PD-1 expression in head and neck squamous cell carcinomas derives primarily from functionally anergic CD4⁺ TILs in the presence of PD-L1⁺ TAMs

Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cavity tumor. In this study, we examined the basis for the activity of programmed cell death protein (PD-1)-based immune checkpoint therapy that is being explored widely in head and neck cancers. Using multispectral imaging, we systematically investigated the OTSCC tumor microenvironment (TME) by evaluating the frequency of PD-1 expression in CD8⁺, CD4⁺, and FoxP3⁺ tumor-infiltrating lymphocytes (TIL). We also defined the cellular sources of PD-1 ligand (PD-L1) to evaluate the utility of PD-1:PD-L1 blocking antibody therapy in this patient population. PD-L1 was expressed in 79% of the OTSCC specimens examined within the TME. Expression of PD-L1 was associated with moderate to high levels of CD4⁺ and CD8⁺ TILs. We found that CD4⁺ TILs were present in equal or greater frequencies than CD8⁺ TILs in 94% of OTSCC and that CD4⁺FOXP3neg TILs were colocalized with PD-1/PD-L1/ CD68 more frequently than CD8⁺ TILs. Both CD4⁺PD1⁺ and CD8⁺PD1⁺ TILs were anergic in the setting of PD-L1 expression. Overall, our results highlight the importance of CD4⁺ TILs as pivotal regulators of PD-L1 levels and in determining the responsiveness of OTSCC to PD1-based immune checkpoint therapy.