key role in the progression to heart failure in patients with systemic hypertension (HTN). The present study aimed to identify a set of cytokines that are associated with early left ventricular (LV) remodeling and dysfunction as captured by echocardiography in patients with HTN in a cross-sectional case-control study nested within the FLEMish study on ENvironment, Genes and Health Outcome. We identified three groups of participants from the cohort: normotensive subjects (normotension; n = 30), HTN with normal LV structure and function (HTN [LV-]; n = 30), and HTN with evidence of adverse LV remodeling (HTN [LV+]; n = 50). We measured cytokines using a 63-plex Luminex platform. Using partial least squares-discriminant analysis, we constructed three latent variables from the measured cytokines that explained 35%-45% of the variance between groups. We identified five common cytokines (interleukin 18, monokine induced by gamma interferon, hepatocyte growth factor, epithelial neutrophil-activating peptide 78, and vascular endothelial growth factor D) with a stable signal which had a major impact on the construction of the latent variables. Among these cytokines, after adjustment for confounders, interleukin 18 remained significantly different between HTN participants with and without LV involvement (P = .02). Moreover, granulocyte-macrophage colony-stimulating factor and leptin showed a consistent upward trend in all HTN patients compared with normotensive subjects. In conclusion, in HTN patients with LV remodeling or/and dysfunction, we identified a set of cytokines strongly associated with LV maladaptation. We also found a distinct profile of inflammatory biomarkers that characterize HTN.

    View details for DOI 10.1016/j.jash.2015.10.003

    View details for PubMedID 26565110

  • Large-Scale and Comprehensive Immune Profiling and Functional Analysis of Normal Human Aging PLOS ONE Whiting, C. C., Siebert, J., Newman, A. M., Du, H., Alizadeh, A. A., Goronzy, J., Weyand, C. M., Krishnan, E., Fathman, C. G., Maecker, H. T. 2015; 10 (7)


    While many age-associated immune changes have been reported, a comprehensive set of metrics of immune aging is lacking. Here we report data from 243 healthy adults aged 40-97, for whom we measured clinical and functional parameters, serum cytokines, cytokines and gene expression in stimulated and unstimulated PBMC, PBMC phenotypes, and cytokine-stimulated pSTAT signaling in whole blood. Although highly heterogeneous across individuals, many of these assays revealed trends by age, sex, and CMV status, to greater or lesser degrees. Age, then sex and CMV status, showed the greatest impact on the immune system, as measured by the percentage of assay readouts with significant differences. An elastic net regression model could optimally predict age with 14 analytes from different assays. This reinforces the importance of multivariate analysis for defining a healthy immune system. These data provide a reference for others measuring immune parameters in older people.

    View details for DOI 10.1371/journal.pone.0133627

    View details for Web of Science ID 000358547600123

  • Immunologic Network and Response to Intramyocardial CD34(+) Stem Cell Therapy in Patients With Dilated Cardiomyopathy JOURNAL OF CARDIAC FAILURE Haddad, F., Sever, M., Poglajen, G., Lezaic, L., Yang, P., Maecker, H., Davis, M., Kuznetsova, T., Wu, J. C., Vrtovec, B. 2015; 21 (7): 572-582


    Although stem cell therapy (SCT) is emerging as a potential treatment for patients with dilated cardiomyopathy (DCM), clinical response remains variable. Our objective was to determine whether baseline differences in circulating immunologic and nonimmunologic biomarkers may help to identify patients more likely to respond to intramyocardial injection of CD34(+)-based SCT.We enrolled from January 3, 2011 to March 5, 2012 37 patients with longstanding DCM (left ventricular ejection fraction [LVEF] <40%, New York Heart Association functional class III) who underwent peripheral CD34(+) stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) and collection by means of apheresis. CD34(+) cells were labeled with (99m)Tc-hexamethylpropyleneamine oxime to allow assessment of stem cell retention at 18 hours. Response to SCT was predefined as an increase in LVEF of ≥5% at 3 months. The majority (84%) of patients were male with an overall mean LVEF of 27 ± 7% and a median N-terminal pro-B-type natriuretic peptide (NT-proBNP) level of 2,774 pg/mL. Nineteen patients (51%) were responders to SCT. There was no significant difference between responders and nonresponders regarding to age, sex, baseline LVEF, NT-proBNP levels, or 6-minute walking distance. With the use of a partial least squares (PLS) predictive model, we identified 9 baseline factors that were associated with both stem cell response and stem cell retention (mechanistic validation). Among the baseline factors positively associated with both clinical response and stem cell retention were G-CSF, SDF-1, LIF, MCP-1, and MCP-3. Among baseline factors negatively associated with both clinical response and retention were IL-12p70, FASL, ICAM-1, and GGT. A decrease in G-CSF at 3-month follow-up was also observed in responders compared with nonresponders (P = .02).If further validated, baseline immunologic and nonimmunologic biomarkers may help to identify patients with DCM who are more likely to respond to CD34(+)-based SCT.

    View details for DOI 10.1016/j.cardfail.2015.03.011

    View details for Web of Science ID 000358105900007

    View details for PubMedID 25863169

  • Cytokine-Stimulated Phosphoflow of Whole Blood Using CyTOF Mass Cytometry. Bio-protocol Fernandez, R., Maecker, H. 2015; 5 (11)


    The ability to assess the function of a range of cytokine, antigen receptor, and Toll-like receptor (TLR) signaling pathways in a range of immune cells could provide a kind of fingerprint of the state of the human immune system. The mass cytometry or CyTOF, platform allows for the parallel application of about 40 labeled antibodies to a single sample, creating the possibility to read out many cell types and signaling pathways in a single small blood sample. We developed such a mass cytometry panel, consisting of 22 antibodies to cell surface lineage markers and 8 antibodies to phospho-specific epitopes of signaling proteins. These antibodies were chosen to discriminate all major white blood cell lineages, to a level of detail that includes subsets such as naïve, central memory, effector memory, and late effector CD4+ and CD8+T cells, naïve, transitional, and switched memory B cells, plasmablasts, myeloid and plasmacytoid dendritic cells, CD16+ and CD16+CD56+ NK cells, CD16+ and classical monocytes etc. 32 such cell subsets are defined in our standard gating scheme. The eight phospho-specific antibodies were chosen to represent major signaling nodes responsive to cytokine, TLR, and antigen receptor signaling. This antibody panel is used with 8 standard stimulation conditions (unstimulated, IFNa, IL-6, IL-7, IL-10, IL-21, LPS, PMA+ ionomycin), although other stimuli can be added. Comparison of healthy controls to subjects with immune deficiencies of unknown etiology may help elucidate the mechanisms of such deficiencies. Phosphorylation of tyrosine, serine, and threonine residues is critical for the control of protein activity involved in various cellular events. An assortment of kinases and phosphatases regulate intracellular protein phosphorylation in many different cell signaling pathways, such as T and B cell signaling, those regulating apoptosis, growth and cell cycle control, plus those involved with cytokine, chemokine, and stress responses. Phosphoflow assays combine phospho-specific antibodies with the power of flow cytometry to enhance phospho protein study. In our assay, peripheral blood mononuclear cells are stimulated by cytokines, fixed, surface-stained with a cocktail of antibodies labeled with MAXPAR (Brand Name) metal-chelating polymers and permeabilized with methanol. They are then stained with intracellular phospho-specific antibodies. We use a CyTOF™ mass cytometer to acquire the ICP-MS data. The current mass window selected is approximately AW 103-203, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type. Median values are reported to quantitate the level of phosphorylation of each protein in response to stimulation. Comparing the level of phosphorylation between samples can offer insight to the status of the immune system. Whole blood stimulation is the closest to the in vivo condition and it allows for assessment of granulocyte population as well as lymphocytes and monocytes.

    View details for PubMedID 27135045

  • Cytokine-stimulated Phosphoflow of PBMC Using CyTOF Mass Cytometry. Bio-protocol Fernandez, R., Maecker, H. 2015; 5 (11)


    Phosphorylation of tyrosine, serine, and threonine residues is critical for the control of protein activity involved in various cellular events. An assortment of kinases and phosphatases regulate intracellular protein phosphorylation in many different cell signaling pathways. These pathways include T and B cell signaling, regulating growth and cell cycle control, plus cytokine, chemokine, and stress responses. Phosphoflow assays combine phosphoprotein-specific antibodies with the power of flow cytometry to enhance phosphoprotein study. In our assay, peripheral blood mononuclear cells are stimulated by cytokines, fixed, surface-stained with a cocktail of antibodies labeled with MAXPAR (brand name) metal-chelating polymers and permeabilized with methanol. They are then stained with intracellular phospho-specific antibodies. We use a CyTOF(™) mass cytometer to acquire the ICP-MS (inductively coupled plasma mass spectrometry) data. The current mass window selected is approximately AW 103-203, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type. Median values are reported to quantitate the level of phosphorylation of each protein in response to stimulation. Comparing the level of phosphorylation between samples can offer insight to the status of the immune system.

    View details for PubMedID 27446979

  • Mass cytometry as a platform for the discovery of cellular biomarkers to guide effective rheumatic disease therapy ARTHRITIS RESEARCH & THERAPY Nair, N., Mei, H. E., Chen, S., Hale, M., Nolan, G. P., Maecker, H. T., Genovese, M., Fathman, C. G., Whiting, C. C. 2015; 17


    The development of biomarkers for autoimmune diseases has been hampered by a lack of understanding of disease etiopathogenesis and of the mechanisms underlying the induction and maintenance of inflammation, which involves complex activation dynamics of diverse cell types. The heterogeneous nature and suboptimal clinical response to treatment observed in many autoimmune syndromes highlight the need to develop improved strategies to predict patient outcome to therapy and personalize patient care. Mass cytometry, using CyTOF®, is an advanced technology that facilitates multiparametric, phenotypic analysis of immune cells at single-cell resolution. In this review, we outline the capabilities of mass cytometry and illustrate the potential of this technology to enhance the discovery of cellular biomarkers for rheumatoid arthritis, a prototypical autoimmune disease.

    View details for DOI 10.1186/s13075-015-0644-z

    View details for Web of Science ID 000354850500001

    View details for PubMedID 25981462

    View details for PubMedCentralID PMC4436107

  • flowCL: ontology-based cell population labelling in flow cytometry BIOINFORMATICS Courtot, M., Meskas, J., Diehl, A. D., Droumeva, R., Gottardo, R., Jalali, A., Taghiyar, M. J., Maecker, H. T., McCoy, J. P., Ruttenberg, A., Scheuermann, R. H., Brinkman, R. R. 2015; 31 (8): 1337-1339


    Finding one or more cell populations of interest, such as those correlating to a specific disease, is critical when analysing flow cytometry data. However, labelling of cell populations is not well defined, making it difficult to integrate the output of algorithms to external knowledge sources.We developed flowCL, a software package that performs semantic labelling of cell populations based on their surface markers and applied it to labelling of the Federation of Clinical Immunology Societies Human Immunology Project Consortium lyoplate populations as a use case.By providing automated labelling of cell populations based on their immunophenotype, flowCL allows for unambiguous and reproducible identification of standardized cell types.Code, R script and documentation are available under the Artistic 2.0 license through Bioconductor ( data are available at Bioinformatics online.

    View details for DOI 10.1093/bioinformatics/btu807

    View details for Web of Science ID 000354453700035

    View details for PubMedID 25481008

    View details for PubMedCentralID PMC4393520

  • Cytomegalovirus infection enhances the immune response to influenza. Science translational medicine Furman, D., Jojic, V., Sharma, S., Shen-Orr, S. S., Angel, C. J., Onengut-Gumuscu, S., Kidd, B. A., Maecker, H. T., Concannon, P., Dekker, C. L., Thomas, P. G., Davis, M. M. 2015; 7 (281): 281ra43-?


    Cytomegalovirus (CMV) is a β-herpesvirus present in a latent form in most people worldwide. In immunosuppressed individuals, CMV can reactivate and cause serious clinical complications, but the effect of the latent state on healthy people remains elusive. We undertook a systems approach to understand the differences between seropositive and negative subjects and measured hundreds of immune system components from blood samples including cytokines and chemokines, immune cell phenotyping, gene expression, ex vivo cell responses to cytokine stimuli, and the antibody response to seasonal influenza vaccination. As expected, we found decreased responses to vaccination and an overall down-regulation of immune components in aged individuals regardless of CMV status. In contrast, CMV-seropositive young adults exhibited enhanced antibody responses to influenza vaccination, increased CD8(+) T cell sensitivity, and elevated levels of circulating interferon-γ compared to seronegative individuals. Experiments with young mice infected with murine CMV also showed significant protection from an influenza virus challenge compared with uninfected animals, although this effect declined with time. These data show that CMV and its murine equivalent can have a beneficial effect on the immune response of young, healthy individuals, which may explain the ubiquity of CMV infection in humans and many other species.

    View details for DOI 10.1126/scitranslmed.aaa2293

    View details for PubMedID 25834109

  • Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry. Journal of immunology Mei, H. E., Leipold, M. D., Schulz, A. R., Chester, C., Maecker, H. T. 2015; 194 (4): 2022-2031


    Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 Ab conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and it reduces wet work and Ab consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45 barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and it should be applicable to fluorescence flow cytometry as well.

