Researchers identify a unique population of pathologic CD4+ T cells in both blood and synovial fluid using mass cytometry

In this video, Dr. Deepak Rao, MD, PhD, of Brigham and Women’s Hospital presents his recent research using mass cytometry and multidimensional flow cytometry to identify key pathologic T cell populations in the synovial fluid of subjects with rheumatoid arthritis.

Determining the pathologic functions of T cells that infiltrate target tissues remains a central challenge in autoimmune diseases. In this video, Dr. Deepak Rao, MD, PhD, of Brigham and Women’s Hospital describes recent work, published in Nature, identifying a unique population of pathologic CD4⁺ T cells, called T peripheral helper (T_PH) cells, that is markedly expanded in the joints of subjects with rheumatoid arthritis (RA). Rao and colleagues used mass cytometry and multidimensional flow cytometry to interrogate T cell populations in synovial tissue and blood from research subjects with RA, a chronic immune-mediated arthritis that affects up to 1% of the population.

Mass cytometric analysis of RA synovial tissue cells revealed a strikingly expanded population of PD-1^hiCXCR5^-CD4⁺ T cells, which constituted ~25% of synovial CD4⁺ T cells. Surprisingly, these cells are not exhausted, but instead highly express factors enabling B cell help, including IL-21, CXCL13, ICOS, SAP and MAF. Like T follicular helper (T_FH) cells, PD-1^hiCXCR5^- cells from synovium and blood induce plasma cell differentiation in vitro via IL 21. However, RNA-seq transcriptomics robustly separates PD-1^hiCXCR5^- cells from T_FH cells, with altered expression of Bcl6 and Blimp-1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1 and CCR5, in PD-1^hiCXCR5^- cells.

Rao and colleagues propose that PD-1^hiCXCR5^- T cells represent a T_PH cell population, analogous to T_FH cells, that supports B cell responses in pathologically inflamed nonlymphoid tissues. Given their marked expansion in RA joints, these cells may be important in driving pathologic B cell responses and autoantibody production within the inflamed target tissue.

Michelle Poulin, PhD, of Fluidigm provides a brief overview of mass cytometry, the high-parameter, single-cell analysis technology used by Rao in his research.

Speakers

Dr. Deepak Rao, MD, PhD   
Rheumatologist   
Co-Director, Human Immunology Center    
Brigham and Women's Hospital 
Boston, MA

Michelle Poulin, PhD
Applications Scientist
Fluidigm Canada Inc. 
Markham, ON, Canada

fluidigm

For Research Use Only. Not for use in diagnostic procedures.

Cheryl Kim of La Jolla Institute for Allergy and Immunology describes the value of mass cytometry for researchers at her institution.

Enabling multiparameter profiling of precious samples, mass cytometry has been instrumental in achieving research breakthroughs around the world. Hear Cheryl Kim, director of the Flow Cytometry Core Facility at La Jolla Institute for Allergy and Immunology, discuss the ways mass cytometry is helping researchers at her institution get the most information from limited samples.

Mass Cytometry in Focus

For Research Use Only. Not for use in diagnostic procedures.

Presented at the American Association for Cancer Research (AACR) 2017 meeting, this poster compares a targeted gene expression profiling approach using high-throughput qPCR readout and a nontargeted approach using mRNA sequencing.

Presented at the American Association for Cancer Research (AACR) 2017 meeting, this poster describes the use of the Clontech SMART-Seq v4 kit with the C1 system, and the resultant gene expression sensitivity and overall performance.

Evan Lind, PhD, of Oregon Health & Science University, explains how mass cytometry enables better understanding of immune systems

Enabling multiparameter profiling of precious samples, mass cytometry has been instrumental in achieving new research breakthroughs around the world. Hear how Evan Lind uses mass cytometry to research the leukemic microenvironment in acute myeloid leukemia (AML) patients in search of better targeted therapies.

Mass Cytometry in Focus

For Research Use Only. Not for use in diagnostic procedures.

