Use of a 50-marker mass cytometry intracellular cytokine staining panel reveals unforeseen functional diversity within cell therapy products and unmasks potential efficacy 'saboteurs'

Cell-based therapies have proven to be highly effective against certain cancers, yet improvements are needed to extend these successes to a wider variety of malignancies. Single-cell functional assessment of cell therapy products is customarily limited to Th1 cytokines (such as IFN-gamma) and the cell survival/proliferative mediator IL-2, overlooking the cellular heterogeneity of CAR T products. This heterogeneity includes diverse antigen-experienced alphabeta T cells previously differentiated into distinct lineages (for example, Type 1, 2, 17, Treg) and innate T cell populations (iNKT, MAIT, gammadelta T), with many of the latter pre-programmed to exert immunosuppressive and/or cell repair effector functions. 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 stimulation that act in direct opposition to the therapeutic intention of the drug. To assess this, we developed a 50-marker CyTOF intracellular cytokine staining (ICS) panel with 24 functional readouts. This enabled clear detection of individual human T cells producing immunosuppressive cytokines such as IL-10, IL-13 and TGF-beta, and measured the composite cytokine responses of both T cells in peripheral blood mononuclear cells (PBMC) and cell therapy products. Our data revealed an unforeseen prevalence of T cells that cross dogmatic lineage paradigms when stimulated. For example, after mitogen stimulation, approximately 30% of T cells from both peripheral blood and a cell therapy product produced the TGF-beta-inducing cytokine amphiregulin (AREG), often concurrently with IFN-gamma within the same cells. In addition, using the CyTOF ICS panel on TCR-stimulated T cell populations revealed peptide-specific production of AREG, IL-13, IL-4 and MIP-1beta by CD8 T cells in PBMC, suggesting CAR T products may release unmeasured cytokines in response to specific CAR antigen binding. From these results, we anticipate application of CyTOF ICS technology to cell therapy testing and modification will transform our understanding of these therapies and ultimately enhance their efficacy.