    View details for DOI 10.4049/jimmunol.1402661

    View details for PubMedID 25609839

    View details for PubMedCentralID PMC4323739

  • Variation in the human immune system is largely driven by non-heritable influences. Cell Brodin, P., Jojic, V., Gao, T., Bhattacharya, S., Angel, C. J., Furman, D., Shen-Orr, S., Dekker, C. L., Swan, G. E., Butte, A. J., Maecker, H. T., Davis, M. M. 2015; 160 (1-2): 37-47


    There is considerable heterogeneity in immunological parameters between individuals, but its sources are largely unknown. To assess the relative contribution of heritable versus non-heritable factors, we have performed a systems-level analysis of 210 healthy twins between 8 and 82 years of age. We measured 204 different parameters, including cell population frequencies, cytokine responses, and serum proteins, and found that 77% of these are dominated (>50% of variance) and 58% almost completely determined (>80% of variance) by non-heritable influences. In addition, some of these parameters become more variable with age, suggesting the cumulative influence of environmental exposure. Similarly, the serological responses to seasonal influenza vaccination are also determined largely by non-heritable factors, likely due to repeated exposure to different strains. Lastly, in MZ twins discordant for cytomegalovirus infection, more than half of all parameters are affected. These results highlight the largely reactive and adaptive nature of the immune system in healthy individuals.

    View details for DOI 10.1016/j.cell.2014.12.020

    View details for PubMedID 25594173

    View details for PubMedCentralID PMC4302727

  • Immune monitoring technology primer: flow and mass cytometry. Journal for immunotherapy of cancer Maecker, H. T., Harari, A. 2015; 3: 44-?

    View details for DOI 10.1186/s40425-015-0085-x

    View details for PubMedID 26380089

    View details for PubMedCentralID PMC4570613

  • Thinking outside the gate: single-cell assessments in multiple dimensions. Immunity Kvistborg, P., Gouttefangeas, C., Aghaeepour, N., Cazaly, A., Chattopadhyay, P. K., Chan, C., Eckl, J., Finak, G., Hadrup, S. R., Maecker, H. T., Maurer, D., Mosmann, T., Qiu, P., Scheuermann, R. H., Welters, M. J., Ferrari, G., Brinkman, R. R., Britten, C. M. 2015; 42 (4): 591–92

    View details for PubMedID 25902473

  • Intracellular Cytokine Staining on PBMCs Using CyTOF™ Mass Cytometry. Bio-protocol Lin, D., Gupta, S., Maecker, H. T. 2015; 5 (1)


    In this protocol, we use a CyTOF™ mass cytometry to collect single-cell data on a large number of cytokines/chemokines as well as cell-surface proteins that characterize T cells and other immune cells. The current selected mass window in AW 103-203 includes the lanthanides used for most antibody labeling, along with iridium and rhodium for DNA intercalators. The output data are in the format as .txt and .fcs files, which is compatible with many analysis programs. This protocol could be adapted to include tetramers into the staining panel, but we have not optimized for that purpose. The principal steps of intracellular cytokine staining are as follows: First, cells are activated for a few hours using either a specific peptide or a non-specific activation cocktail. An inhibitor of protein transport (e.g. Brefeldin A) is added to retain the cytokines within the cell. Next, EDTA is added to remove adherent cells from the activation vessel. After washing, antibodies to cell surface markers are added to the cells. The cells are then fixed in paraformaldehyde and permeabilized. We use a gentle detergent, saponin, as the permealization buffer because it is less destructive to surface and intracellular epitopes compared to harsh detergents or methanol. After permeabilization, the metal-conjugated anti-cytokine antibodies are added into the cell suspension. The stained cells are then sequentially introduced into the mass cytometry for signal intensity analysis.

    View details for PubMedID 29104886

  • Predictors of clinical response to immunotherapy with or without radiotherapy. Journal of radiation oncology Hiniker, S. M., Maecker, H. T., Knox, S. J. 2015; 4: 339-345


    Success with recent immunotherapies has resulted in previously unattainable response rates, as well as durable responses in diseases with historically poor prognoses. The combination of radiation therapy and immunotherapy has been a recent area of active investigation, with exciting results in a subset of patients. However, patient characteristics predictive of probable benefit from therapy and clinically meaningful biomarkers indicative of the early development of an antitumor immune response have yet to be identified. What is needed is a better way to predict which patients are likely to benefit from therapy, which would allow those patients unlikely to benefit from immunotherapy to be spared potentially futile therapies, thereby avoiding unnecessary risks of toxicity and costly treatment. Here, we summarize the early data on predictors of clinical response to immunotherapy, and to immunotherapy in combination with radiation.

    View details for PubMedID 26709361

  • Phenotyping of Live Human PBMC using CyTOF™ Mass Cytometry. Bio-protocol Leipold, M. D., Maecker, H. T. 2015; 5 (2)


    Single-cell analysis has become an method of importance in immunology. Fluorescence flow cytometry has been a major player. However, due to issues such as autofluorescence and emission spillover between different fluorophores, alternative techniques are being developed. In recent years, mass cytometry has emerged, wherein antibodies labeled with metal ions are detected by ICP-MS. In order for a cell to be seen, a metal in the mass window must be present; there is no analogous parameter to forward or side scatter. The current mass window selected is approximately AW 103-196, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators. In this protocol, we use a cocktail of antibodies labeled with MAXPAR metal-chelating polymers to surface-stain live PBMC that have been previously cryopreserved. Many of these markers were taken from a standard fluorescence phenotyping panel (Maecker et al., 2012). No intracellular antibodies are used. We use a CyTOF™ (Cytometry by Time-Of-Flight) mass cytometer to acquire the ICP-MS data. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type.

    View details for PubMedID 27390767

  • Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry. Methods in molecular biology (Clifton, N.J.) Leipold, M. D., Newell, E. W., Maecker, H. T. 2015; 1343: 81-95


    The standard for single-cell analysis of phenotype and function in recent decades has been fluorescence flow cytometry. Mass cytometry is a newer technology that uses heavy metal ions, rather than fluorochromes, as labels for probes such as antibodies. The binding of these ion-labeled probes to cells is quantitated by mass spectrometry. This greatly increases the number of phenotypic and functional markers that can be probed simultaneously. Here, we review topics that must be considered when adapting existing flow cytometry panels to mass cytometry analysis. We present a protocol and representative panels for surface phenotyping and intracellular cytokine staining (ICS) assays.

    View details for DOI 10.1007/978-1-4939-2963-4_7

    View details for PubMedID 26420710

    View details for PubMedCentralID PMC4748856

  • Mass cytometry as a platform for the discovery of cellular biomarkers to guide effective rheumatic disease therapy. Arthritis research & therapy Nair, N., Mei, H. E., Chen, S., Hale, M., Nolan, G. P., Maecker, H. T., Genovese, M., Fathman, C. G., Whiting, C. C. 2015; 17: 127-?


    The development of biomarkers for autoimmune diseases has been hampered by a lack of understanding of disease etiopathogenesis and of the mechanisms underlying the induction and maintenance of inflammation, which involves complex activation dynamics of diverse cell types. The heterogeneous nature and suboptimal clinical response to treatment observed in many autoimmune syndromes highlight the need to develop improved strategies to predict patient outcome to therapy and personalize patient care. Mass cytometry, using CyTOF®, is an advanced technology that facilitates multiparametric, phenotypic analysis of immune cells at single-cell resolution. In this review, we outline the capabilities of mass cytometry and illustrate the potential of this technology to enhance the discovery of cellular biomarkers for rheumatoid arthritis, a prototypical autoimmune disease.

    View details for DOI 10.1186/s13075-015-0644-z

    View details for PubMedID 25981462

  • Algorithmic Tools for Mining High-Dimensional Cytometry Data. Journal of immunology (Baltimore, Md. : 1950) Chester, C., Maecker, H. T. 2015; 195 (3): 773–79


    The advent of mass cytometry has led to an unprecedented increase in the number of analytes measured in individual cells, thereby increasing the complexity and information content of cytometric data. Although this technology is ideally suited to the detailed examination of the immune system, the applicability of the different methods for analyzing such complex data is less clear. Conventional data analysis by manual gating of cells in biaxial dot plots is often subjective, time consuming, and neglectful of much of the information contained in a highly dimensional cytometric dataset. Algorithmic data mining has the promise to eliminate these concerns, and several such tools have been applied recently to mass cytometry data. We review computational data mining tools that have been used to analyze mass cytometry data, outline their differences, and comment on their strengths and limitations. This review will help immunologists to identify suitable algorithmic tools for their particular projects.

    View details for PubMedID 26188071

  • mTOR inhibition improves immune function in the elderly SCIENCE TRANSLATIONAL MEDICINE Mannick, J. B., Del Giudice, G., Lattanzi, M., Valiante, N. M., Praestgaard, J., Huang, B., Lonetto, M. A., Maecker, H. T., Kovarik, J., Carson, S., Glass, D. J., Klickstein, L. B. 2014; 6 (268)


    Inhibition of the mammalian target of rapamycin (mTOR) pathway extends life span in all species studied to date, and in mice delays the onset of age-related diseases and comorbidities. However, it is unknown if mTOR inhibition affects aging or its consequences in humans. To begin to assess the effects of mTOR inhibition on human aging-related conditions, we evaluated whether the mTOR inhibitor RAD001 ameliorated immunosenescence (the decline in immune function during aging) in elderly volunteers, as assessed by their response to influenza vaccination. RAD001 enhanced the response to the influenza vaccine by about 20% at doses that were relatively well tolerated. RAD001 also reduced the percentage of CD4 and CD8 T lymphocytes expressing the programmed death-1 (PD-1) receptor, which inhibits T cell signaling and is more highly expressed with age. These results raise the possibility that mTOR inhibition may have beneficial effects on immunosenescence in the elderly.

    View details for DOI 10.1126/scitranslmed.3009892

    View details for Web of Science ID 000346844100005

    View details for PubMedID 25540326

  • In utero arsenic exposure and fetal immune repertoire in a US pregnancy cohort CLINICAL IMMUNOLOGY Nadeau, K. C., Li, Z., Farzan, S., Koestler, D., Robbins, D., Fei, D. L., Malipatlolla, M., Maecker, H., Enelow, R., Korrick, S., Karagas, M. R. 2014; 155 (2): 188-197


    Arsenic has wide-ranging effects on human health and there is evidence that it alters the immune response by influencing CD4+/CD8+ T cell ratios, IL-2 cytokine levels, and the expression of immune-response genes. We investigated the impact of in utero environmental arsenic exposure on immune development and function in newborns participating in a pregnancy cohort in New Hampshire, U.S., where arsenic levels have exceeded the current EPA maximum contaminant level of 10 μg/L. Our results showed that maternal urinary arsenic concentrations were inversely related to absolute total CD45RA+ CD4+ cord blood CD69+ T cell counts (N=116, p=0.04) and positively associated with CD45RA+ CD69- CD294+ cell counts (p=0.01). In placental samples (N=70), higher in utero urinary arsenic concentrations were positively associated with the expression of IL1β (p=0.03). These data provide evidence that relatively low-level arsenic exposure in utero may alter the fetal immune system and lead to immune dysregulation.

    View details for DOI 10.1016/j.clim.2014.09.004

    View details for Web of Science ID 000346114300004

    View details for PubMedID 25229165

    View details for PubMedCentralID PMC4309995

  • The Split Virus Influenza Vaccine rapidly activates immune cells through Fc gamma receptors VACCINE O'Gorman, W. E., Huang, H., Wei, Y., Davis, K. L., Leipold, M. D., Bendall, S. C., Kidd, B. A., Dekker, C. L., Maecker, H. T., Chien, Y., Davis, M. M. 2014; 32 (45): 5989-5997


    Seasonal influenza vaccination is one of the most common medical procedures and yet the extent to which it activates the immune system beyond inducing antibody production is not well understood. In the United States, the most prevalent formulations of the vaccine consist of degraded or "split" viral particles distributed without any adjuvants. Based on previous reports we sought to determine whether the split influenza vaccine activates innate immune receptors-specifically Toll-like receptors. High-dimensional proteomic profiling of human whole-blood using Cytometry by Time-of-Flight (CyTOF) was used to compare signaling pathway activation and cytokine production between the split influenza vaccine and a prototypical TLR response ex vivo. This analysis revealed that the split vaccine rapidly and potently activates multiple immune cell types but yields a proteomic signature quite distinct from TLR activation. Importantly, vaccine induced activity was dependent upon the presence of human sera indicating that a serum factor was necessary for vaccine-dependent immune activation. We found this serum factor to be human antibodies specific for influenza proteins and therefore immediate immune activation by the split vaccine is immune-complex dependent. These studies demonstrate that influenza virus "splitting" inactivates any potential adjuvants endogenous to influenza, such as RNA, but in previously exposed individuals can elicit a potent immune response by facilitating the rapid formation of immune complexes.

