Effector and Regulatory T Cell Assays to Assess Your Autoimmune-Targeted Therapeutic

Autoimmune disease is characterized by the presence of autoreactive T and/or B cells which help drive a destructive inflammatory immune response against our own tissues.

T cells offer a tractable target to allow researchers to re-program the immune system and to shift the balance towards regulation and homeostasis, rather than inappropriate activation and inflammation; for example, by inhibiting inflammatory T cell types or by driving the generation of suppressive regulatory T cells. Current therapies in the clinic used to modulate T cell function in the context of autoimmune disease include:

  • Global immunosuppressants such as Cyclosporin A and Rapamycin
  • Therapeutics inhibiting T cell activation via blocking co-stimulatory pathways (e.g., CTLA-4 fusion proteins)
  • Inhibitors of T cell differentiation or effector molecules, which target cytokine signaling (e.g., Jak/STAT pathways) and antibodies (e.g., TNFα and IL-17)

By increasing specificity of a therapy and targeting the specific T cell populations which are driving disease the rest of the immune system is left intact and the patient is not globally immuno-suppressed and left susceptible to infection.

So, controlling or limiting a specific T cell response is an effective strategy to control the autoimmune disease process. Tools like our inflammation and autoimmune T cell assays evaluate your therapy’s efficacy using human T cells in an in vitro system. This is especially critical, as a thorough investigation of a drug that targets T cell function requires the study of human T cells and animal models of human disease. Using data from both systems accounts for the differences in mouse and human T cell populations (e.g., in terms of balance between memory and naïve T cell subsets).


Evaluating Autoimmune Disease Targeted Therapies That Reprogram the Immune System

microbiome, immune system and inflammation models in mice

In this webinar, our experts explain how combining disease-relevant models and in vitro inflammation and autoimmune T cell assays can provide a clear picture for your therapy to reprogram the immune system.

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Improve your understanding of how your therapeutic modulates T cell function with guidance from our team. We’ll help you select the best approach to model autoimmune disease processes from our broad portfolio of validated and custom T cell assays.

T cells as targets for therapeutic development

T cells play a critical role in autoimmune and inflammatory diseases and can act to drive or suppress immune responses, depending on their phenotype.

  • CD4+ function

    CD4+ T cells polarize into different helper T cell subsets depending on the context of the conditions under which they are activated. These subsets include:

    • Inflammatory subsets (Th1, Th17)
    • Th2 cells, which provide B cell help and are specialized in fighting extracellular pathogens
    • TFH cells which help different aspects of the B cell response within secondary lymphoid organs
  • Regulatory T cells population

    Regulatory T cells (Treg), which are a heterogeneous population characterized by their almost unique ability to suppress immune responses.

  • CD8+ T cells role

    CD8+ T cells, which mediate anti-tumor and anti-viral defence and are also associated with GVHD and can facilitate aspects of autoimmune disease.

    The function of these T cell populations can be defined in validated or custom designed in vitro T cell assays to help define and understand drug efficacy with regard to regulating T cell function.

Inflammation and autoimmune T cell assays

  • Polyclonal T cell activation assays
  • Antigen-specific T cell activation assays
  • Mixed lymphocyte reaction (MLR) assays
  • Analysis of naïve or memory T cell populations
  • Expansion and functional analysis of rare antigen-specific T cells
  • T cell differentiation
  • Natural or inducible Regulatory T Cell (Treg) functional assays
  • Immune synapse formation
  • Polyclonal or antigen-specific in vitro T cell models of human naïve or memory T cell responses

    Antigen recall assays, which reactivate the T cell memory pool in an antigen-specific manner, allow you to model how your therapeutic may influence existing auto-reactive T cell populations.

    As an illustration here, we use the seasonal influenza vaccine, an antigen to which most healthy individuals respond, to stimulate T cells and determine the ability of a test agent to modify this recall response.

