Choosing T-Cell Assays for Cancer Immunotherapy: A Guide to Selection

Introduction

Cancer has long been a formidable adversary, challenging scientists and clinicians alike to develop innovative strategies for treatment and management. Immuno-oncology, a field focused on harnessing the power of the immune system to fight cancer, has emerged as a promising avenue in the quest for effective therapies. Among the various tools and techniques in immuno-oncology, T cell assays play a crucial role in understanding and evaluating immune responses against cancer prior to clinical trials. In this blog post, we will delve into the basics, methods, and applications of immuno-oncology T cell assays, highlighting their significance and citing relevant examples.

Basics of Immuno-Oncology T Cell Assays

Immuno-oncology T cell assays are designed to measure and characterize the function and potency of T cells, which are pivotal in mounting an immune response against cancer. These assays provide valuable insights into the interaction between cancer cells and the immune system, enabling researchers and clinicians to evaluate the efficacy of immunotherapeutic interventions. By quantifying and analyzing T cell responses, these assays can provide information about T cell activation, proliferation, cytokine secretion, cytotoxicity, and immune checkpoint blockade.

Types of Immuno-Oncology T Cell Assays

Cytotoxicity Assays: Cytotoxicity assays assess the ability of T cells to directly kill cancer cells. Cytotoxic T cells require recognition of specific antigens on tumor cells.  Tumor cells are killed by cytotoxic granules released by T cells that disrupt cell surface membranes, leading to cell death.  Assays to detect tumor cell death typically involve co-culturing T cells with cancer cells and measuring the extent of target cancer cell lysis. Various methods are employed to quantify cytotoxicity, such as flow cytometry-based assays using fluorescently labeled target cancer cells or surrogate markers of cell death. Cytotoxicity assays help evaluate the potency of drug candidates in enhancing T cell-mediated killing of tumor cells.

 

    Table 1: Examples of T-cell cytotoxicity assays

Type of T-cell Cytotoxicity AssayPrinciple
Cell Membrane Integrity AssaysEvaluate cell membrane integrity, which is often compromised during cell death or cytotoxicity. Example: The lactate dehydrogenase (LDH) release assay measures the release of LDH, an enzyme present in the cytosol of cells, into the supernatant upon target cell lysis.
Flow Cytometry based AssaysLive/dead cell flow cytometry assays utilize combinations of fluorescent dyes to discriminate between live and dead cells.
ATP-based AssaysATP (adenosine triphosphate) is an essential energy molecule present in all viable cells. ATP-based luminescence assays measure cell viability by quantifying the amount of ATP in a sample.
Chromium Release AssayThe chromium release assay is a classic and widely used method for measuring T-cell cytotoxicity; however, this assay has been replaced by more efficient and safer technologies.

 

T Cell Activation Assays: T cell activation assays measure the ability of drug candidates to stimulate T cell activation and proliferation. These assays typically involve exposing T cells to candidate drugs or antigen-presenting cells presenting tumor-specific antigens. T cell activation markers, such as CD25 and CD69, can be measured using flow cytometry to assess the drug’s impact on T cell activation. Additionally, cytokine secretion assays, such as ELISA or ELISpot, can be employed to measure the release of cytokines like IFN-γ or IL-2, indicating T cell activation.

 

Table 2: Types of T-cell activation assays

Type of T-cell Activation AssayPrinciple
Proliferation AssaysProliferation assays measure the ability of T cells to undergo cell division in response to antigenic stimulation.
Cytokine Secretion AssaysCytokine secretion assays measure the production and release of cytokines by activated T cells.
Flow Cytometry AssaysFlow cytometry can measure changes in protein expression on cells after activation. For example, cell surface proteins CD25 and CD69 are upregulated upon T cell activation.
Calcium Flux AssaysCalcium flux assays assess increases in intracellular calcium concentrations, which occur upon T cell activation.

 

Immune Checkpoint Assays: Immune checkpoint assays evaluate the impact of drug candidates on immune checkpoint molecules that inhibit T cell activity.  The same assays that are used for T cell activation may be utilized, but in the presence of checkpoint inhibitors. Flow cytometry assays can be used to measure the expression levels of inhibitory checkpoint molecules (e.g., PD-1, CTLA-4) on T cells. Additionally, functional assays can assess the ability of drug candidates to inhibit checkpoint signaling pathways, such as measuring the release of cytokines or proliferation of T cells upon checkpoint blockade.

T Cell Proliferation Assays: T cell proliferation assays assess the capacity of drug candidates to induce, or reduce T cell expansion. These assays typically involve labeling T cells with fluorescent dyes, such as CFSE, and stimulating the T cells with antigen-presenting cells or tumor cell lines, and observing the effect of drug candidates. The extent of T cell proliferation can be quantified by measuring the dilution of fluorescent dyes using flow cytometry. Proliferation assays provide insights into the antigen-specific response and the potential of drug candidates to enhance T cell expansion.

