Functional and Molecular Characterization of PD1+ Tumor-Infiltrating Lymphocytes From Lung Cancer Patients

Abstract Background The majority of infiltrating T-cells (TILs) in lung cancer are contained in the memory compartment and overexpress PD1 and have been associated with dysfunction. Antibody-mediated cancer immunotherapy targets inhibitory surface molecules, such as PD1, PD-L1, and CTLA-4, aiming to re-invigorate dysfunctional T cells.Methods Using fluorescence-activated cell sorting (FACS), we purified CD45RO+ memory CD8+ and CD4+ TILs and their patient-matched non-tumor counterparts from treatment-naïve NSCLC patient biopsies to better evaluate the effect of PD1 expression on the functional and molecular profile of tumor-resident T cells. Moreover, we compared the functional, molecular, and clonal composition of TIL preparations after TCR-dependent in vitro expansion with their freshly isolated counterparts in matched patients.Results We show that PD1+CD8+ TILs have elevated expression of the transcriptional regulator ID3 and that the overall cytotoxic potential of CD8 T cells can be improved by knocking down ID3, defining it as a potential regulator of T cell effector function. PD1+CD4+ memory TILs remain functionally intact and despite overexpressing key transcriptional activators known to negatively regulate CD8 function such as TOX and TOX2, display transcriptional patterns consistent with both follicular helper and regulator function and robustly facilitate B cell activation and expansion in response to TCR-dependent stimulation. Furthermore, we show that expanding ex vivo-prepared TILs in vitro in a TCR-dependent manner broadly preserves their functionality with respect to tumor cell killing, expansion and activation of B cells, and TCR repertoire. Although purified PD1+CD8+ TILs generally maintain an exhausted phenotype upon expansion in vitro, transcriptional analysis reveals a downregulation of markers of T cell dysfunction, including the co-inhibitory molecules PD1 and CTLA-4 and the transcription factors ID3, TOX and TOX2, while genes involved in cell cycle and DNA repair are upregulated. We find reduced expression of WNT signaling components to be a hallmark of PD1+CD8+ exhausted T cells in vivo and in vitro and demonstrate that restoring WNT signaling, by pharmacological blockade of GSK3β, can improve effector function. Conclusions These data unveil novel targets for tumor immunotherapy and have promising implications for development of a personalized adoptive TIL-based cell therapy for lung cancer.