    View details for DOI 10.1016/j.vaccine.2014.07.115

    View details for Web of Science ID 000343629900016

    View details for PubMedCentralID PMC4191649

  • Monitoring the immune competence of cancer patients to predict outcome CANCER IMMUNOLOGY IMMUNOTHERAPY Chang, S., Kohrt, H., Maecker, H. T. 2014; 63 (7): 713-719


    A new era of cancer immunotherapy has brought not only successful cancer vaccines but also immunomodulators, such as those that target checkpoint blockade in order to induce endogenous host immune responses. However, the immune system of cancer patients can be compromised through multiple means, including immune suppression by the tumor and by prior therapies such as chemotherapy and radiation. Therefore, a comprehensive means of assessing patient immunocompetence would seem helpful for determining whether patients are ready to benefit from immunotherapy, and perhaps even which immunotherapy might be most appropriate for them. Unfortunately, there are no standardized tests for immune competence, nor is there agreement on what to measure and what will be predictive of outcome. In this review, we will discuss the technologies and assays that might be most useful for this purpose. We argue for a comprehensive approach that should maximize the chances of developing predictive biomarkers for eventual clinical use.

    View details for DOI 10.1007/s00262-014-1521-3

    View details for Web of Science ID 000339873300006

    View details for PubMedID 24487923

    View details for PubMedCentralID PMC4058358

  • IFN Priming Is Necessary but Not Sufficient To Turn on a Migratory Dendritic Cell Program in Lupus Monocytes JOURNAL OF IMMUNOLOGY Rodriguez-Pla, A., Patel, P., Maecker, H. T., Rossello-Urgell, J., Baldwin, N., Bennett, L., Cantrell, V., Baisch, J., Punaro, M., Gotte, A., Nassi, L., Wright, T., Palucka, A. K., Banchereau, J., Pascual, V. 2014; 192 (12): 5586-5598


    Blood monocytes from children with systemic lupus erythematosus (SLE) behave similar to dendritic cells (DCs), and SLE serum induces healthy monocytes to differentiate into DCs in a type I IFN-dependent manner. In this study, we found that these monocytes display significant transcriptional changes, including a prominent IFN signature, compared with healthy controls. Few of those changes, however, explain DC function. Exposure to allogeneic T cells in vitro reprograms SLE monocytes to acquire DC phenotype and function, and this correlates with both IFN-inducible (IP10) and proinflammatory cytokine (IL-1β and IL6) expression. Furthermore, we found that both IFN and SLE serum induce the upregulation of CCR7 transcription in these cells. CCR7 protein expression, however, requires a second signal provided by TLR agonists such as LPS. Thus, SLE serum "primes" a subset of monocytes to readily (<24 h) respond to TLR agonists and acquire migratory DC properties. Our findings might explain how microbial infections exacerbate lupus.

    View details for DOI 10.4049/jimmunol.1301319

    View details for Web of Science ID 000337172100018

    View details for PubMedID 24829414

  • Effects of serum and plasma matrices on multiplex immunoassays. Immunologic research Rosenberg-Hasson, Y., Hansmann, L., Liedtke, M., Herschmann, I., Maecker, H. T. 2014; 58 (2-3): 224-233


    Multiplexed fluorescence or electrochemiluminescence immunoassays of soluble cytokines are commonly performed in the context of human serum or plasma, to look for disease biomarkers and to monitor the immune system in a simple and minimally invasive way. These assays provide challenges due to the complexities of the matrix (serum or plasma) and the presence of many cytokines near the limit of detection of the assay. Here, we compare the readout of matched serum and plasma samples, which are generally correlated. However, a subset of cytokines usually have higher levels in serum, and the non-specific background is significantly increased in serum versus plasma. Presumably as a result of this non-specific background, disease-related decreases in low-abundance cytokines can sometimes be detected in plasma but not in serum. We further show, through spike recovery experiments, that both serum and plasma inhibit the readout of many cytokines, with some variability between donors, but with serum causing greater inhibition than plasma in many cases. Standard diluents from different vendors can partially reverse this inhibition to varying degrees. Dilution of samples can also partly overcome the inhibitory effect of the matrix. We also show that dilution is nonlinear and differentially affects various cytokines. Together, these data argue that (1) plasma is a more sensitive matrix for detecting changes in certain low-abundance cytokines; (2) calculation of concentrations in serum or plasma matrices is inherently inaccurate; and (3) dilution of samples should not be assumed to be linear, i.e., all comparisons need to be made among similarly diluted samples.

    View details for DOI 10.1007/s12026-014-8491-6

    View details for PubMedID 24522699

  • Vitamin D Deficiency in a Multiethnic Healthy Control Cohort and Altered Immune Response in Vitamin D Deficient European-American Healthy Controls PLOS ONE Ritterhouse, L. L., Lu, R., Shah, H. B., Robertson, J. M., Fife, D. A., Maecker, H. T., Du, H., Fathman, C. G., Chakravarty, E. F., Scofield, R. H., Kamen, D. L., Guthridge, J. M., James, J. A. 2014; 9 (4)


    In recent years, vitamin D has been shown to possess a wide range of immunomodulatory effects. Although there is extensive amount of research on vitamin D, we lack a comprehensive understanding of the prevalence of vitamin D deficiency or the mechanism by which vitamin D regulates the human immune system. This study examined the prevalence and correlates of vitamin D deficiency and the relationship between vitamin D and the immune system in healthy individuals.Healthy individuals (n = 774) comprised of European-Americans (EA, n = 470), African-Americans (AA, n = 125), and Native Americans (NA, n = 179) were screened for 25-hydroxyvitamin D [25(OH)D] levels by ELISA. To identify the most noticeable effects of vitamin D on the immune system, 20 EA individuals with severely deficient (<11.3 ng/mL) and sufficient (>24.8 ng/mL) vitamin D levels were matched and selected for further analysis. Serum cytokine level measurement, immune cell phenotyping, and phosphoflow cytometry were performed.Vitamin D sufficiency was observed in 37.5% of the study cohort. By multivariate analysis, AA, NA, and females with a high body mass index (BMI, >30) demonstrate higher rates of vitamin D deficiency (p<0.05). Individuals with vitamin D deficiency had significantly higher levels of serum GM-CSF (p = 0.04), decreased circulating activated CD4+ (p = 0.04) and CD8+ T (p = 0.04) cell frequencies than individuals with sufficient vitamin D levels.A large portion of healthy individuals have vitamin D deficiency. These individuals have altered T and B cell responses, indicating that the absence of sufficient vitamin D levels could result in undesirable cellular and molecular alterations ultimately contributing to immune dysregulation.

    View details for DOI 10.1371/journal.pone.0094500

    View details for Web of Science ID 000336736200083

    View details for PubMedID 24727903

    View details for PubMedCentralID PMC3984168

  • Cytokine and chemokine patterns across 100 days after hematopoietic stem cell transplantation in children. Biology of blood and marrow transplantation DiCarlo, J., Agarwal-Hashmi, R., Shah, A., Kim, P., Craveiro, L., Killen, R., Rosenberg-Hasson, Y., Maecker, H. 2014; 20 (3): 361-369


    We mapped the cytokine response to hematopoietic stem cell transplantation (HSCT) by assaying 51 cytokines and chemokines each week for 100 days in 51 children receiving allogeneic (n = 44) or autologous HSCT (n = 7). Assay values were reported as mean fluorescence intensity (MFI). Log transformation converted MFI to clinically relevant measures (ie, pg/mL). We searched for potential markers of transplant complications by using mixed treatment by subject analysis of variance. Global cytokine secretion in HSCT recipients was significantly lower than in concurrent control patients (n = 11). Coincident with the nadir in WBC count, the concentration of many cytokines declined further by the second and third week. All analytes (except monokine induced by gamma interferon [MIG]) subsequently rebounded by week 4 (coincident with engraftment and recovery of WBC count) but often still remained well below control levels. Concurrent with the collective nadir of multiple cytokines, monocyte chemoattractant protein 1 (MCP-1), growth-regulated oncogene alpha (GRO-a), and leptin surged during weeks 2 to 4. High levels of leptin persisted throughout the 100 post-transplant days. Also during weeks 2 to 4, hepatocyte growth factor (HGF) and IL-6 surged in children with complications but not in those without complications. The peak in HGF was more pronounced in veno-occlusive disease (VOD). HGF and IL-6 secretion rose at least 2 weeks before the clinical diagnosis of VOD or graft-versus-host disease (GVHD). From week 4 onward in all groups, the MFI of the cytokine resistin increased to 5 to 15 times above concurrent control. HGF has now emerged in 3 or more biomarker discovery efforts for GVHD (and in our population for VOD as well). HGF (with or without IL-6) should be investigated as a potential predictive biomarker of VOD or GVHD. Alternatively, the hyperinflammatory "signature" provided by a multicytokine assay may be predictive.

    View details for DOI 10.1016/j.bbmt.2013.11.026

    View details for PubMedID 24316459

  • Influence of Frequent Infectious Exposures on General and Varicella-Zoster Virus-Specific Immune Responses in Pediatricians CLINICAL AND VACCINE IMMUNOLOGY Ogunjimi, B., Smits, E., Heynderickx, S., Van Den Bergh, J., Bilcke, J., Jansens, H., Malfait, R., Ramet, J., Maecker, H. T., Cools, N., Beutels, P., Van Damme, P. 2014; 21 (3): 417-426


    Reexposure to viruses is assumed to strengthen humoral and cellular immunity via the secondary immune response. We studied the effects of frequent exposure to viral infectious challenges on immunity. Furthermore, we assessed whether repetitive exposures to varicella-zoster virus (VZV) elicited persistently high immune responses. Blood samples from 11 pediatricians and matched controls were assessed at 3 time points and 1 time point, respectively. Besides the assessment of general immunity by means of measuring T-cell subset percentages, antibody titers and gamma interferon (IFN-γ)/interleukin 2 (IL-2)-producing T-cell percentages against adenovirus type 5 (AdV-5), cytomegalovirus (CMV), tetanus toxin (TT), and VZV were determined. Pediatricians had lower levels of circulating CD4(+)-naive T cells and showed boosting of CD8(+) effector memory T cells. Although no effect on humoral immunity was seen, repetitive exposures to VZV induced persistently higher percentages of IFN-γ-positive T cells against all VZV antigens tested (VZV glycoprotein E [gE], VZV intermediate-early protein 62 [IE62], and VZV IE63) than in controls. T cells directed against latency-associated VZV IE63 benefitted the most from natural exogenous boosting. Although no differences in cellular or humoral immunity were found between the pediatricians and controls for AdV-5 or TT, we did find larger immune responses against CMV antigens in pediatricians. Despite the high infectious burden, we detected a robust and diverse immune system in pediatricians. Repetitive exposures to VZV have been shown to induce a stable increased level of VZV-specific cellular but not humoral immunity. Based on our observations, VZV IE63 can be considered a candidate for a zoster vaccine.

    View details for DOI 10.1128/CVI.00818-13

    View details for Web of Science ID 000332036900019

    View details for PubMedID 24429070

  • Peanut oral immunotherapy results in increased antigen-induced regulatory T-cell function and hypomethylation of forkhead box protein 3 (FOXP3). journal of allergy and clinical immunology Syed, A., Garcia, M. A., Lyu, S., Bucayu, R., Kohli, A., Ishida, S., Berglund, J. P., Tsai, M., Maecker, H., O'Riordan, G., Galli, S. J., Nadeau, K. C. 2014; 133 (2): 500-510


    The mechanisms contributing to clinical immune tolerance remain incompletely understood. This study provides evidence for specific immune mechanisms that are associated with a model of operationally defined clinical tolerance.Our overall objective was to study laboratory changes associated with clinical immune tolerance in antigen-induced T cells, basophils, and antibodies in subjects undergoing oral immunotherapy (OIT) for peanut allergy.In a phase 1 single-site study, we studied participants (n = 23) undergoing peanut OIT and compared them with age-matched allergic control subjects (n = 20) undergoing standard of care (abstaining from peanut) for 24 months. Participants were operationally defined as clinically immune tolerant (IT) if they had no detectable allergic reactions to a peanut oral food challenge after 3 months of therapy withdrawal (IT, n = 7), whereas those who had an allergic reaction were categorized as nontolerant (NT; n = 13).Antibody and basophil activation measurements did not statistically differentiate between NT versus IT participants. However, T-cell function and demethylation of forkhead box protein 3 (FOXP3) CpG sites in antigen-induced regulatory T cells were significantly different between IT versus NT participants. When IT participants were withdrawn from peanut therapy for an additional 3 months (total of 6 months), only 3 participants remained classified as IT participants, and 4 participants regained sensitivity along with increased methylation of FOXP3 CpG sites in antigen-induced regulatory T cells.In summary, modifications at the DNA level of antigen-induced T-cell subsets might be predictive of a state of operationally defined clinical immune tolerance during peanut OIT.