    • Alongside this, we can model your compounds’ ability to modify naïve T cell differentiation using a novel antigen to which individuals will not have been exposed, such as keyhole limpet hemocyanin (KLH).
    • During inflammation and autoimmune T cell assays. We can detect T cell responses by examining proliferation by cell trace violet (CTV) dilution or tritiated thymidine incorporation and cytokine production by Luminex® technology or intracellular cytokine staining.

     

    Inflammation and autoimmune T cell assays by flow cytometry: Analysis of antigen-specific naïve or memory T cell proliferation

    Assessing effects of a therapeutic on human naïve and memory T cell responses to model aspects of autoimmune disease in vitro
    PBMC from healthy donors were stimulated with seasonal influenza virus vaccine (Flu; Memory) or keyhole limpet hemocyanin (KLH; Naïve) for 7 days and proliferation tracked by CFSE dilution. Proliferation of CD4+ T cells was tracked by staining with anti-CD4 in combination with a viability dye (7AAD) to allow exclusion of non-viable cells from the analysis. Compound X inhibited naïve T cell proliferation but had little effect on memory T cell proliferation.

  • Expansion and functional analysis of rare antigen-specific T cells

    Naïve T cells or antigen-specific memory T cell pools are often present at low frequency in peripheral blood which makes these populations hard to study in terms of phenotype and functionality directly ex vivo:

    • Sorting of cells using pentamer/peptide strategies and subsequent expansion using in vitro T cell assays allows us to assess a therapy’s ability to modulate the function and phenotype of a population of T cells with a specificity relevant to the autoimmune disease of interest.
    • Alternatively, in early clinical trials traditionally associated with safety assessment, analyzing specific T cell subsets may identify PD biomarkers that indicate a therapy’s on-target effects in man.

     

    Expansion and assessment of functionality of rare antigen specific T cells using inflammation and autoimmune T cell assay

    Expansion of rare antigen specific T cell populations and assessment of T cell function using a combination of peptide/pentamer sorting and flow cytometric and ELISpot readouts of effector function as determined by expression of effector molecules and production of cytokines.
    A) Rare CD8 T cell populations were FACS sorted by direct peptide/pentamer staining of CD8 T cells. After two weeks of polyclonal expansion, cells were tested for pentamer/peptide specificity (B) Expanded T cells were re-stimulated using APC loaded with peptide and stained for LAMP1 or isotype control antibody along with other markers to assess cytotoxic function. C) Expanded CD8 T cells were assessed for peptide specificity by ELISpot measurement of IFNɣ production in response to peptide stimulation.

  • Helper T cell differentiation assays

    Helper T cells (Th cells) are CD4+ T cells which constitute a critical component of the adaptive immune system.

    Th subsets include Th1, Th2, Th17, TFH, thymically-derived natural regulatory T cells (nTreg), and inducible regulatory T cells (iTreg), each of which generates a distinct immune response.

    • Th1 cells generate strong immune responses against intracellular pathogens and are identified by expression of interferon gamma (IFNɣ).
    • n/iTregs suppress T cell proliferation and are involved in immune tolerance.

    Therapeutic intervention targeted to altering Th subset skewing of CD4 T cells could be targeted either directly to the T cell during differentiation, or by treating antigen presenting cells (APCs) to alter APC signaling to the T cell.

    The following example shows a T cell assay that models CD4 T cell polarization in vitro to assess a therapeutic’s ability to modulate T cell polarization away from a pro-inflammatory phenotype and drive the cells towards a regulatory phenotype.

     

    CD4 Th1 and iTreg (inducible regulatory T cell) differentiation assays

    Th1 and iTreg differentiation assays studied using flow cytometry
    Using one of our inflammation and autoimmune T cell assays, naive CD4 T cells were purified from human PBMC and differentiated towards Th1 or iTreg cells using differentiation cocktails consisting of cytokines and antibodies. After up to 7 days of differentiation, cells were stained and analyzed by flow cytometry. Th1 cells were identified as IFNɣ-expressing CD4 T cells, whereas inducible regulatory T cells were identified as FoxP3hi/CD127lo-expressing CD4 T cells.

