T Cell Assays

Super agonists such as Staphylococcal enterotoxin B (SEB) bind APC expressing MHC-II to TCR driving robust T cell proliferative and cytokine responses. In vitro SEB driven T cell assays can be used to assess the ability of immunomodulators including immune checkpoint blockade (ICB) therapeutics to enhance T cell function. For example, PD-1 and CTLA-4 blocking therapeutics, pembrolizumab, and ipilimumab respectively, act individually and synergistically to enhance T cell release of pro-inflammatory cytokines.

(Figure 1) Checkpoint inhibitors enhance pro-inflammatory cytokine levels individually and synergistically in T cell assays. PBMC were stimulated with SEB in the presence of checkpoint inhibitors and IL-2 and IFNγ release measured by Luminex. T cell assays can be performed with total PBMC (left) or purified CD4s co-cultured with autologous APC.

In vitro tumor killing assays offer the ability to recapitulate aspects of cellular and humoral interactions found in tumors. Immune mediated tumor cell killing is kinetically assessed by combining fluorescently labelled target cells with PBMC in the presence of an apoptosis reporter. Immune checkpoint blockade (ICB) or immuno-stimulating therapeutics enhance T cell killing over three days as measured by a decrease in the number of target cells and increase in the percentage of target cells dying by apoptosis. Immune subtype depletion facilitates identification of the functional cellular target in a mixed PBMC population.

Further complexity can be added to model elements seen in vivo via culturing cells in 3D, supplementing the media with immunosuppressive factors or introducing cell types harbored by the tumor microenvironment (e.g., TAMs, CAFs, and MDSCs).

Tumor cell line TKA

(Figure 2) Checkpoint inhibitors enhance target cell killing by PBMC. Fluorescent tumor cell targets were plated for 24 hours. PBMC or CD8+ depleted PBMC from a healthy donor were added to tumor cells in the presence of an isotype IgG, checkpoint inhibitors (CPI) or IL-2. A caspase-3/7 reporter dye was included to monitor tumor cell apoptosis. Co-cultures were imaged every 2 hours by IncuCyte and the number of tumor cells present over time or the percentage of those dying by apoptosis was determined. CD8+ T cells are a critical PBMC immune subset required to mediate target cell apoptosis.

PDX cell line TKA

Consider using patient derived xenograph T cell assay designs that require a minimal phenotypic/mutational drift from original human tumors or where cell lines do not recapitulate antigen expression patterns.

Antigen recall assays assess the capacity of memory T cells to respond to their cognate antigen. As the frequency of T cells responding to tumor antigens within a healthy individual is very low, to model antigen-specific T cell activation, we utilize a panel of peptides derived from viruses or pathogens to which many people will have been vaccinated against or have been infected with. The phenotype of responding T cells can be assessed by their proliferative responses, flow cytometry after staining for the degranulation marker CD107a, and cytokine production. Immune modulating biologics or small molecules can dose-dependently enhance antigen-specific CD8 responses to peptide stimulation.

(Figure 3) Anti-LAG3 antibody dose-dependently enhances CD8+ T cell activation. PBMC from a healthy donor were stimulated with CEFT peptides with or without check-point inhibitors. T cells were stained for markers of activation, the percentage of CD8 cells expressing the degranulation marker CD107a and the pro-inflammatory cytokine IFNγ was determined.

In a mixed lymphocyte reaction (MLR) T cells recognize an HLA mismatch on allogenic cells and respond by proliferation and pro-inflammatory cytokine release. In the presence of CPI, T cells have a higher magnitude of proliferation and release greater amounts of IL-2 and IFNγ cytokines.

(Figure 4) Pembrolizumab dose-dependently enhances MLR driven T cell proliferation and pro-inflammatory cytokine release. MLR CD4+ proliferation and cytokine release was determined in the presence or absence of the PD-1 inhibitor Pembrolizumab (0, 0.2, 3, and 20 µg/ml). Lower levels of proliferation and IFNγ release at 20 µg/ml Pembrolizumab likely reflect the kinetics of T cell activation.