    View details for DOI 10.1016/j.jaci.2013.12.1037

    View details for PubMedID 24636474

  • Vitamin d deficiency in a multiethnic healthy control cohort and altered immune response in vitamin D deficient European-American healthy controls. PloS one Ritterhouse, L. L., Lu, R., Shah, H. B., Robertson, J. M., Fife, D. A., Maecker, H. T., Du, H., Fathman, C. G., Chakravarty, E. F., Scofield, R. H., Kamen, D. L., Guthridge, J. M., James, J. A. 2014; 9 (4)


    In recent years, vitamin D has been shown to possess a wide range of immunomodulatory effects. Although there is extensive amount of research on vitamin D, we lack a comprehensive understanding of the prevalence of vitamin D deficiency or the mechanism by which vitamin D regulates the human immune system. This study examined the prevalence and correlates of vitamin D deficiency and the relationship between vitamin D and the immune system in healthy individuals.Healthy individuals (n = 774) comprised of European-Americans (EA, n = 470), African-Americans (AA, n = 125), and Native Americans (NA, n = 179) were screened for 25-hydroxyvitamin D [25(OH)D] levels by ELISA. To identify the most noticeable effects of vitamin D on the immune system, 20 EA individuals with severely deficient (<11.3 ng/mL) and sufficient (>24.8 ng/mL) vitamin D levels were matched and selected for further analysis. Serum cytokine level measurement, immune cell phenotyping, and phosphoflow cytometry were performed.Vitamin D sufficiency was observed in 37.5% of the study cohort. By multivariate analysis, AA, NA, and females with a high body mass index (BMI, >30) demonstrate higher rates of vitamin D deficiency (p<0.05). Individuals with vitamin D deficiency had significantly higher levels of serum GM-CSF (p = 0.04), decreased circulating activated CD4+ (p = 0.04) and CD8+ T (p = 0.04) cell frequencies than individuals with sufficient vitamin D levels.A large portion of healthy individuals have vitamin D deficiency. These individuals have altered T and B cell responses, indicating that the absence of sufficient vitamin D levels could result in undesirable cellular and molecular alterations ultimately contributing to immune dysregulation.

    View details for DOI 10.1371/journal.pone.0094500

    View details for PubMedID 24727903

    View details for PubMedCentralID PMC3984168

  • Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry. Science translational medicine Horowitz, A., Strauss-Albee, D. M., Leipold, M., Kubo, J., Nemat-Gorgani, N., Dogan, O. C., Dekker, C. L., Mackey, S., Maecker, H., Swan, G. E., Davis, M. M., Norman, P. J., Guethlein, L. A., Desai, M., Parham, P., Blish, C. A. 2013; 5 (208): 208ra145-?


    Natural killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 37 parameters, including 28 NK cell receptors, on peripheral blood NK cells from 5 sets of monozygotic twins and 12 unrelated donors of defined human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6000 to 30,000 phenotypic populations within an individual and >100,000 phenotypes in the donor panel. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation.

    View details for DOI 10.1126/scitranslmed.3006702

    View details for PubMedID 24154599

  • A harmonized approach to intracellular cytokine staining gating: Results from an international multiconsortia proficiency panel conducted by the Cancer Immunotherapy Consortium (CIC/CRI). Cytometry. Part A : the journal of the International Society for Analytical Cytology McNeil, L. K., Price, L., Britten, C. M., Jaimes, M., Maecker, H., Odunsi, K., Matsuzaki, J., Staats, J. S., Thorpe, J., Yuan, J., Janetzki, S. 2013; 83 (8): 728-738


    Previous results from two proficiency panels of intracellular cytokine staining (ICS) from the Cancer Immunotherapy Consortium and panels from the National Institute of Allergy and Infectious Disease and the Association for Cancer Immunotherapy highlight the variability across laboratories in reported % CD8+ or % CD4+ cytokine-positive cells. One of the main causes of interassay variability in flow cytometry-based assays is due to differences in gating strategies between laboratories, which may prohibit the generation of robust results within single centers and across institutions. To study how gating strategies affect the variation in reported results, a gating panel was organized where all participants analyzed the same set of Flow Cytometry Standard (FCS) files from a four-color ICS assay using their own gating protocol (Phase I) and a gating protocol drafted by consensus from the organizers of the panel (Phase II). Focusing on analysis removed donor, assay, and instrument variation, enabling us to quantify the variability caused by gating alone. One hundred ten participating laboratories applied 110 different gating approaches. This led to high variability in the reported percentage of cytokine-positive cells and consequently in response detection in Phase I. However, variability was dramatically reduced when all laboratories used the same gating strategy (Phase II). Proximity of the cytokine gate to the negative population most impacted true-positive and false-positive response detection. Recommendations are provided for the (1) placement of the cytokine-positive gate, (2) identification of CD4+ CD8+ double-positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and 6) proper adjustment of the biexponential scaling. © 2013 International Society for Advancement of Cytometry.

    View details for DOI 10.1002/cyto.a.22319

    View details for PubMedID 23788464

  • Experimental pain and opioid analgesia in volunteers at high risk for obstructive sleep apnea. PloS one Doufas, A. G., Tian, L., Padrez, K. A., Suwanprathes, P., Cardell, J. A., Maecker, H. T., Panousis, P. 2013; 8 (1)


    Obstructive sleep apnea (OSA) is characterized by recurrent nocturnal hypoxia and sleep disruption. Sleep fragmentation caused hyperalgesia in volunteers, while nocturnal hypoxemia enhanced morphine analgesic potency in children with OSA. This evidence directly relates to surgical OSA patients who are at risk for airway compromise due to postoperative use of opioids. Using accepted experimental pain models, we characterized pain processing and opioid analgesia in male volunteers recruited based on their risk for OSA.After approval from the Intitutional Review Board and informed consent, we assessed heat and cold pain thresholds and tolerances in volunteers after overnight polysomnography (PSG). Three pro-inflammatory and 3 hypoxia markers were determined in the serum. Pain tests were performed at baseline, placebo, and two effect site concentrations of remifentanil (1 and 2 µg/ml), an μ-opioid agonist. Linear mixed effects regression models were employed to evaluate the association of 3 PSG descriptors [wake after sleep onset, number of sleep stage shifts, and lowest oxyhemoglobin saturation (SaO(2)) during sleep] and all serum markers with pain thresholds and tolerances at baseline, as well as their changes under remifentanil.Forty-three volunteers (12 normal and 31 with a PSG-based diagnosis of OSA) were included in the analysis. The lower nadir SaO(2) and higher insulin growth factor binding protein-1 (IGFBP-1) were associated with higher analgesic sensitivity to remifentanil (SaO(2), P = 0.0440; IGFBP-1, P = 0.0013). Other pro-inflammatory mediators like interleukin-1β and tumor necrosis factor-α (TNF-α) were associated with an enhanced sensitivity to the opioid analgesic effect (IL-1β, P = 0.0218; TNF-α, P = 0.0276).Nocturnal hypoxemia in subjects at high risk for OSA was associated with an increased potency of opioid analgesia. A serum hypoxia marker (IGFBP-1) was associated with hypoalgesia and increased potency to opioid analgesia; other pro-inflammatory mediators also predicted an enhanced opioid NCT00672737.

    View details for DOI 10.1371/journal.pone.0054807

    View details for PubMedID 23382975

    View details for PubMedCentralID PMC3558510

  • Daily cytokine fluctuations, driven by leptin, are associated with fatigue severity in chronic fatigue syndrome: evidence of inflammatory pathology. Journal of translational medicine Stringer, E. A., Baker, K. S., Carroll, I. R., Montoya, J. G., Chu, L., Maecker, H. T., Younger, J. W. 2013; 11: 93-?


    Chronic fatigue syndrome (CFS) is a debilitating disorder characterized by persistent fatigue that is not alleviated by rest. The lack of a clearly identified underlying mechanism has hindered the development of effective treatments. Studies have demonstrated elevated levels of inflammatory factors in patients with CFS, but findings are contradictory across studies and no biomarkers have been consistently supported. Single time-point approaches potentially overlook important features of CFS, such as fluctuations in fatigue severity. We have observed that individuals with CFS demonstrate significant day-to-day variability in their fatigue severity.Therefore, to complement previous studies, we implemented a novel longitudinal study design to investigate the role of cytokines in CFS pathophysiology. Ten women meeting the Fukuda diagnostic criteria for CFS and ten healthy age- and body mass index (BMI)-matched women underwent 25 consecutive days of blood draws and self-reporting of symptom severity. A 51-plex cytokine panel via Luminex was performed for each of the 500 serum samples collected. Our primary hypothesis was that daily fatigue severity would be significantly correlated with the inflammatory adipokine leptin, in the women with CFS and not in the healthy control women. As a post-hoc analysis, a machine learning algorithm using all 51 cytokines was implemented to determine whether immune factors could distinguish high from low fatigue days.Self-reported fatigue severity was significantly correlated with leptin levels in six of the participants with CFS and one healthy control, supporting our primary hypothesis. The machine learning algorithm distinguished high from low fatigue days in the CFS group with 78.3% accuracy.Our results support the role of cytokines in the pathophysiology of CFS.

    View details for DOI 10.1186/1479-5876-11-93

    View details for PubMedID 23570606

  • Apoptosis and other immune biomarkers predict influenza vaccine responsiveness. Molecular systems biology Furman, D., Jojic, V., Kidd, B., Shen-Orr, S., Price, J., Jarrell, J., Tse, T., Huang, H., Lund, P., Maecker, H. T., Utz, P. J., Dekker, C. L., Koller, D., Davis, M. M. 2013; 9: 659-?


    Despite the importance of the immune system in many diseases, there are currently no objective benchmarks of immunological health. In an effort to identifying such markers, we used influenza vaccination in 30 young (20-30 years) and 59 older subjects (60 to >89 years) as models for strong and weak immune responses, respectively, and assayed their serological responses to influenza strains as well as a wide variety of other parameters, including gene expression, antibodies to hemagglutinin peptides, serum cytokines, cell subset phenotypes and in vitro cytokine stimulation. Using machine learning, we identified nine variables that predict the antibody response with 84% accuracy. Two of these variables are involved in apoptosis, which positively associated with the response to vaccination and was confirmed to be a contributor to vaccine responsiveness in mice. The identification of these biomarkers provides new insights into what immune features may be most important for immune health.

    View details for DOI 10.1038/msb.2013.15

    View details for PubMedID 23591775

    View details for PubMedCentralID PMC3658270

  • The phenotypic distribution and functional profile of tuberculin-specific CD4 T-cells characterizes different stages of TB infection. Cytometry. Part B, Clinical cytometry Streitz, M., Fuhrmann, S., Thomas, D., Cheek, E., Nomura, L., Maecker, H., Martus, P., Aghaeepour, N., Brinkman, R. R., Volk, H., Kern, F. 2012; 82 (6): 360-368


    Recent publications have suggested that altered proportions of functional CD4 T-cell subsets correlate with active pulmonary TB. Also, CD27-expression on tuberculin-activated IFN-γ(+) CD4 T-cells is known to differ significantly between patients with active pulmonary TB and healthy TB-unexposed BCG vaccinees. Here, we explore links between CD4 T-cell phenotype, multiple functional subsets, and control of TB.We examined ex-vivo overnight tuberculin activated CD4 T-cells in regards to CD27-expression and the activation markers, CD154 upregulation, IFN-γ, TNF-α, IL-2, and degranulation in 44 individuals, including cases of clinically active pulmonary TB, and hospital staff with prolonged TB exposure, some of whom had latent TB.Active pulmonary TB generally showed an excess of TNF-α(+) subsets over IFN-γ(+) subsets, paralleled by decreased CD27 expression on activated IFN-γ(+) or CD154(+) CD4 T-cells. The single subset distinguishing best between active pulmonary TB and high TB exposure was CD154(+) /TNF-α(+) / IFN-γ(-) /IL-2(-) /degranulation(-) (AUROC 0.90). The ratio between the frequencies of TNF-α(+) /IFN-γ(+) CD4 T-cells was an effective alternative parameter (AUROC 0.87).Functional subsets and phenotype of tuberculin induced CD4 T-cells differ between stages of TB infection. Predominance of TNF-α(+) CD4 T-cells in active infection suggests an increased effort of the immune system to contain disease.

    View details for DOI 10.1002/cyto.b.21041

    View details for PubMedID 22961735

  • T Cell Assays and MIATA: The Essential Minimum for Maximum Impact IMMUNITY Britten, C. M., Janetzki, S., Butterfield, L. H., Ferrari, G., Gouttefangeas, C., Huber, C., Kalos, M., Levitsky, H. I., Maecker, H. T., Melief, C. J., O'Donnell-Tormey, J., Odunsi, K., Old, L. J., Ottenhoff, T. H., Ottensmeier, C., Pawelec, G., Roederer, M., Roep, B. O., Romero, P., van der Burg, S. H., Walter, S., Hoos, A., DAVIS, M. M. 2012; 37 (1): 1-2

    View details for DOI 10.1016/j.immuni.2012.07.010

    View details for Web of Science ID 000307133200001

    View details for PubMedID 22840835

  • New tools for classification and monitoring of autoimmune diseases NATURE REVIEWS RHEUMATOLOGY Maecker, H. T., Lindstrom, T. M., Robinson, W. H., Utz, P. J., Hale, M., Boyd, S. D., Shen-Orr, S. S., Fathman, C. G. 2012; 8 (6): 317-328


    Rheumatologists see patients with a range of autoimmune diseases. Phenotyping these diseases for diagnosis, prognosis and selection of therapies is an ever increasing problem. Advances in multiplexed assay technology at the gene, protein, and cellular level have enabled the identification of 'actionable biomarkers'; that is, biological metrics that can inform clinical practice. Not only will such biomarkers yield insight into the development, remission, and exacerbation of a disease, they will undoubtedly improve diagnostic sensitivity and accuracy of classification, and ultimately guide treatment. This Review provides an introduction to these powerful technologies that could promote the identification of actionable biomarkers, including mass cytometry, protein arrays, and immunoglobulin and T-cell receptor high-throughput sequencing. In our opinion, these technologies should become part of routine clinical practice for the management of autoimmune diseases. The use of analytical tools to deconvolve the data obtained from use of these technologies is also presented here. These analyses are revealing a more comprehensive and interconnected view of the immune system than ever before and should have an important role in directing future treatment approaches for autoimmune diseases.