  • Assessing regulatory T cell function: nTreg suppression assays

    Naturally occurring regulatory T cells (nTregs) suppress the immune response and play an essential role in maintaining immune tolerance. This balance is disturbed in autoimmune disease by as yet largely undiscovered mechanisms.

    • Natural regulatory T cells are present at low frequency in human blood and are identified as CD4+ T cells expressing the master regulator transcription factor, FoxP3, and lacking CD127 expression.
    • When natural regulatory T cells are co-cultured with responsive cell types, such as CD4+ responder T cells in the presence of a TCR stimulus in a T cell assay, the immune response of the responder cells is suppressed; as can be assessed by proliferation, the T cell activation status of the cells and cytokines produced.

    Therapeutic interventions targeting regulatory T cells may be a promising strategy in the treatment of autoimmune and inflammatory diseases.

     

    Identification and isolation of natural regulatory T cells in peripheral blood and testing their functional capacity in a natural regulatory T cell - nTreg suppression assay

    nTreg suppression assay results by flow cytometry.
    nTregs and CD4+ responder T cell isolation and use in T Cell suppression assays. nTreg and CD4+ responder T cells were purified from human PBMC and stained with a panel of antibodies for flow cytometric analysis of purity; natural regulatory T cells were identified as CD4+ FoxP3+ CD127- and responders were identified as CD4+. Natural regulatory T cells and responders were then seeded at three ratios (nTregs: responders) and activated using CD3/CD28 stimulation beads in the presence or absence of test compounds. Cells were cultured for 5 days before pulsing with tritiated thymidine to determine levels of proliferation and ability of regulatory T cells to inhibit responder proliferation

  • Immune synapse formation - modifying disease processes early in the development of autoimmune disease

    The immune synapse is the interface between an antigen presenting cell (APC) and a T cell. This interaction allows the exchange of signals between the two cell types and is critical in driving the T cell response.

    An antigen presenting cell, such as a dendritic cell (DC) presents peptides largely derived from intracellular proteins or cross-presented extracellular proteins on MHC class I or II.

    In the case of pathogens, these can be virally-derived peptides, or in the case of autoimmune disease, self-derived peptides. These peptides presented on MHC interact with the cognate T cell receptor, and if the APC has been activated co-stimulation and cytokine production provide signals 2 & 3 to fully activate a naïve T cell response.

    The nature of these three signals determines the type and quality of response generated and these interactions can be manipulated to drive immune tolerance in autoimmune disease. Therapeutic agents which modify the binding affinity or prevent cell-cell interactions may be tested in T cell assays that assess immune synapse formation.

     

    Analysis of T cell/APC interaction frequencies using one of our inflammation and autoimmune T cell assays to assess immune synapse formation

    Microscope images of co-cultures of T Cell and DC and graph
    Immune synapse formation is inhibited by an LFA-1 antagonist (Lifitegrast). Immature DC were co-cultured with autologous T cells and stimulated with SEB for 20 minutes to provide MHC/TCR cross linking before being fixed and stained for immunohistochemistry. Representative images of co-cultures with staining for anti-CD3 (red) to indicate T cells and anti-ICAM-1 (green) to indicate DCs. Nuclei are counterstained with DAPI (blue). White arrows indicate APC interacting T cells. Graph shows the quantification of the percentage of interacting T cells following 20 minutes stimulation. Data displayed are the mean of four donors and error bars represent +/- SEM. Statistics displayed as **** when p<0.0001.

After you assess the ability of your therapeutic to target human T cell differentiation and/or function, (and depending on your therapeutic target), studies using complementary disease models can provide information on in vivo efficacy in your disease of interest. Relevant in vivo disease and PD models could include imiquimod- induced psorisasis (Th17/iTreg axis), EAE and EAU (iTreg/Th1/Th17 axis), adoptive transfer colitis (iTreg/Th1/Th17 axis), dermatitis models (Th1/Th2 axis),or immuno-oncology T cell assays.