Watch this video to learn about a novel approach to improving checkpoint blockade therapy in cancer under investigation in early-phase trials by Joshua Brody, MD, and colleagues at the Icahn School of Medicine at Mount Sinai.

Enhancing Checkpoint Blockade in Lymphoma with In Situ Vaccination

Checkpoint blockade therapy for cancer has had tremendous impact on clinical outcomes, yet only a subset of patients respond. Recent studies show that response to checkpoint blockade does not always correlate with tumor-associated antigen (TAA) load and so must be determined by factors beyond mutational burden. This suggests that checkpoint blockade is limited by suboptimal cross-presentation of TAA by activated dendritic cells (DC) and will be potentiated by recruitment, loading and activation of cross-presenting DC at the tumor site.

To test this hypothesis, an early-phase trial (NCT01976585) in low-grade lymphoma was carried out testing a unique in situ vaccine (ISV) combining: 1) fms-like tyrosine kinase 3 ligand (FLT3L) to recruit DC, 2) radiotherapy to load FLT3L-mobilized DC with TAA, and 3) toll-like receptor agonist (TLRa) to activate TAA-loaded DC for cross-presentation. Strikingly, Brody and colleagues found partial and complete systemic tumor regressions at distant, untreated tumors. They also found specific elimination of malignant B cells with sparing of healthy B cells, suggesting a systemic anti-tumor immune response.

This data prompted a new trial in which anti-PD1 monoclonal antibody (mAb) and ISV are combined with a novel immune-monitoring approach involving co-administration of "surrogate antigens (Ag)" at the ISV site. The approach is being applied to multiple cancer types, including breast, head and neck, melanoma and sarcoma.

In this webinar, Brody discusses these studies, including the key role mass cytometry played in defining the effects of treatment on the intratumoral and systemic immune repertoire with high resolution and in profiling the distinct checkpoint/co-activator molecules on TAA-specific, surrogate-Ag-specific and bulk CD8 T cells.

Michelle Poulin, PhD, Fluidigm Field Applications Scientist, gives a brief overview of mass cytometry, the high-parameter, single-cell technology used by Brody in his research.

Mass cytometry, or cytometry by time-of-flight, the basis of Fluidigm® CyTOF® technology, uniquely enables high-dimensional single-cell proteomic analysis for systems-level discovery and comprehensive functional profiling applications. This brief overview will describe the basic principles and workflow of the technology, including recent advancements.

Speakers

Joshua Brody, MD
Assistant Professor, Hematology and Medical Oncology
Director, Lymphoma Immunotherapy Program
Icahn School of Medicine at Mount Sinai
New York, NY

Michelle Poulin, PhD
Field Applications Scientist
Fluidigm Corporation
South San Francisco, CA

For Research Use Only. Not for use in diagnostic procedures.

Watch this video to hear Dr. Stephen Oh, MD, PhD, of Washington University detail his findings using mass cytometry to investigate altered signaling networks and cellular cytokine expression in subjects with myelofibrosis.

Myeloproliferative neoplasms including myelofibrosis (MF) are characterized by anemia, splenomegaly, bone marrow fibrosis and inflammatory cytokine production. JAK2 inhibition with ruxolitinib improves symptoms and lowers circulating plasma cytokine levels but does not ameliorate anemia, fibrosis or malignant clonal burden. The goal of this study is to better define the relationship between dysregulated cytokines and downstream signaling in MF and to determine how these pathways can be effectively manipulated for therapeutic benefit.

To interrogate altered signaling in MF, we have employed mass cytometry, a novel technology that enables the quantitative analysis of more than 40 parameters at the single-cell level. We identified NF-kB pathway hyperactivation distributed across multiple hematopoietic cell populations, including T cells. Plasma TNFα levels were elevated in these subjects, suggesting that excessive production of TNFα may lead to broad activation of NF-κB in a non-cell-autonomous fashion.