 

Table 3: Types of T-cell proliferation assays

Type of T-cell Proliferation AssayPrinciple
CFSE (Carboxyfluorescein Succinimidyl Ester) and Cell Trace Violet (CTV) AssaysThe CFSE and CTV assays are widely used methods to track T cell proliferation. They are fluorescent dyes that are retained in the cytoplasm. Upon cell division, daughter cells have half the fluorescent intensity.
BrdU (Bromodeoxyuridine) Incorporation AssayThe BrdU incorporation assay measures T cell proliferation by detecting the incorporation of BrdU, a thymidine analog, into the DNA of dividing cells.
Ki-67 StainingThe Ki-67 staining assay measures the expression of the Ki-67 protein, a nuclear antigen present during all phases of the cell cycle except for G0. Ki-67 staining is often used as a marker of cellular proliferation.
[3H] Thymidine Incorporation AssayThe [3H] thymidine incorporation into DNA is a classic and was a widely used method to assess T cell proliferation. More recent technology is easier and more robust.

 

Cytokine Secretion Assays: Cytokine secretion assays measure the ability of drug candidates to modulate the production of immune regulatory cytokines by T cells. These assays can employ techniques like ELISA, ELISpot, or intracellular cytokine staining (ICS) to detect and quantify cytokines secreted by activated T cells. By assessing the impact of drug candidates on cytokine profiles, these assays provide information on the functional consequences of T cell activation and the potential for immunomodulation.

 

Table 4: Types of cytokine secretion assays

Type of Cytokine Secretion AssayPrinciple
Enzyme-Linked Immunosorbent Assay (ELISA)ELISA is a widely used method to measure levels of secreted proteins. Target-specific antibodies are coated onto a microplate, and the amount of protein bound to the antibody is measured.
Enzyme-Linked Immunospot Assay (ELISpot)ELISpot assays are designed to enumerate the number of individual cytokine-secreting cells.
Intracellular Cytokine Staining (ICS)ICS assays measure the intracellular production of cytokines in response to stimuli, primarily by flow cytometry.
Multiplex ImmunoassaysMultiplex immunoassays use various techniques, such as bead-based or microarray-based platforms, to simultaneously measure multiple different proteins in a single sample.

 

Co-culture Assays: Co-culture assays involve the co-incubation of T cells with tumor cells or antigen-presenting cells to evaluate the functional interactions and anti-tumor immune responses. These assays can measure T cell activation, cytokine secretion, cytotoxicity, and other functional endpoints to assess the effects of drug candidates on T cell-mediated anti-tumor immune responses. Co-culture assays provide a more physiologically relevant setting to evaluate the drug’s impact on T cell behavior.

 

 Table 5: Types of T-cell co-culture assays / T-cell killing assays

Type of T-cell Co-culture AssayPrinciple
Tumor Cell Co-culture Assays / T-cell Killing AssaysIn tumor cell co-culture assays, T cells are co-cultured with tumor cells to evaluate anti-tumor immune responses and cytotoxicity.
Dendritic Cell (DC) Co-culture AssaysCo-culturing T cells with dendritic cells (DCs) is commonly used to evaluate antigen-specific T cell responses.
T Cell-B Cell Co-culture AssaysCo-culturing T cells with B cells allows the examination of T cell-B cell interactions and immune responses.
T Cell-Macrophage Co-culture AssaysCo-culturing T cells with macrophages allows the study of T cell-macrophage interactions and the role of macrophages in modulating T cell responses.
T Cell-Endothelial Cell Co-culture AssaysCo-culturing T cells with endothelial cells enables the investigation of T cell-endothelial cell interactions and their role in immune cell trafficking and inflammation.

 

Conclusion

Immuno-oncology T cell assays represent powerful tools for evaluating and understanding the immune response against cancer. By measuring T cell functionality, cytokine secretion, and proliferation, these assays provide valuable insights into immunotherapies, vaccine development, and facilitate biomarker discovery for use in clinical trials. The integration of T cell assays into clinical trials and research studies has the potential to enhance treatment outcomes and guide personalized cancer therapy decisions. As the field of immuno-oncology continues to advance, these assays will undoubtedly play a crucial role in unlocking the full potential of the immune system in the fight against cancer.

 

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1.  Development of a Pharmacokinetic (PK) Mouse Serum GLP ELISA for an Anti–CD19–AntiCD3 Diabody
bioRxiv 2025.03.19.644217; doi: https://doi.org/10.1101/2025.03.19.644217
2. Cell-Based Potency Assay for Anti-CD3-Anti-CD19 Diabody. bioRxiv 2025.04.15.648836v1 https://www.biorxiv.org/content/10.1101/2025.04.15.648836v1
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