    View details for DOI 10.1038/nrrheum.2012.66

    View details for PubMedID 22647780

  • The Stanford Data Miner: a novel approach for integrating and exploring heterogeneous immunological data JOURNAL OF TRANSLATIONAL MEDICINE Siebert, J. C., Munsil, W., Rosenberg-Hasson, Y., Davis, M. M., Maecker, H. T. 2012; 10


    Systems-level approaches are increasingly common in both murine and human translational studies. These approaches employ multiple high information content assays. As a result, there is a need for tools to integrate heterogeneous types of laboratory and clinical/demographic data, and to allow the exploration of that data by aggregating and/or segregating results based on particular variables (e.g., mean cytokine levels by age and gender).Here we describe the application of standard data warehousing tools to create a novel environment for user-driven upload, integration, and exploration of heterogeneous data. The system presented here currently supports flow cytometry and immunoassays performed in the Stanford Human Immune Monitoring Center, but could be applied more generally.Users upload assay results contained in platform-specific spreadsheets of a defined format, and clinical and demographic data in spreadsheets of flexible format. Users then map sample IDs to connect the assay results with the metadata. An OLAP (on-line analytical processing) data exploration interface allows filtering and display of various dimensions (e.g., Luminex analytes in rows, treatment group in columns, filtered on a particular study). Statistics such as mean, median, and N can be displayed. The views can be expanded or contracted to aggregate or segregate data at various levels. Individual-level data is accessible with a single click. The result is a user-driven system that permits data integration and exploration in a variety of settings. We show how the system can be used to find gender-specific differences in serum cytokine levels, and compare them across experiments and assay types.We have used the tools and techniques of data warehousing, including open-source business intelligence software, to support investigator-driven data integration and mining of diverse immunological data.

    View details for DOI 10.1186/1479-5876-10-62

    View details for Web of Science ID 000304554800001

    View details for PubMedID 22452993

  • NK cells are dysfunctional in human chronic myelogenous leukemia before and on imatinib treatment and in BCR-ABL-positive mice LEUKEMIA Chen, C., Koschmieder, S., KERSTIENS, L., Schemionek, M., Altvater, B., Pscherer, S., Gerss, J., Maecker, H. T., Berdel, W. E., Juergens, H., Lee, P. P., Rossig, C. 2012; 26 (3): 465-474


    Although BCR-ABL+ stem cells in chronic myeloid leukemia (CML) resist elimination by targeted pharmacotherapy in most patients, immunological graft-versus-leukemia effects can cure the disease. Besides cytotoxic T cells, natural killer (NK) cells may have a role in immune control of CML. Here, we explored the functionality of NK cells in CML patients and in a transgenic inducible BCR-ABL mouse model. Compared with controls, NK-cell proportions among lymphocytes were decreased at diagnosis of CML and did not recover during imatinib-induced remission for 10-34 months. Functional experiments revealed limited in vitro expansion of NK cells from CML patients and a reduced degranulation response to K562 target cells both at diagnosis and during imatinib therapy. Consistent with the results in human CML, relative numbers of NK1.1+ NK cells were reduced following induction of BCR-ABL expression in mice, and the defects persisted after BCR-ABL reversion. Moreover, target-induced degranulation by expanded BCR-ABL+ NK cells was compromised. We conclude that CML is associated with quantitative and functional defects within the NK-cell compartment, which is reproduced by induced BCR-ABL expression in mice. Further work will aim at identifying the mechanisms of NK-cell deficiency in CML and at developing strategies to exploit NK cells for immunotherapy.

    View details for DOI 10.1038/leu.2011.239

    View details for Web of Science ID 000301290300012

    View details for PubMedID 21904381

  • Standardizing immunophenotyping for the Human Immunology Project NATURE REVIEWS IMMUNOLOGY Maecker, H. T., McCoy, J. P., Nussenblatt, R. 2012; 12 (3): 191-200


    The heterogeneity in the healthy human immune system, and the immunological changes that portend various diseases, have been only partially described. Their comprehensive elucidation has been termed the 'Human Immunology Project'. The accurate measurement of variations in the human immune system requires precise and standardized assays to distinguish true biological changes from technical artefacts. Thus, to be successful, the Human Immunology Project will require standardized assays for immunophenotyping humans in health and disease. A major tool in this effort is flow cytometry, which remains highly variable with regard to sample handling, reagents, instrument setup and data analysis. In this Review, we outline the current state of standardization of flow cytometry assays and summarize the steps that are required to enable the Human Immunology Project.

    View details for DOI 10.1038/nri3158

    View details for Web of Science ID 000300790600013

    View details for PubMedID 22343568

    View details for PubMedCentralID PMC3409649

  • Mass cytometry: protocol for daily tuning and running cell samples on a CyTOF mass cytometer. Journal of visualized experiments : JoVE Leipold, M. D., Maecker, H. T. 2012: e4398-?


    In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS. Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes. Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples. Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells. Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

    View details for DOI 10.3791/4398

    View details for PubMedID 23149654

    View details for PubMedCentralID PMC3499083

  • Vitamin D Deficient Healthy Individuals Have Decreased Activated T Cells and Altered Lymphocyte Responses to Cytokine Stimulation 75th Annual Scientific Meeting of the American-College-of-Rheumatology/46th Annual Scientific Meeting of the Association-of-Rheumatology-Health-Professionals (ARHP) Ritterhouse, L. L., Maecker, H. T., Du, H., Fathman, C. G., Guthridge, J., James, J. A. WILEY-BLACKWELL. 2011: S19–S19
  • Recommendations from the iSBTc-SITC/FDA/NCI Workshop on Immunotherapy Biomarkers CLINICAL CANCER RESEARCH Butterfield, L. H., Palucka, A. K., Britten, C. M., Dhodapkar, M. V., Hakansson, L., Janetzki, S., Kawakami, Y., Kleen, T., Lee, P. P., Maccalli, C., Maecker, H. T., Maino, V. C., Maio, M., Malyguine, A., Masucci, G., Pawelec, G., Potter, D. M., Rivoltini, L., Salazar, L. G., Schendel, D. J., Slingluff, C. L., Song, W., Stroncek, D. F., Tahara, H., Thurin, M., Trinchieri, G., van der Burg, S. H., Whiteside, T. L., Wigginton, J. M., Marincola, F., Khleif, S., Fox, B. A., Disis, M. L. 2011; 17 (10): 3064-3076


    To facilitate development of innovative immunotherapy approaches, especially for treatment concepts exploiting the potential benefits of personalized therapy, there is a need to develop and validate tools to identify patients who can benefit from immunotherapy. Despite substantial effort, we do not yet know which parameters of antitumor immunity to measure and which assays are optimal for those measurements.The iSBTc-SITC (International Society for Biological Therapy of Cancer-Society for Immunotherapy of Cancer), FDA (Food and Drug Administration), and NCI (National Cancer Institute) partnered to address these issues for immunotherapy of cancer. Here, we review the major challenges, give examples of approaches and solutions, and present our recommendations.Although specific immune parameters and assays are not yet validated, we recommend following standardized (accurate, precise, and reproducible) protocols and use of functional assays for the primary immunologic readouts of a trial; consideration of central laboratories for immune monitoring of large, multi-institutional trials; and standardized testing of several phenotypic and functional potential potency assays specific to any cellular product. When reporting results, the full QA (quality assessment)/QC (quality control) should be conducted and selected examples of truly representative raw data and assay performance characteristics should be included. Finally, to promote broader analysis of multiple aspects of immunity, and gather data on variability, we recommend that in addition to cells and serum, RNA and DNA samples be banked (under standardized conditions) for later testing. We also recommend that sufficient blood be drawn to allow for planned testing of the primary hypothesis being addressed in the trial, and that additional baseline and posttreatment blood is banked for testing novel hypotheses (or generating new hypotheses) that arise in the field.

    View details for DOI 10.1158/1078-0432.CCR-10-2234

    View details for Web of Science ID 000290610000003

    View details for PubMedID 21558394

    View details for PubMedCentralID PMC3096674

  • Tuberculin-Specific T Cells Are Reduced in Active Pulmonary Tuberculosis Compared to LTBI or Status Post BCG Vaccination JOURNAL OF INFECTIOUS DISEASES Streitz, M., Fuhrmann, S., Powell, F., Quassem, A., Nomura, L., Maecker, H., Martus, P., Volk, H., Kern, F. 2011; 203 (3): 378-382


    Functional characteristics of tuberculosis (TB)-specific CD4 T cells were studied in clinically active pulmonary TB (n = 21) and high TB exposure including LTBI (n = 17). Following tuberculin stimulation, activated CD4 T cells were identified by flow-cytometry (CD154 up-regulation, degranulation, interferon γ [IFN-γ], tumor necrosis factor α [TNF-α], and interleukin 2 [IL-2\ production). Interestingly, CD154 up-regulation accounted for ∼80% of activated CD4 T cells in the active TB group but just 40% in the controls, whereas IFN-γ accounted for only ∼50% of activated cells in each group. The frequencies of CD4 T cells displaying at least 1 activation marker discriminated better between the groups than those displaying degranulation or IFN-γ production alone.

    View details for DOI 10.1093/infdis/jiq065

    View details for Web of Science ID 000286611800016

    View details for PubMedID 21186260

  • Quality assurance of intracellular cytokine staining assays: Analysis of multiple rounds of proficiency testing JOURNAL OF IMMUNOLOGICAL METHODS Jaimes, M. C., Maecker, H. T., Yan, M., Maino, V. C., Hanley, M. B., Greer, A., Darden, J. M., D'Souza, M. P. 2011; 363 (2): 143-157


    When evaluating candidate prophylactic HIV and cancer vaccines, intracellular cytokine staining (ICS) assays that measure the frequency and magnitude of antigen-specific T-cell subsets are one tool to monitor immunogen performance and make product advancement decisions. To assess the inter-laboratory assay variation among multiple laboratories testing vaccine candidates, the NIH/NIAID/DAIDS in collaboration with BD Biosciences implemented an ICS Quality Assurance Program (QAP). Seven rounds of testing have been conducted in which 16 laboratories worldwide participated. In each round, IFN-γ, IL-2 and/or TNF-α responses in CD4+ and CD8+ T-cells to CEF or CMV pp65 peptide mixes were tested using cryopreserved peripheral blood mononuclear cells (PBMC) from CMV seropositive donors. We found that for responses measured above 0.2%, inter-laboratory %CVs were, on average, 35%. No differences in inter-laboratory variation were observed if a 4-color antibody cocktail or a 7-color combination was used. Moreover, the data allowed identification of important sources of variability for flow cytometry-based assays, including: number of collected events, gating strategy and instrument setup and performance. As a consequence, in this multi-site study we were able to define pass and fail criteria for ICS assays, which will be adopted in the subsequent rounds of testing and could be easily extrapolated to QAP for other flow cytometry-based assays.

    View details for DOI 10.1016/j.jim.2010.08.004

    View details for Web of Science ID 000287176100006

    View details for PubMedID 20727897

  • Multiparameter intracellular cytokine staining. Methods in molecular biology (Clifton, N.J.) Lovelace, P., Maecker, H. T. 2011; 699: 165-178


    Intracellular cytokine staining (ICS) is a popular method for visualizing cellular responses, most often T-cell responses to antigenic or mitogenic stimulation. It can be coupled with staining for other functional markers, such as upregulation of CD107 or CD154, as well as phenotypic markers that define specific cellular subsets, e.g. effector and memory T-cell compartments. Recent advances in multicolor flow cytometry instrumentation and software have allowed the routine combination of 8-12 (or more) markers in combination, creating technical and analytical challenges along the way, and exposing a need for standardization in the field. Here, we will review best practices for antibody panel design and procedural variables for multicolor ICS, and present an optimized protocol with variations designed for use with specific markers and sample types.

    View details for DOI 10.1007/978-1-61737-950-5_8

    View details for PubMedID 21116983

  • Minimal information about T cell assays: the process of reaching the community of T cell immunologists in cancer and beyond CANCER IMMUNOLOGY IMMUNOTHERAPY Britten, C. M., Janetzki, S., van der Burg, S. H., Huber, C., Kalos, M., Levitsky, H. I., Maecker, H. T., Melief, C. J., O'Donnell-Tormey, J., Odunsi, K., Old, L. J., Pawelec, G., Roep, B. O., Romero, P., Hoos, A., DAVIS, M. M. 2011; 60 (1): 15-22


    Many assays to evaluate the nature, breadth, and quality of antigen-specific T cell responses are currently applied in human medicine. In most cases, assay-related protocols are developed on an individual laboratory basis, resulting in a large number of different protocols being applied worldwide. Together with the inherent complexity of cellular assays, this leads to unnecessary limitations in the ability to compare results generated across institutions. Over the past few years a number of critical assay parameters have been identified which influence test performance irrespective of protocol, material, and reagents used. Describing these critical factors as an integral part of any published report will both facilitate the comparison of data generated across institutions and lead to improvements in the assays themselves. To this end, the Minimal Information About T Cell Assays (MIATA) project was initiated. The objective of MIATA is to achieve a broad consensus on which T cell assay parameters should be reported in scientific publications and to propose a mechanism for reporting these in a systematic manner. To add maximum value for the scientific community, a step-wise, open, and field-spanning approach has been taken to achieve technical precision, user-friendliness, adequate incorporation of concerns, and high acceptance among peers. Here, we describe the past, present, and future perspectives of the MIATA project. We suggest that the approach taken can be generically applied to projects in which a broad consensus has to be reached among scientists working in fragmented fields, such as immunology. An additional objective of this undertaking is to engage the broader scientific community to comment on MIATA and to become an active participant in the project.

