Combined PD-L1 and TIM3 blockade improves expansion of fit human CD8(+) antigen-specific T cells for adoptive immunotherapy

Antigen-specific T cell expansion ex vivo followed by adoptive transfer enables targeting of a multitude of microbial and cancer antigens. However, clinical-scale T cell expansion from rare precursors requires repeated stimulation, which may lead to T cell dysfunction and limited therapeutic potential. We used a clinically compliant protocol to expand Epstein-Barr virus (EBV) and Wilms tumor 1 (WT1) antigen-specific CD8(+) T cells, and leveraged T cell exhaustion-associated inhibitory receptor blockade to improve T cell expansion. Several inhibitory receptors were expressed early by ex vivo-expanded antigen-specific CD8(+) T cells, including PD-1 and TIM3, with co-expression matching evidence of T cell dysfunction as the cultures progressed. Introduction of anti-PD-L1 and anti-TIM3 blockade in combination (but not individually) to the culture led to markedly improved antigen-specific T cell expansion without inducing T cell dysfunction. Single-cell RNA sequencing (RNA-seq) and T cell receptor (TCR) repertoire profiling revealed that double blockade does not impart specific transcriptional programs in T cells or alterations in TCR repertoires. However, combined blockade may affect gene expression in a minority of clonotypes in a donor-specific fashion. We conclude that antigen-specific CD8(+) T cell manufacturing can be improved by using TIM3 and PD-L1/PD-1 axis blockade in combination. This approach is readily applicable to several adoptive immunotherapy strategies.