Immune checkpoint NKG2A marks repeatedly stimulated CD8 T cells whereas PD-1 is indicative for recent activation

The surface inhibitory receptor NKG2A forms heterodimers with the invariant CD94 chain and is expressed on a subset of activated CD8 T cells. As antibodies to NKG2A are currently tested in several efficacy trials in different tumor indications, we were interested in the characteristics of the NKG2A+ CD8 T cell population in the context of other inhibitory receptors. Here we demonstrate in a well-controlled synchronized culture system that NKG2A is a late inhibitory receptor, expressed after repeated TCR stimulations. In contrast, PD-1, TIGIT and Lag-3 are already induced hours after the first stimulation and are down regulated again in each resting phase. This late, but stable expression kinetics of NKG2A was most similar with that of TIM-3 and CD39 and these receptors were often co-expressed in the same CD8 T cell cluster of human cancers, as revealed by single cell transcriptomics of TILs. Furthermore, NKG2A expression was associated with proliferating cells and was promoted by TGF-ß in vitro. Despite that this suppressive cytokine is overtly present in the tumor microenvironment, CD8 T cells defective in TGF-ß signal transduction did not show a different profile of inhibitory receptors in a mouse tumor model. Together, our results portray NKG2A as a late inhibitory receptor, stably expressed on CD8 T cells after multiple TCR stimulations and, together with TIM-3 and CD39 marks activated and tumor-specific TILs.