Daan Pieren

133 Regulatory KIR+RA+ CD8+ T cells accumulate with age and KIR receptors. ( E ) Comparison of expression of selected genes associated with cellular regulation, co-inhibition/exhaustion, senescence, and cytotoxicity between KIR + RA + T cells and NKG2A + RA + T cells. For each of these analyses, an absolute log2 Fold Change > 1.0; -Log10 p value of > 1.5 corresponding to p <0.05 at a false discovery rate (FDR) of 0.10 was used. TIGIT Hi CD226 Low KIR + RA + T cells contribute to age-related TIGIT expression in CD8 + T cells The co-inhibitory molecule TIGIT was one of the molecules that we identified in the transcriptome of KIR + RA + T cells and has previously been described as a marker for regulation [39,40], exhaustion [41,42], and has recently been linked to aging [20]. We therefore questioned whether the accumulation of KIR + RA + T cells may be linked to the aging-related increase in TIGIT + CD8 + T cells. Consistent with previous findings [20], the frequency of TIGIT + cells of total CD8 + T cells increased with age ( Figure 3A,B ). Strikingly, the proportion of KIR + RA + T cells showed a strong positive correlation with the frequency of TIGIT + CD8 + T cells ( Figure 3C ). Among all memory T-cell subsets in our study, this correlation was strongest for the KIR + RA + T cells ( Supplementary Figure 4A ). KIR + RA + T cells indeed showed the highest frequency of TIGIT + cells and median expression per cell (MFI) compared to all other CD8 + T cell subsets ( Figure 3D, Supplementary Figure 4B ). These findings show that KIR + RA + T cells contribute to the accumulation of TIGIT + CD8 + T cells with age. The co-stimulatory receptor CD226 competes with TIGIT for interaction with their shared ligands [42]. The ratio between CD226 and TIGIT on a CD8 + T cell determines the threshold for its proliferative and activation potential [41-44]. Combining analyses of TIGIT and CD226 expression resulted in a separation of the six T-cell subsets ( Figure 3E ). KIR + RA + T cells expressed the highest frequency of TIGIT + cells and the lowest frequency of CD226 + cells, and clearly separated from NKG2A + RA + T cells based on these two markers ( Figure 3F ). The ratio between expression of TIGIT and CD266 by KIR + RA + T cells showed that the balance between these markers is strongly skewed towards TIGIT in this subset ( Figure 3G ). Thus, KIR + RA + T cells are characterized by TIGIT Hi CD226 Low expression and contribute to age-related TIGIT expression by CD8 + T cells. 5