To further elucidate the etiology of systemic NF-κB hyperactivation, we extended our mass cytometry approach to study the cellular distribution of cytokine production in MF. Intracellular levels of several cytokines (including TNFα) were constitutively elevated in MF subjects compared to healthy controls. Supranormal cytokine expression was accentuated by stimulation with PMA/ionomycin or TLR ligands R-848 or Pam3CSK4. PMA/ionomycin increased production of TNFα from MF CD34+ cells and CD33+ immature myeloid cells.

These findings imply that multiple cell populations in MF overexpress inflammatory cytokines and are hypersensitive to potentiating stimuli. Future experiments will identify signaling effects of multiple elevated cytokines, which may underlie MF features that persist despite JAK2 inhibitor therapy.

Jennifer Frahm, PhD, of Fluidigm also presents a brief overview of mass cytometry, the high-parameter, single-cell analysis technology used by Dr. Oh in his research.

Speakers

Dr. Stephen Oh, MD, PhD   
Assistant Professor of Medicine
Division of Hematology 
Washington University School of Medicine
St. Louis, MO

Jennifer Frahm, PhD
Field Applications Scientist
Fluidigm Corporation
South San Francisco, CA

fluidigm

For Research Use Only. Not for use in diagnostic procedures.

Virginie Rozot, PhD, of the University of Cape Town describes the value of mass cytometry for translational studies.

Enabling multiparameter profiling of precious samples, mass cytometry has been instrumental in achieving new research breakthroughs around the world. Hear how Virginie Rozot and colleagues from the South African Tuberculosis Vaccine Initiative are using mass cytometry to gain fresh insights about the immune response to tuberculosis.

Mass Cytometry in Focus

For Research Use Only. Not for use in diagnostic procedures.

Mass cytometry holds great promise for multiparameter interrogation of complex cellular microenvironments from precious translational research samples. Here, Jonathan Irish, PhD, describes the use of mass cytometry to provide a systems-level characterization of cells in healthy human tissues and solid tumors. The state of the art in single-cell cancer biology is discussed, including tissue collection, technical and biological quality controls, analysis of cell signaling (phospho-flow) and computational approaches.

Watch the webinar, "Decoding human tumors and healthy tissue with mass cytometry," to:

• Hear the value of mass cytometry for deep profiling of single cells isolated from healthy tissue and tumors.
• Learn proven techniques for single-cell isolation, cell processing for mass cytometry and quality control.
• Explore the use of mass cytometry along with other technology platforms to implement a systems-level approach to tissue characterization.
• fluidigm

About the presenter:

Jonathan M. Irish, PhD
Assistant Professor of Cancer Biology
Assistant Professor of Pathology, Microbiology, and Immunology
Vanderbilt University

The main focus of Jonathan Irish’s research is to understand how changes at the single-cell level alter signaling in healthy cells and lead to therapy-resistant populations in human diseases. His experimental approach has focused on measuring signaling events in individual cells from primary tissues, including human tumors.

Natural killer (NK) cells play critical roles in immune defense and reproduction but remain the most poorly understood major lymphocyte population. In this webinar, Dr. Blish describes her work using mass cytometry to reveal the vast and previously unappreciated diversity of the NK repertoire and how deep profiling of NK cell populations may be clinically useful in predicting outcomes of viral exposure. Michelle Poulin also gives a brief overview of mass cytometry, the high-parameter, single-cell technology utilized by Dr. Blish in her NK cell analyses.

Watch the webinar to:

• Learn about the functional diversity of NK cells revealed by mass cytometric analysis.
• Hear how immune experience diversifies and specializes the NK repertoire.
• Evaluate data linking NK diversity with HIV acquisition rates in a patient population.

About the presenters:

Catherine Blish, MD, PhD
Assistant Professor, Department of Medicine and Immunology,
Stanford University School of Medicine
Assistant Director of the Stanford Medical Scientist Training Program

Michelle Poulin, PhD
Field Applications Scientist
Fluidigm

For Research Use Only. Not for use in diagnostic procedures.