    View details for DOI 10.1007/s00262-010-0940-z

    View details for Web of Science ID 000286662100002

    View details for PubMedID 21080166

  • Disturbed NK Cell Compartment In Human CML and Bcr-Abl Positive Mice. 52nd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Chen, C. I., Koschmieder, S., Kamp, L., Altvater, B., Pscherer, S., Maecker, H., Berdel, W. E., Juergens, H., Lee, P. P., Rossig, C. AMER SOC HEMATOLOGY. 2010: 518–18
  • A model for harmonizing flow cytometry in clinical trials. Nature immunology Maecker, H. T., McCoy, J. P., Amos, M., Elliott, J., Gaigalas, A., Wang, L., Aranda, R., Banchereau, J., Boshoff, C., Braun, J., Korin, Y., Reed, E., Cho, J., Hafler, D., Davis, M., Fathman, C. G., Robinson, W., Denny, T., Weinhold, K., Desai, B., Diamond, B., Gregersen, P., Di Meglio, P., Nestle, F. O., Peakman, M., Villanova, F., Ferbas, J., Field, E., Kantor, A., Kawabata, T., Komocsar, W., Lotze, M., Nepom, J., Ochs, H., O'Lone, R., Phippard, D., Plevy, S., Rich, S., Roederer, M., Rotrosen, D., Yeh, J. 2010; 11 (11): 975-978


    Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.

    View details for DOI 10.1038/ni1110-975

    View details for PubMedID 20959798

    View details for PubMedCentralID PMC3400260

  • New technologies for autoimmune disease monitoring CURRENT OPINION IN ENDOCRINOLOGY DIABETES AND OBESITY Maecker, H. T., Nolan, G. P., Fathman, C. G. 2010; 17 (4): 322-328


    This article will review new technologies used to characterize the immune phenotype of cells and serum for potential use in studies of autoimmunity.One area of recent development in studies of immune phenotyping is the contrast between cells of the immune system at rest and following activation. This simply involves comparing these cells at rest and following ligand-induced activation and measuring signaling system activation (phosphoepitope identification) or intracellular cytokine production or activation-induced gene expression. Preliminary data using these techniques have begun to identify signatures of disease (biomarkers) that are only seen when using these activation-induced assays. One of the most exciting new technologies, cytometry by time-of-flight mass spectrometry, couples a flow cytometer to a mass spectrometer, allowing many more parameters to be analyzed per cell, and without spillover between assay reagents, compared to conventional optical flow cytometry (heavy ions, mass, replaces fluorophore readout). Another new technology to analyze soluble proteins, bead-based immunoassays, can simultaneously measure up to 75 soluble analytes in a multiplexed array. Other technologies provide similar innovations in terms of analytical content, throughput, and miniaturization.We believe that new cellular genomic and protein-based technologies can provide key insights into autoimmune disease pathogenesis, progression, and therapy, and that these assays need to be applied in a systematic way to samples from patients with autoimmune diseases.

    View details for DOI 10.1097/MED.0b013e32833ada91

    View details for Web of Science ID 000285063800003

    View details for PubMedID 20531181

  • "MIATA"-Minimal Information about T Cell Assays IMMUNITY Janetzki, S., Britten, C. M., Kalos, M., Levitsky, H. I., Maecker, H. T., Melief, C. J., Old, L. J., Romero, P., Hoos, A., Davis, M. M. 2009; 31 (4): 527-528


    Immunotherapy, especially therapeutic vaccination, has a great deal of potential in the treatment of cancer and certain infectious diseases such as HIV (Allison et al., 2006; Fauci et al., 2008; Feldmann and Steinman, 2005). Numerous vaccine candidates have been tested in patients with a variety of tumor types and chronic viral diseases. Often, the best way to assess the clinical potential of these vaccines is to monitor the induced T cell response, and yet there are currently no standards for reporting these results. This letter is an effort to address this problem.

    View details for DOI 10.1016/j.immuni.2009.09.007

    View details for Web of Science ID 000271403900001

    View details for PubMedID 19833080

  • Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium CANCER IMMUNOLOGY IMMUNOTHERAPY Britten, C. M., Janetzki, S., Ben-Porat, L., Clay, T. M., Kalos, M., Maecker, H., Odunsi, K., Pride, M., Old, L., Hoos, A., Romero, P. 2009; 58 (10): 1701-1713


    The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay.Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry. The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis.We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions.Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedures.

    View details for DOI 10.1007/s00262-009-0681-z

    View details for Web of Science ID 000268294400016

    View details for PubMedID 19259668

  • Multiparameter flow cytometry monitoring of T cell responses. Methods in molecular biology (Clifton, N.J.) Maecker, H. T. 2009; 485: 375-391


    HIV vaccine research increasingly uses polychromatic flow cytometry as a tool to monitor T cell responses. The use of this technology allows for the analysis of highly defined subsets of cells with unique phenotypes and functions. Ultimately, such studies may identify surrogate markers of protection from disease progression. However, this powerful technology comes with a number of technical hurdles, and there is a need to standardize the assays and protocols used in clinical trial monitoring. Here an optimized protocol, with variations for specific circumstances, is presented. This protocol covers the analysis of multiple cytokines, cell surface markers, and other functional markers such as perforin, CD107, and CD154. While the protocol can be adapted to various numbers of fluorescence parameters, optimized panels of 8-10 colors are presented.

    View details for DOI 10.1007/978-1-59745-170-3_25

    View details for PubMedID 19020838

  • A systematic approach to biomarker discovery; Preamble to "the iSBTc-FDA taskforce on immunotherapy biomarkers" JOURNAL OF TRANSLATIONAL MEDICINE Butterfield, L. H., Disis, M. L., Fox, B. A., Lee, P. P., Khleif, S. N., Thurin, M., Trinchieri, G., Wang, E., Wigginton, J., Chaussabel, D., Coukos, G., Dhodapkar, M., Hakansson, L., Janetzki, S., Kleen, T. O., Kirkwood, J. M., Maccalli, C., Maecker, H., Maio, M., Malyguine, A., Masucci, G., Palucka, A. K., Potter, D. M., Ribas, A., Rivoltini, L., Schendel, D., Seliger, B., Selvan, S., Slingluff, C. L., Stroncek, D. F., Streicher, H., Wu, X., Zeskind, B., Zhao, Y., Zocca, M., Zwierzina, H., Marincola, F. M. 2008; 6


    The International Society for the Biological Therapy of Cancer (iSBTc) has initiated in collaboration with the United States Food and Drug Administration (FDA) a programmatic look at innovative avenues for the identification of relevant parameters to assist clinical and basic scientists who study the natural course of host/tumor interactions or their response to immune manipulation. The task force has two primary goals: 1) identify best practices of standardized and validated immune monitoring procedures and assays to promote inter-trial comparisons and 2) develop strategies for the identification of novel biomarkers that may enhance our understating of principles governing human cancer immune biology and, consequently, implement their clinical application. Two working groups were created that will report the developed best practices at an NCI/FDA/iSBTc sponsored workshop tied to the annual meeting of the iSBTc to be held in Washington DC in the Fall of 2009. This foreword provides an overview of the task force and invites feedback from readers that might be incorporated in the discussions and in the final document.

    View details for DOI 10.1186/1479-5876-6-81

    View details for Web of Science ID 000263537200001

    View details for PubMedID 19105846

    View details for PubMedCentralID PMC2630944

  • Standardization and optimization of multiparameter intracellular cytokine staining. Cytometry. Part A : the journal of the International Society for Analytical Cytology Nomura, L., Maino, V. C., Maecker, H. T. 2008; 73 (11): 984-991


    Intracellular cytokine staining (ICS) is a common method for rapid quantitation of cytokine-producing antigen-specific T cells. T cell production of IFNgamma in particular, and more recently IL-2 as well, is often taken as a measure of vaccine immunogenicity in experimental vaccine trials. As more fluorochromes become available for use in ICS and other applications detecting intracellular markers, the selection of optimal fluorochrome combinations becomes correspondingly more complicated. Additionally, as more sophisticated flow cytometers become available, more attention is being paid to potential result variability from one instrument to another. This review summarizes an oral presentation given at MASIR 2008, January 30-Feb 1, 2008, in La Plagne, France. We focus on issues associated with multiparameter (>four color) flow cytometry, including matching antibody specificities with available fluorochromes and techniques to optimize fluorochrome combinations. We examine issues specific to intracellular staining as well as broader topics such as instrument setup, experimental controls, sample management, and analysis of multiparameter data sets. Particular emphasis is placed on the use of lyophilized cells, antibodies, beads, peptides, etc. (collectively known as "lyoplates"), which can decrease experiment-to-experiment variability as well as processing time. Most clinical trials compile results from multiple testing sites, using data that was acquired on-site in each location. We present data from two different ongoing multi-laboratory standardization studies, one involving 15 laboratories and one involving nine. We identify issues of variability and, where possible, offer solutions.

    View details for DOI 10.1002/cyto.a.20602

    View details for PubMedID 18612990

  • Development and dynamics of robust T-cell responses to CML under imatinib treatment patients BLOOD Chen, C. I., Maecker, H. T., Lee, P. P. 2008; 111 (11): 5342-5349


    Novel molecular targeted therapies, such as imatinib for chronic myelogenous leukemia (CML), represent the first agents that inhibit cancer cells more than other dividing cells, such as immune cells. We hypothesize that imatinib may create a window in which the immune response is partially restored while apoptotic leukemic cells are present, thus rendering leukemic cells immunogenic as patients enter remission. To detect and quantify antileukemia immune responses in an antigen-unbiased way, we used cryopreserved autologous pretreatment blood samples (representing predominantly leukemic cells) as stimulators to detect antileukemia T-cell responses in CML patients in remission on imatinib. We studied patients over time to address the dynamics of such responses. Our data show that antileukemia T-cell responses develop in the majority of CML patients (9 of 14) in remission and that CD4(+) T cells producing tumor necrosis factor-alpha (median 17.6%) represent the major response over interferon-gamma. This confirms the immune system's ability to respond to leukemia under certain conditions. Such responses may be further amplified as a potential therapy that synergizes with imatinib for improved control of CML.

    View details for DOI 10.1182/blood-2007-12-128397

    View details for Web of Science ID 000256336500016

    View details for PubMedID 18326818

    View details for PubMedCentralID PMC2396727

  • An analytical workflow for investigating cytokine profiles. Cytometry. Part A : the journal of the International Society for Analytical Cytology Siebert, J. C., Inokuma, M., Waid, D. M., Pennock, N. D., Vaitaitis, G. M., Disis, M. L., Dunne, J. F., Wagner, D. H., Maecker, H. T. 2008; 73 (4): 289-298


    Understanding cytokine profiles of disease states has provided researchers with great insight into immunologic signaling associated with disease onset and progression, affording opportunities for advancement in diagnostics and therapeutic intervention. Multiparameter flow cytometric assays support identification of specific cytokine secreting subpopulations. Bead-based assays provide simultaneous measurement for the production of ever-growing numbers of cytokines. These technologies demand appropriate analytical techniques to extract relevant information efficiently. We illustrate the power of an analytical workflow to reveal significant alterations in T-cell cytokine expression patterns in type 1 diabetes (T1D) and breast cancer. This workflow consists of population-level analysis, followed by donor-level analysis, data transformation such as stratification or normalization, and a return to population-level analysis. In the T1D study, T-cell cytokine production was measured with a cytokine bead array. In the breast cancer study, intracellular cytokine staining measured T cell responses to stimulation with a variety of antigens. Summary statistics from each study were loaded into a relational database, together with associated experimental metadata and clinical parameters. Visual and statistical results were generated with custom Java software. In the T1D study, donor-level analysis led to the stratification of donors based on unstimulated cytokine expression. The resulting cohorts showed statistically significant differences in poststimulation production of IL-10, IL-1 beta, IL-8, and TNF beta. In the breast cancer study, the differing magnitude of cytokine responses required data normalization to support statistical comparisons. Once normalized, data showed a statistically significant decrease in the expression of IFN gamma on CD4+ and CD8+ T cells when stimulated with tumor-associated antigens (TAAs) when compared with an infectious disease antigen stimulus, and a statistically significant increase in expression of IL-2 on CD8+ T cells. In conclusion, the analytical workflow described herein yielded statistically supported and biologically relevant findings that were otherwise unapparent.

