Daan Pieren

135 Regulatory KIR+RA+ CD8+ T cells accumulate with age KIR + RA + T cells are responsive to stimulation but have low proliferative capacity We next characterized the functionality of KIR + RA + T cells. Upon non- specific stimulation with PMA/ionomycin, KIR + RA + T cells highly expressed CD107a compared to the other T-cell subsets, suggesting enhanced cellular degranulation ( Figure 4A ). The frequency of KIR + RA + T cells producing IL-2 was similar to levels found in T EMRA and NKG2A + RA + T cells ( Figure 4B ). Frequencies of KIR + RA + T cells producing IFN- γ and TNF- α or combination of these cytokines, did not significantly differ from those found in the other subsets ( Figure 4C,D , Supplementary Figure 5A ). These findings show that the KIR + RA + T-cell subset is not hampered in production of cytokines. We further assessed responsiveness of the KIR + RA + T cells by assessing their capacity to upregulate the activation marker CD69 and proliferate in response to anti-CD3 stimulation of PBMCs for three days. CD8 + T cells expressing KIR showed the highest frequency of CD69 + cells compared to cells that expressed NKG2A or cells that did not express KIR or NKG2A ( Figure 4E ). However, sorted KIR + RA + T cells did not show increased frequencies of CD69 + cells compared to sorted KIR - NKG2A - and NKG2A + RA + T cells ( Supplementary Figure 5B ). This suggests that KIR + RA + T cells may depend on other cells and/or cell-derived cytokines for their activation. Furthermore, we observed that the proliferative capacity of the overall CD8 + T-cell population declined with age ( Figure 4F ). When we analyzed correlations of CD8 + T-cell proliferation with proportions of the different T-cell subsets ( Figure 4G and Supplementary Figure 5C ), we found that this overall decline of proliferation strongly correlated with increased proportions of KIR + RA + T cells present ex vivo ( Figure 4G ). This negative correlation may be explained by non-proliferative KIR + RA + T cells, despite their higher activation status. We therefore assessed proliferation in KIR + NKG2A - and the KIR - T-cell populations after anti-CD3-mediated stimulation of PBMCs. CD8 + T cells expressing KIR showed the lowest proliferation, compared to the populations that did not express KIR( Figure 4H ), suggesting that the correlation between decreased CD8 + T-cell proliferation and increased proportion of KIR + RA + T cells can be explained by low proliferative capacity of KIR + RA + T cells. Indeed, the proportion of T cells that were KIR + did not expand whereas the proportion of cells that expressed NKG2A did ( Figure 4I ). Together, these findings show that despite high activation potential of KIR + RA + T cells, KIR + RA + T cells have low proliferative capacity. Since the proportion of KIR + RA + T cells among the total CD8 + T-cell pool accumulates with age, their lack of proliferative capacity may contribute to the decreased proliferation of the total CD8 + T-cell pool at older age. 5