T cell stimulation via soluble or immobilized anti-CD3 polyclonally activates T cells without the need for antigen or accessory cells. Bespoke assay design through tailoring co-stimulation, cytokine supplement, and fine tuning the strength of stimulus enables therapeutic enhancement of T cell phenotype and function.

T cell exhaustion can occur due to chronic or suboptimal stimulation. This state was first described in the context of viral infection, but the same problem chronic stimulation can apply for tumor specific T cells. Tex cells are characterized by a loss of proliferative capacity under continued stimulation and poor proliferation upon re-stimulation. The Tex cell phenotype can be modeled through polyclonal stimulation which generates cells expressing inhibitory molecules. These are also expressed on exhausted cells such as PD-1, CTLA-4, LAG-3, TIM-3, and TIGIT.

The ability of therapeutics to inhibit the exhausted state or reverse it can be assessed in T cell assays. For example, IL-2 treatment in combination with TCR re-stimulation is able to reverse exhaustion and restore the proliferative capacity of Tex cells. However, despite PD-1 and CTLA-4 expression, Tex cells appear refractory to checkpoint inhibition in this assay format.

(Figure 5) T cell assay to model reversal of T cell exhaustion. CD4+ cells were purified from healthy donor PBMC and polyclonally stimulated for up to 10 days to induce a hypo-proliferative state (monitored by 3H-thymidine incorporation). At day 10 post stimulation, expression of inhibitory receptors associated with the exhausted state is high. Upon re-stimulation with anti-CD3, exogenous IL-2 is sufficient to break exhaustion and drive proliferation while checkpoint inhibitors targeting PD-1 and CTLA-4 cannot.

T cell proliferation is easily monitored in multiple ways including, dye dilution (eg CTV, CFSE), Tritiated thymidine incorporation, MTT or CTG assays, and DNA binding dyes. An example of a dye dilution experiment is shown below where purified CD8 T cells were stained with a cell proliferation dye and polyclonally stimulated with anti-CD3. Each gate represents a T cell division where the dye is diluted by 2 and many rounds of division can be tracked over multiple generations.

(Figure 6) T cell proliferation assays using flow cytometry. CD8+ T cells were purified from a healthy donor PBMC, stained with cell proliferation dye and polyclonally stimulated for 6 days. Typically, 5-6 rounds of divisions can be determined. By multiparameter staining, the ability of a therapeutic to modulate proliferation of specific T cell subsets and the co-expression of other surface and intracellular markers or effector molecules can be assessed.

Phospho-flow can be used to assess whether signaling pathways have been engaged in different cell subsets. The STAT assessed for phosphorylation will depend on the cytokine of interest. Phospho-flow staining and flow cytometry was carried out to assess IL-2 signaling by determining phosphorylation of STAT5 (or pSTAT3 not shown) in a range of immune subsets (Regulatory T cells, conventional CD4 cells, CD8 T cells, NK cells) in response to rhIL-2. The potency (EC50 values) of rhIL-2 to drive pSTAT5 expression by different immune cells in a mixed PBMC population can be determined from human (shown) and murine PBMC.

(Figure 7) Utilizing flow cytometry to determine cellular responses in a heterogeneous population. PBMC from a healthy donor were stimulated with a range of IL-2 doses to evoke IL-2 signaling, measured by pSTAT5 expression. PBMC were then stained with a panel of phenotypic markers to identify CD4+ T cells, CD8+ T cells, Treg, and NK cells. The gating strategy used enables the expression of pSTAT5 to be determined for each subpopulation. IL-2 potency varies between immune subsets with Tregs responding most sensitively, as reported in the literature, most likely due to robust expression of the high affinity IL-2 receptor.

We offer a range of immune mediated tumor killing assays (TKA).

In recent years, cellular therapeutics have emerged resulting in a paradigm shift. However, much is still to be learned to improve efficacy and safety. We offer assessments for a wide range of oncology cellular therapeutic functionality and on/off target responses.

