Daan Pieren

129 Regulatory KIR+RA+ CD8+ T cells accumulate with age RESULTS KIR + RA + T cells accumulate with age, whereas NKG2A + RA + T cells decline with age Virtual memory cells identified as CD45RA + CD8 + T cells expressing KIR and/ or NKG2A accumulate with age in PBMCs of healthy individuals (n=50) ( Figure 1A ), which is in line with previous reports [21-23]. However, the accumulation of CD45RA + CD8 + T cells positive for KIR or NKG2A differed between young and aged individuals ( Figure 1B, Supplementary Figure 1A for gating strategy). We observed an increase in the proportion of KIR + NKG2A - cells with age ( Figure 1C ), whereas the proportion of KIR - NKG2A + cells decreased with age ( Figure 1D ). CD45RA + KIR + NKG2A - cells were abundantly present, on average 30%, within the classically defined terminally differentiated effector cell subset (T EMRA ) defined by CD27 - and CD45RA + ( Figure 1E ). Based on these findings we hypothesize that the so-called “virtual memory cell” subset comprises two different subsets; KIR + NKG2A - CD45RA + T cells and KIR - NKG2A + CD45RA + T cells, which we will conveniently address as KIR + RA + T cells and NKG2A + RA + T cells, respectively. By regarding KIR + RA + T cells and NKG2A + RA + T cells as separate CD8 + T-cell subsets, the CD8 + T-cell population can be divided into six different subsets ( Figure 1F, Supplementary Figure 1A ): naive T cells (T NAIVE , CD45RA + CD27 + KIR - NKG2A - ), central memory T cells (T CM , CD45RA - CD27 + ), effector memory T cells (T EM , CD45RA - CD27 - ), T EMRA cells (CD45RA + CD27 - KIR - NKG2A - ), KIR + RA + T cells (CD45RA + KIR + NKG2A - ), and NKG2A + RA + T cells (CD45RA + KIR - NKG2A + ). Of these subsets, the proportions of T NAIVE and NKG2A + RA + T cells decline with age, the proportion of T CM remains stable, whereas the T EM , T EMRA , and KIR + RA + T cells increase with age ( Figure 1G, Supplementary Figure 1B ). Thus, different proportions of KIR + RA + and NKG2A + RA + T cells with progressing age may indicate that these are distinct cell subsets. 5