1:IDIBAPS; 2:Hospital Clinic Barcelona; 3:CNAG-CRG

CAR-T cell immunotherapy has achieved remarkable success in hematologic malignancies but remains ineffective in solid tumors. CAR-T cell dysfunction in the tumor microenvironment is a major hurdle for CAR-T therapy. Here we have established and validated an in vivo mouse model to study the T-cell intrinsic mechanistic pathways leading to CAR-T cell unresponsiveness. We have isolated tumor infiltrating T cells at two different phases: 1) after T cell activation that leads to tumor regression (named as “Effective CAR-T cells”) and 2) at a second phase in which persisting antigen leads to loss of T-cell functions and tumor progression (named as “Dysfunctional CAR-T cells”).

Dysfunctional TILs recapitulated features of T cell exhaustion including high level of PD-1 inhibitory marker expression and loss of Ki67 proliferation marker. Effective but not dysfunctional TILs displayed cytotoxic activity and released IFNγ when co-cultured with tumor cells ex vivo.

RNAseq analysis revealed extensive differences between effective and dysfunctional TILs with more than 2.000 differentially expressed genes. Dysfunctional CAR-TILs displayed a transcriptional profile overlapping with gene sets described in models of chronic viral infections and in dysfunctional human TILs. Surprisingly, while effective TILs retained their effector functions and were able to induce tumor regression in mice, T cell exhaustion gene signatures were also early enriched in effective TILs. Gene clusters and pathways possibly implicated in the dysregulated gene expression signature will be discussed. Altogether, our results suggest that CAR-T cell dysfunction needs to be addressed to unleash the full potential of CAR-T cell therapy in solid tumors.