Regulatory T cells (Treg) are a heterogeneous population characterized by their ability to suppress immune responses. They are comprised of several different lineages but these can be split into natural nTreg (Foxp3+), which are thymically derived, and iTreg, of which there are several types that are induced under certain conditions in the periphery. In the TME Treg can limit effector T cell function and support tumor cell evasion and growth. Therapeutics can be assessed for their ability to inhibit nTreg suppression of responder T cells with the aim of enhancing T cell responses.

(Figure 8) In vitro functional nTreg assay. nTregs and CD4+ responders were purified from human PBMC and stained with a panel of antibodies for flow cytometric analysis. nTregs were identified as CD4+ CD25+CD127- and responders were identified as CD4+. nTregs 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 cocultured then pulsed with tritiated thymidine to determine levels of proliferation.

Naive CD4+ cells can be polarized into different subsets dependent on the conditions in which they are activated. This enables the ability of compounds to subvert their polarization to be tested.

(Figure 9) In vitro phenotypic iTreg assay. Naïve CD4+ cells were purified by magnetic selection and their differentiation skewed towards Tr1 or iTreg cells using a defined cocktail of immunomodulators. Their phenotype is determined by flow cytometry. The ability of therapeutics to skew this phenotype away from a regulatory phenotype and towards a more pro-inflammatory phenotype can be assessed.

T cells that recognize tumor antigens naturally circulate in the blood but typically at very low frequencies. These rare cells can be enriched and expanded ex vivo for phenotypic and functional assays. Specificity and cytotoxic capacity are assessed by co-culture with antigen expressing target cells and subsequent target cell death. This approach has value for therapeutics looking to enhance antigen-specific T cell activation, expansion, and functional capacity. At each stage a therapeutic effect is monitored using appropriate readouts including phenotyping, proliferation, cytokine release, and target cell viability.

(Figure 10) Purification and expansion of functional tumor antigen (TAA) specific CD8 T cells from blood. TAA specific populations were FACS sorted by direct peptide/pentamer staining of CD8 cells. After two weeks of polyclonal expansion, cells were tested for pentamer/peptide specificity. Expanded CD8 cells were assessed for their functional ability to kill TAA expressing tumor cells in a cytotoxicity assay.

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 IO, tumor associated antigens. 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 T cells. The nature of these three signals determines the type and quality of response generated and these interactions can be manipulated to drive immune activation in IO. Therapeutic agents that modify the binding affinity or enhance cell-cell interactions may be tested in T cell assays that assess immune synapse formation.

(Figure 11) Analysis of T cell/APC interaction to assess immune synapse formation. 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) and anti-ICAM-1 (green) (DC). 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.

Antigen-specific human T cell activation or polyclonal T cell activation (for example by Super antigen) can be used to drive T cell priming and activation. Assessing immune synapse formation can provide insights into priming by antigen presenting cells. iTreg and CD4 Th cell differentiation are T cell assays developed to observe activation and polarization of T cell subsets. Treg assays assess the ability of a therapeutic to inhibit Treg function, a major readout is the proliferation of the responder T cell subset.

Exhausted T cells (Tex) persistently activated by tumor antigens develop deficits in proliferation, pro-inflammatory cytokine release, and cytotoxic capability. To model exhaustion in vitro, T cells can be activated polyclonally or in an antigen-specific manner.

Polyclonal T cell stimulation offers a reliable method to activate all CD3+ cells in a controlled and reproducible way leading to a high percentage of hypoproliferative cells expressing a range of exhaustion markers (PD-1, TIM-3, LAG-3 and CTLA-4). Polyclonal Tex assays incorporating proliferative and cytokine readouts are a suitable model to test therapeutic intervention to either prevent or reverse T cell exhaustion. Modeling antigen-specific driven exhaustion can be achieved by using antigens from viruses or pathogens to which most people will have been infected by or vaccinated against. This means that most healthy donors will have a repertoire of memory T cells to these antigens, which can then be re-stimulated and driven to have aspects of exhaustion in vitro.

T cell chemotaxis, T cell/DC adhesion, transwell assays, and Trans-endothelial migration are T cell assays developed to observe T cell trafficking.

2D T cell-mediated cytotoxicity and 3D spheroid T cell-mediated cytotoxicity can be used to assess the ability of a therapeutic to enhance immune mediated tumor killing.