• Tumor Microenvironment
• Cancer Immunotherapy
• Cell Exhaustion
• Immune Checkpoint Receptor
• Cellular Activation Status

Programmed Death 1 (PD-1) and T cell Ig and mucin domain-3 protein (Tim-3) are two immune checkpoint receptors (ICR) highly co-expressed on tumor infiltrating T lymphocytes (TIL). PD-1 has been shown to inhibit T cell activation and type 1 T cell responses, while Tim-3 has been proposed as a further marker of exhaustion on TIL [1, 2], leading us to investigate the phenotypic and functional characteristics of TIL with differential PD-1 and Tim-3 expression from head and neck cancer (HNC) patients. Our data showed that PD-1⁺Tim-3⁺ CD8⁺ and Foxp3^- CD4⁺ TILs manifested high phosphorylated signal transducers and activators of transcription 1 (p-STAT1) and the associated Th1 transcription factor T-bet, which might correlate with T cell exhaustion, both at baseline and upon TCR stimulation. Moreover, the sorted PD-1⁺Tim-3⁺ CD8⁺ TILs expressed the lowest IFN-γ and TNF-α transcripts and the least amount of secreted IFN-γ upon TCR stimulation, indicating they are the most dysfunctional T cells in the tumor microenvironment (TME). Among CD4⁺CD25^lo/- TIL subsets, PD-1^hiTim-3^- cells are more defective in terms of IFN-γ expression. Sorted PD-1^intTim-3^- CD8⁺ and CD4⁺CD25^lo/- TILs showed higher TCR-stimulated expression of IFN-γ and TNF-α transcripts and secretion of IFN-γ, suggesting they are the most activated subsets. In addition, sorted PD-1⁺Tim-3⁺ and PD-1^hiTim-3^- TIL were less proliferative than other subsets, concomitant with lower expression of phosphorylated S6 (p-S6), while PD-1^intTim-3^-, PD-1^-Tim-3⁺ and PD-1^-Tim-3^- TIL retained p-S6 activation or proliferation, suggesting that high expression of PD-1 on T cells interferes with TCR or Tim-3 signaling and associated cellular activation status. Taken together, PD-1⁺Tim-3⁺ and PD-1^hiTim-3^- TIL are most dysfunctional, while PD-1^intTim-3^- TIL are more activated in terms of both Th1 cytokine production and proliferation. These results provide a better understanding of the functional status of TIL subsets and roles of PD-1 and Tim-3 in regulating anti-tumor T cell response, as targets for cancer immunotherapy.