Use of a 50-Marker Mass Cytometry Intracellular Cytokine Staining Panel Reveals Unforeseen Functional Diversity Within Cell Therapy Products 2258158

Abstract Introduction Despite significant investment, most cancer therapeutics, including CAR T cell therapies, fail in the clinic. Functional assessment of CAR T cells is typically limited to Th1 cytokines (for example, IFNγ), overlooking the known heterogeneity of the T cell subsets that make up the products, including populations with immunosuppressive capacity. This diversity introduces potential “saboteur” cells with off-target effects that may undermine therapeutic efficacy. Surface marker profiling is insufficient for functional characterization, yet traditional flow cytometry lacks resolution for certain intracellular targets, particularly immunosuppressive cytokines. Mass cytometry (CyTOF™ technology) overcomes this technical limitation, providing superior intracellular signal resolution in the context of large panel analyses, a prerequisite of deep functional assessment of single cells. We hypothesized that there are sizeable proportions of saboteur T cells in cell therapy products that exert functions after CAR stimulation that act in direct opposition to the therapeutic intention of the drug. Methods To assess this, we developed a 50-marker CyTOF intracellular cytokine staining (ICS) panel with 24 functional readouts, enabling clear detection of individual human T cells producing immunosuppressive cytokines such as IL-10, IL-13 and TGF-b in human PBMC and cell therapy products. Results Our data revealed an unforeseen prevalence of T cells that cross dogmatic lineage paradigms when stimulated. For example, approximately 30% of T cells from both peripheral blood and a cell therapy product produced the TGF-b-inducing cytokine amphiregulin and often did so simultaneously with IFNg within individual T cells. Conclusion From these results, we predict that application of this novel CyTOF ICS panel for cell therapy development protocols could prove transformative in the treatment of many cancers and other diseases. Funding Source Standard Biotools Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)