    View details for DOI 10.1002/cyto.a.20509

    View details for PubMedID 18163472

  • Precision and linearity targets for validation of an IFN gamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides BMC IMMUNOLOGY Maecker, H. T., Hassler, J., Payne, J. K., Summers, A., Comatas, K., Ghanayem, M., Morse, M. A., Clay, T. M., Lyerly, H. K., Bhatia, S., Ghanekar, S. A., Maino, V. C., delaRosa, C., Disis, M. L. 2008; 9


    Single-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNgamma-based ELISPOT and cytokine flow cytometry (CFC), as well as tetramer assays.Precision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen.These data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.

    View details for DOI 10.1186/1471-2172-9-9

    View details for Web of Science ID 000254604100001

    View details for PubMedID 18366814

  • Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI) CANCER IMMUNOLOGY IMMUNOTHERAPY Janetzki, S., Janetzki, S., Panageas, K. S., Ben-Porat, L., Boyer, J., Britten, C. M., Clay, T. M., Kalos, M., Maecker, H. T., Romero, P., Yuan, J., Kast, W. M., Hoos, A. 2008; 57 (3): 303-315


    The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels.

    View details for DOI 10.1007/s00262-007-0380-6

    View details for Web of Science ID 000251794100003

    View details for PubMedID 17721781

  • Bacillus Calmette-Guerin vaccination of human newborns induces T cells with complex cytokine and phenotypic profiles JOURNAL OF IMMUNOLOGY Soares, A. P., Scriba, T. J., Joseph, S., Harbacheuski, R., Murray, R. A., Gelderbloem, S. J., Hawkridge, A., Hussey, G. D., Maecker, H., Kaplair, G., Hanekom, W. A. 2008; 180 (5): 3569-3577


    The immune response to vaccination with bacillus Calmette-Guérin (BCG), the only tuberculosis vaccine available, has not been fully characterized. We used multiparameter flow cytometry to examine specific T cell cytokine production and phenotypic profiles in blood from 10-wk-old infants routinely vaccinated with BCG at birth. Ex vivo stimulation of whole blood with BCG for 12 h induced expression of predominantly IFN-gamma, IL-2, and TNF-alpha in CD4+ T cells in seven distinct cytokine combinations. IL-4 and IL-10 expression was detected in CD4+ T cells at low frequencies and only in cells that did not coexpress type 1 cytokines. Specific CD8+ T cells were less frequent than CD4+ T cells and produced mainly IFN-gamma and/or IL-2 and less TNF-alpha, IL-4, and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-gamma. The predominant phenotype of BCG-specific type 1 T cells was that of effector cells, i.e., CD45RA-CCR7-CD27+, which may reflect persistence of Mycobacterium bovis BCG in infants until 10 wk of age. Among five phenotypic patterns of CD4+ T cells, central memory cells were more likely to be IL-2+ and effector cells were more likely to be IFN-gamma+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-gamma production alone underestimates the magnitude and complexity of the host cytokine response to BCG vaccination and may not be an optimal readout in studies of BCG and novel tuberculosis vaccination.

    View details for Web of Science ID 000256730000099

    View details for PubMedID 18292584

  • Poor predictive value of cytomegalovirus (CMV)-specific T cell assays for the development of CMV retinitis in patients with AIDS CLINICAL INFECTIOUS DISEASES Jacobson, M. A., Tan, Q. X., Girling, V., Poon, C., Van Natta, M., Jabs, D. A., Inokuma, M., Maecker, H. T., Bredt, B., Sinclair, E. 2008; 46 (3): 458-466


    We examined the potential clinical utility of using a cytomegalovirus (CMV)-specific T cell immunoassay to determine the risk of developing new-onset CMV retinitis (CMVR) in patients with acquired immunodeficiency syndrome (AIDS).CMV-specific T cell assays were performed by multiparameter flow cytometry using stored peripheral blood mononuclear cells that had been obtained in an observational study 2-6 months before new-onset CMVR was diagnosed in case patients (at a study visit during which a dilated ophthalmologic examination revealed no evidence of CMVR) and at the same study visit in control subjects (matched by absolute CD4(+) T cell count at entry) who did not subsequently develop retinitis during 1-6 years of study follow-up.There were no significant differences in CMV-specific CD4(+) or CD8(+) T cell interferon-gamma or interleukin-2 expression in peripheral blood mononuclear cells from case patients and control subjects. Although there were trends toward lower percentages and absolute numbers of CMV-specific, cytokine-expressing CD8(+) T cells with a "late memory" phenotype (CD27(-)CD28(-)) as well as with an "early memory" phenotype (CD27(+)CD28(+)CD45RA(+)) in case patients than in control subjects, these differences were not statistically significant.Many studies have reported that CMV-specific CD4(+) and CD8(+) T cell responses distinguish patients with active CMVR (i.e., who lack CMV-protective immunity) from those with inactive CMVR after immune restoration by antiretroviral treatment (i.e., who have CMV-protective immunity). However, the multiple CMV-specific immune responses we measured do not appear to have clinical utility for predicting the risk for patients with AIDS of developing new-onset CMVR with sufficient accuracy to be used in guiding therapeutic management.

    View details for DOI 10.1086/525853

    View details for Web of Science ID 000252221200018

    View details for PubMedID 18173357

  • Functional T cell responses to tumor antigens in breast cancer patients have a distinct phenotype and cytokine signature JOURNAL OF IMMUNOLOGY Inokuma, M., dela Rosa, C., Schmitt, C., Haaland, P., Siebert, J., Petry, D., Tang, M., Suni, M. A., Ghanekar, S. A., Gladding, D., Dunne, J. F., Maino, V. C., Disis, M. L., Maecker, H. T. 2007; 179 (4): 2627-2633


    The overall prevalence with which endogenous tumor Ags induce host T cell responses is unclear. Even when such responses are detected, they do not usually result in spontaneous remission of the cancer. We hypothesized that this might be associated with a predominant phenotype and/or cytokine profile of tumor-specific responses that is different from protective T cell responses to other chronic Ags, such as CMV. We detected significant T cell responses to CEA, HER-2/neu, and/or MAGE-A3 in 17 of 21 breast cancer patients naive to immunotherapy. The pattern of T cell cytokines produced in response to tumor-associated Ags (TAAs) in breast cancer patients was significantly different from that produced in response to CMV or influenza in the same patients. Specifically, there was a higher proportion of IL-2-producing CD8(+) T cells, and a lower proportion of IFN-gamma-producing CD4(+) and/or CD8(+) T cells responding to TAAs compared with CMV or influenza Ags. Finally, the phenotype of TAA-responsive CD8(+) T cells in breast cancer patients was almost completely CD28(+)CD45RA(-) (memory phenotype). CMV-responsive CD8(+) T cells in the same patients were broadly distributed among phenotypes, and contained a high proportion of terminal effector cells (CD27(-)CD28(-)CD45RA(+)) that were absent in the TAA responses. Taken together, these results suggest that TAA-responsive T cells are induced in breast cancer patients, but those T cells are phenotypically and functionally different from CMV- or influenza-responsive T cells. Immunotherapies directed against TAAs may need to alter these T cell signatures to be effective.

    View details for Web of Science ID 000248959200069

    View details for PubMedID 17675526

  • Loss of Receptor on Tuberculin-Reactive T-Cells Marks Active Pulmonary Tuberculosis PLOS ONE Streitz, M., Tesfa, L., Yildirim, V., Yahyazadeh, A., Ulrichs, T., Lenkei, R., Quassem, A., Liebetrau, G., Nomura, L., Maecker, H., Volk, H., Kern, F. 2007; 2 (8)


    Tuberculin-specific T-cell responses have low diagnostic specificity in BCG vaccinated populations. While subunit-antigen (e.g. ESAT-6, CFP-10) based tests are useful for diagnosing latent tuberculosis infection, there is no reliable immunological test for active pulmonary tuberculosis. Notably, all existing immunological tuberculosis-tests are based on T-cell response size, whereas the diagnostic potential of T-cell response quality has never been explored. This includes surface marker expression and functionality of mycobacterial antigen specific T-cells.Flow-cytometry was used to examine over-night antigen-stimulated T-cells from tuberculosis patients and controls. Tuberculin and/or the relatively M. tuberculosis specific ESAT-6 protein were used as stimulants. A set of classic surface markers of T-cell naïve/memory differentiation was selected and IFN-gamma production was used to identify T-cells recognizing these antigens. The percentage of tuberculin-specific T-helper-cells lacking the surface receptor CD27, a state associated with advanced differentiation, varied considerably between individuals (from less than 5% to more than 95%). Healthy BCG vaccinated individuals had significantly fewer CD27-negative tuberculin-reactive CD4 T-cells than patients with smear and/or culture positive pulmonary tuberculosis, discriminating these groups with high sensitivity and specificity, whereas individuals with latent tuberculosis infection exhibited levels in between.Smear and/or culture positive pulmonary tuberculosis can be diagnosed by a rapid and reliable immunological test based on the distribution of CD27 expression on peripheral blood tuberculin specific T-cells. This test works very well even in a BCG vaccinated population. It is simple and will be of great utility in situations where sputum specimens are difficult to obtain or sputum-smear is negative. It will also help avoid unnecessary hospitalization and patient isolation.

    View details for DOI 10.1371/journal.pone.0000735

    View details for Web of Science ID 000207455200012

    View details for PubMedID 17710135

    View details for PubMedCentralID PMC1936433

  • Phenotype and in vitro function of mature MDDC generated from cryopreserved PBMC of cancer patients are equivalent to those from healthy donors. Journal of immune based therapies and vaccines Ghanekar, S. A., Bhatia, S., Ruitenberg, J. J., dela Rosa, C., Disis, M. L., Maino, V. C., Maecker, H. T., Waters, C. A. 2007; 5: 7-?


    Monocyte-derived-dendritic-cells (MDDC) are the major DC type used in vaccine-based clinical studies for a variety of cancers. In order to assess whether in vitro differentiated MDDC from cryopreserved PBMC of cancer patients are functionally distinct from those of healthy donors, we compared these cells for their expression of co-stimulatory and functional markers. In addition, the effect of cryopreservation of PBMC precursors on the quality of MDDC was also evaluated using samples from healthy donors.Using flow cytometry, we compared normal donors and cancer patients MDDC grown in the presence of GM-CSF+IL-4 (immature MDDC), and GM-CSF+IL-4+TNFalpha+IL-1beta+IL-6+PGE-2 (mature MDDC) for (a) surface phenotype such as CD209, CD83 and CD86, (b) intracellular functional markers such as IL-12 and cyclooxygenase-2 (COX-2), (c) ability to secrete IL-8 and IL-12, and (d) ability to stimulate allogeneic and antigen-specific autologous T cells.Cryopreservation of precursors did affect MDDC marker expression, however, only two markers, CD86 and COX-2, were significantly affected. Mature MDDC from healthy donors and cancer patients up-regulated the expression of CD83, CD86, frequencies of IL-12+ and COX-2+ cells, and secretion of IL-8; and down-regulated CD209 expression relative to their immature counterparts. Compared to healthy donors, mature MDDC generated from cancer patients were equivalent in the expression of nearly all the markers studied and importantly, were equivalent in their ability to stimulate allogeneic and antigen-specific T cells in vitro.Our data show that cryopreservation of DC precursors does not significantly affect the majority of the MDDC markers, although the trends are towards reduced expression of co-stimulatory makers and cytokines. In addition, monocytes from cryopreserved PBMC of cancer patients can be fully differentiated into mature DC with phenotype and function equivalent to those derived from healthy donors.

    View details for PubMedID 17477875

  • Protective immunity to cytomegalovirus (CMV) retinitis in AIDS is associated with CMV-specific T cells that express interferon-G and interleukin-2 and have a CD8(+) cell early maturational phenotype JOURNAL OF INFECTIOUS DISEASES Sinclair, E., Tan, Q. X., Sharp, M., Girling, V., Poon, C., Van Natta, M., Jabs, D. A., Inokuma, M., Maecker, H. T., Bredt, B., Jacobson, M. A. 2006; 194 (11): 1537-1546


    To determine potential correlates of immune recovery from AIDS-related cytomegalovirus retinitis (CMVR), multiparameter flow cytometry was used to characterize CMV-specific T cells from subjects with CMVR. Individuals with active retinitis were compared with those who had been clinically immunorestored by antiretroviral therapy and had > or =2 years of ophthalmologic follow-up without anti-CMV therapy or retinitis reactivation or progression. In comparison with patients with active retinitis, immunorestored patients had higher circulating CD4(+) and CD8(+) T cells expressing interleukin-2 and interferon- gamma in response to combined CMV pp65 and IE1 peptide pool stimulation. CD4(+) T cell responses were predominantly to pp65, whereas CD8(+) T cell responses were predominantly to IE. Immunorestored patients, compared with patients with active retinitis, had increased levels of circulating CMV-specific CD8(+) T cells with "early" (CD27(+)CD28(+)CD45RA(+), CD27(+)CD28(+)CD45RA(-)) and "intermediate" (CD27(-)CD28(+)CD45RA(-)) phenotypes. Recovery from AIDS-related CMVR after the initiation of antiretroviral therapy may be mediated by CMV-specific CD4(+) and CD8(+) T cells capable of promoting antigen-specific CD8(+) T cell proliferation.