The following scientific poster presents an autoimmune application of these T cell assays in inflammatory bowel disease (IBD).


Modelling Inflammatory Bowel Disease using a Translational T Cell Assay Platform

Screenshot from IBD poster inflammation and autoimmune T Cell assay and mouse models

Get the data on validated animal models and in vitro human T cell assays used to test immunomodulatory therapeutics for IBD.

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Our team can help you determine which T cell assays best support your program, whether you require a screening assay format or bespoke complex assay design.

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Frequently Asked Questions (FAQs) about Inflammation and Autoimmune T cell Assays

  • How are T cells activated? Is it best to test my therapeutic in a polyclonal or antigen-specific response?

    T cells are activated via recognition of peptides in the context of cognate MHC and co-stimulation. The cytokine milieu helps to determine the nature of this response. During, inflammation and autoimmune T cell assays, T cell activation can be mimicked in vitro, or ‘in the dish’ by using agonistic antibodies against CD3 and CD28. Peptide or antigen stimulation is specific and will only stimulate a subset of T cells which recognize that antigen whereas CD3/28 stimulation is polyclonal, and all T cells are activated at once. Whether it is better to use polyclonal or antigen-specific stimulation for inflammation and autoimmune T cell assays depend on the question you are trying to answer. Polyclonal stimulation is more robust and can be useful in initial T cell assay screens whereas antigen-specific responses are more physiologically relevant and allow you to examine the effect of therapeutic agents on existing memory T cell populations.

  • What are the differences between nTreg and iTreg of inflammation and autoimmune T cell assays?

    Natural regulatory T cells, or nTreg, are a thymically-derived T cell pool and suppress autoreactive T cell responses. Inducible regulatory T cells, or iTreg, are generated in the periphery during a T cell response to a specific antigen. The function of both subsets can be tested in a T cell suppression assay, where a therapeutic’s ability to drive iTreg generation or activate or enhance natural regulatory T cells can be tested. Your compound’s ability to drive T cell differentiation away from an inflammatory TH1/TH17 phenotype and towards iTreg formation in a T cell assay may also be relevant depending on the target of your therapeutic.

    Marco Romano & al. Past, Present, and Future of Regulatory T Cell Therapy in Transplantation and Autoimmunity

  • How can I best measure the effect of my compound on proliferation and function of cells in a T cell assay?

    Key readouts of activated T cell function are proliferation; which is rapid division of the T cell subset following TCR engagement, cytokine production and expression of effector molecules, such as IFNɣ, IL-17 or IL-10, which can be measured by several techniques including flow cytometry and Luminex multiplexing in an inflammation and autoimmune T cell assay. Cytotoxic CD8 T cells can release cytokines such as IFNɣ and TNFα and secrete lytic granules containing granzymes and perforin, which can also be quantified by either Luminex multiplexing or flow cytometry. T cell phenotype can also be assessed by gene expression analysis using qPCR or NanoString analysis.

    Edward J.Moticka A Historical Perspective on Evidence-Based Immunology, chap 27

  • Which T cell assays demonstrate my therapeutic’s potential efficacy in autoimmune disease?

    The first step would typically be a polyclonal stimulation or MLR assay, to assess your therapy’s ability to suppress or alter the T cell response in terms of proliferation and cytokine production. More complex assays downstream could include T cell assay differentiation using positively selected T cells. This assay uses cells that are magnetically isolated by metal beads and bound to an antibody which recognizes the T cell subset of interest, in this case CD45RA. These inflammation and autoimmune T cell assays allow you to determine whether your compound can suppress a Th1/Th17 response or generate increased frequencies or functionality of iTreg. Factors such as IL-2 and IFNɣ produced by Th1 drive cytotoxic CD8+ T cell activation and inhibiting these factors would be beneficial in an autoimmune context. A general switch of the cytokine profile towards a more suppressive phenotype (increased IL-10 and decreased IFNɣ/ IL-17) would be advantageous.

    Valérie Dardalhon & al. Role of Th1 and Th17 cells in organ-specific autoimmunity