    View details for Web of Science ID 000241820800010

    View details for PubMedID 17083038

  • Bacillus Calmette Guerin vaccination of human newborns induces a specific, functional CD8(+) T cell response JOURNAL OF IMMUNOLOGY Murray, R. A., Mansoor, N., Harbacheuski, R., Soler, J., Davids, V., Soares, A., Hawkridge, A., Hussey, G. D., Maecker, H., Kaplan, G., Hanekom, W. A. 2006; 177 (8): 5647-5651


    Mounting evidence points to CD8+ T cells playing an important role in protective immunity against Mycobacterium tuberculosis. The only available vaccine against tuberculosis, bacillus Calmette Guérin (BCG), has traditionally been viewed not to induce these cells optimally. In this study, we show that vaccination of human newborns with BCG does indeed induce a specific CD8+ T cell response. These cells degranulated or secreted IFN-gamma, but not both, when infant blood was incubated with BCG. This stimulation also resulted in proliferation and up-regulation of cytotoxic molecules. Overall, the specific CD8+ T cell response was quantitatively smaller than the BCG-induced CD4+ T cell response. Incubation of whole blood with M. tuberculosis also caused CD8+ T cell IFN-gamma expression. We conclude that BCG induces a robust CD8+ T cell response, which may contribute to vaccination-induced protection against tuberculosis.

    View details for Web of Science ID 000241093100080

    View details for PubMedID 17015753

  • Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry. Part A : the journal of the International Society for Analytical Cytology Maecker, H. T., Trotter, J. 2006; 69 (9): 1037-1042


    A frequent goal of flow cytometric analysis is to classify cells as positive or negative for a given marker, or to determine the precise ratio of positive to negative cells. This requires good and reproducible instrument setup, and careful use of controls for analyzing and interpreting the data. The type of controls to include in various kinds of flow cytometry experiments is a matter of some debate and discussion. In this tutorial, we classify controls in various categories, describe the options within each category, and discuss the merits of each option.

    View details for PubMedID 16888771

  • Immune responses in the draining lymph nodes against cancer: Implications for immunotherapy 1st International Symposium on Cancer Metastasis and the Lymphovascular System Shu, S., Cochran, A. J., Huang, R., Morton, D. L., Maecker, H. T. SPRINGER. 2006: 233–42


    Regional lymph nodes are the first site for melanoma metastases. The sentinel node (SN), on the direct lymphatic drainage pathway, which usually harbors first metastases, demonstrates significant suppression in its ability to respond to antigenic stimulation. This down-regulation of SN immunity is likely the basis of its susceptibility to tumor metastases, suggesting a potential role of the immune system in the control of malignant tumors. Despite immune dysfunction in the SN, phase II trials of systemic post-operative immunotherapy with a polyvalent melanoma vaccine developed at the John Wayne Cancer Institute showed improved 5-year overall survival in patients with melanoma metastatic to regional nodes. However, most immunotherapy clinical trials have failed to demonstrate a significant clinical response, and analyses of immune responses to tumor-associated antigens that correlate clinical responses have not been established. Therefore, refinements in assay methodologies and improvements in vaccine designs are critical to the success of cancer immunotherapy. Antigen presentation by dendritic cells (DCs) is the most potent means to initiate a T cell immunity. Dendritic cell-based immunotherapies have been vigorously attempted in the past decade. To improve the immunogenicity of cancer vaccines, we recently generated heterokaryons of DCs and tumor cells by electrofusion. The fusion hybrids retained their full antigen-presenting capacity and all natural tumor antigens. In pre-clinical animal experiments, a single injection of the DC-tumor fusion hybrids was sufficient to mediate the regression of tumors established in the lung, skin and brain. Most interestingly, successful therapy required the delivery of fusion hybrids directly into lymphoid organs such as lymph nodes. A clinical trial is now being carried out to test the immunogenicity and therapeutic effects of fusion hybrids for the treatment of metastatic melanoma.

    View details for DOI 10.1007/s10555-006-8503-7

    View details for Web of Science ID 000238268800006

    View details for PubMedID 16770535

  • Maximizing the retention of antigen specific lymphocyte function after cryopreservation JOURNAL OF IMMUNOLOGICAL METHODS Disis, M. L., dela Rosa, C., Goodell, V., Kuan, L. Y., Chang, J. C., Kuus-Reichel, K., Clay, T. M., Lyerly, H. K., Bhatia, S., Ghanekar, S. A., Maino, V. C., Maecker, H. T. 2006; 308 (1-2): 13-18


    The ability to cryopreserve lymphocytes in peripheral blood mononuclear cells (PBMC) to retain their function after thawing is critical to the analysis of cancer immunotherapy studies. We evaluated a variety of cryopreservation strategies with the aim of developing an optimized protocol for freezing and thawing PBMC to retain viability and function. We determined several factors which do not affect cell viability after cryopreservation such as shipping frozen samples on dry ice, the length of time and speed at which samples are washed and centrifuged after thawing, and the number of cells frozen per container. Different media additives, however, did impact the viability of the cells after thawing. There was a significant reduction in the viability of the cells after freezing when using human AB serum compared to all other additives tested (p<0.000). A second critical parameter was the temperature of the media used to wash the cells after removal from the cryotubes. When the media was cooled to 4 degrees C prior to washing, the mean viability was 69.7+/-12.5%, at 25 degrees C 92.55+/-3.1%, and at 37 degrees C 95.11+/-2.5%. Finally, we used an optimized cryopreservation protocol with different media additives to determine if functional T cell responses to tetanus toxoid could be preserved. There was a statistically significant correlation between the tetanus specific stimulation index (S.I.) of the non-cryopreserved PBMC and SI obtained from cells frozen with media containing human serum albumin as compared to other additives such as dextran or fetal bovine serum.

    View details for DOI 10.1016/j.jim.2005.09.011

    View details for Web of Science ID 000235285700002

    View details for PubMedID 16337957

  • IL-2 production correlates with effector cell differentiation in HIV-specific CD8+ T cells. AIDS research and therapy Nomura, L. E., Emu, B., Hoh, R., Haaland, P., Deeks, S. G., Martin, J. N., McCune, J. M., Nixon, D. F., Maecker, H. T. 2006; 3: 18-?


    Diminished IL-2 production and lack of effector differentiation have been reported for HIV-specific T cells. In this study, we examined the prevalence of these phenomena using 8-color cytokine flow cytometry, and tested the hypothesis that these two findings were causally related. We analyzed cytokine profiles and memory/effector phenotypes of HIV-specific and CMV-specific T cells using short-term in vitro stimulation with HIV or CMV peptide pools. Nineteen HIV-positive subjects with progressive disease and twenty healthy, HIV-negative subjects were examined.Among HIV-infected subjects, there were significantly fewer CD8+ IL-2+ T cells responding to HIV compared to CMV, with no significant difference in CD4+ IL-2+ T cells. The majority of CMV-specific T cells in both HIV-negative and HIV-positive subjects appeared to be terminally differentiated effector cells (CD8+ CD27- CD28- CD45RA+ or CD8+ CD27- CD28- CD45RA-). In HIV-positive subjects, the most common phenotype of HIV-specific T cells was intermediate in differentiation (CD8+ CD27+ CD28- CD45RA-). These differences were statistically significant, both as absolute cell frequencies and as percentages. There was a significant correlation between the absolute number of HIV-specific CD8+ IL-2+ T cells and HIV-specific CD8+ CD27- CD28- CD45RA+ terminal effector cells.IL-2 production from antigen-specific CD8+ T cells correlates with effector cell differentiation of those cells.

    View details for PubMedID 16859558

  • Ex vivo analysis of T-cell function CURRENT OPINION IN IMMUNOLOGY Suni, M. A., Maino, V. C., Maecker, H. T. 2005; 17 (4): 434-440


    Our ability to analyze T-cell function in vitro has progressed in recent years to include analysis of early signaling events, such as specific protein phosphorylation, intermediate functions, such as degranulation and cytokine production, and later functions, such as proliferation. Many assays are now available to monitor these events, and comparative studies of some of these assays have been published. Major recent developments in this area include the ability to measure T-cell degranulation via cell surface exposure of CD107 and the use of polychromatic flow cytometry to examine multiple phenotypes and functions of responding T cells.

    View details for DOI 10.1016/j.coi.2005.05.002

    View details for Web of Science ID 000230673900016

    View details for PubMedID 15950444

  • Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT BMC IMMUNOLOGY Maecker, H. T., Moon, J., Bhatia, S., Ghanekar, S. A., Maino, V. C., Payne, J. K., Kuus-Reichel, K., Chang, J. C., Summers, A., Clay, T. M., Morse, M. A., Lyerly, H. K., DeLaRosa, C., Ankerst, D. P., Disis, M. L. 2005; 6


    Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC), and ELISPOT. In this study, we compared the performance of these assays on leukapheresed PBMC shipped overnight in medium versus cryopreserved PBMC from matched donors.Using CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p

    View details for DOI 10.1186/1471-2172-6-17

    View details for Web of Science ID 000235714000001

    View details for PubMedID 16026627

    View details for PubMedCentralID PMC1190174

  • Standardization of cytokine flow cytometry assays BMC IMMUNOLOGY Maecker, H. T., Rinfret, A., D'Souza, P., Darden, J., Roig, E., Landry, C., Hayes, P., Birungi, J., Anzala, O., Garcia, M., Harari, A., Frank, I., Baydo, R., Baker, M., Holbrook, J., Ottinger, J., Lamoreaux, L., Epling, C. L., Sinclair, E., Suni, M. A., Punt, K., Calarota, S., El-Bahi, S., Alter, G., Maila, H., Kuta, E., Cox, J., Gray, C., Altfeld, M., Nougarede, N., Boyer, J., Tussey, L., Tobery, T., Bredt, B., Roederer, M., Koup, R., Maino, V. C., Weinhold, K., Pantaleo, G., Gilmour, J., Horton, H., Sekaly, R. P. 2005; 6


    Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online).Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells.ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

    View details for DOI 10.1186/1471-2172-6-13

    View details for Web of Science ID 000235713700001

    View details for PubMedID 15978127

    View details for PubMedCentralID PMC1184077

  • Immune dysfunction and micrometastases in women with breast cancer BREAST CANCER RESEARCH AND TREATMENT Campbell, M. J., Scott, J., Maecker, H. T., Park, J. W., Esserman, L. J. 2005; 91 (2): 163-171


    Cytokines produced by T lymphocytes are critical to the efficacy of a given immune response and dysregulation of immune responses may play a role in cancer progression. We assessed the intracellular cytokine profiles of T cells in the peripheral blood of women with breast cancer and explored the relationship of these responses with the presence of cancer in lymph nodes and bone marrow. Peripheral blood lymphocytes from 84 patients and 26 healthy volunteers were analyzed by 4-color flow cytometry for surface markers and for intracellular cytokines. Bone marrow samples from some of these patients were also collected and analyzed for the presence of epithelial cells (micrometastases) by flow cytometry. The percentages of both CD4(+) and CD8(+) cells producing type1 (IL-2, IFN-gamma or TNF-alpha) and type 2 (IL-4) were significantly lower in patients with breast cancer compared to healthy controls. These results indicate a general immune dysfunction in these patients as opposed to a shift in the balance of type1 and type2 cells. These dysregulated T cell responses did not correlate with age, stage of disease, or nodal status. However, we did observe a correlation between number of micrometastases in the bone marrow and T cell responsiveness.

    View details for DOI 10.1007/s10549-004-7048-0

    View details for Web of Science ID 000228867600008

    View details for PubMedID 15868444

  • Dysfunction of simian immunodeficiency virus/simian human immunodeficiency virus-induced IL-2 expression by central memory CD4+ T lymphocytes JOURNAL OF IMMUNOLOGY Sun, Y., Schmitz, J. E., Acierno, P. M., Santra, S., Subbramanian, R. A., Barouch, D. H., Gorgone, D. A., Lifton, M. A., Beaudry, K. R., Manson, K., Philippon, V., Xu, L., Maecker, H. T., MASCOLA, J. R., Panicali, D., Nabel, G. J., Letvin, N. L. 2005; 174 (8): 4753-4760


    Production of IL-2 and IFN-gamma by CD4+ T lymphocytes is important for the maintenance of a functional immune system in infected individuals. In the present study, we assessed the cytokine production profiles of functionally distinct subsets of CD4+ T lymphocytes in rhesus monkeys infected with pathogenic or attenuated SIV/simian human immunodeficiency virus (SHIV) isolates, and these responses were compared with those in vaccinated monkeys that were protected from immunodeficiency following pathogenic SHIV challenge. We observed that preserved central memory CD4+ T lymphocyte production of SIV/SHIV-induced IL-2 was associated with disease protection following primate lentivirus infection. Persisting clinical protection in vaccinated and challenged monkeys is thus correlated with a preserved capacity of the peripheral blood central memory CD4+ T cells to express this important immunomodulatory cytokine.

    View details for Web of Science ID 000228234600040

    View details for PubMedID 15814700

  • Selecting fluorochrome conjugates for maximum sensitivity. Cytometry. Part A : the journal of the International Society for Analytical Cytology Maecker, H. T., Frey, T., Nomura, L. E., Trotter, J. 2004; 62 (2): 169-173

    View details for PubMedID 15536642

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