Daan Pieren

141 Regulatory KIR+RA+ CD8+ T cells accumulate with age Blood samples from older adults suffering from a common respiratory virus infection (62-83 years of age, n=36) were analyzed at the acute phase (0) (within two days of fever onset), and during follow-up at +2 and +8 weeks. Healthy asymptomatic individuals (61-82 years of age, n=8) from the same cohort were used as control samples. ( A ) Frequency of KIR + RA + T cells within the CD8 + T-cell population and ( B ) the frequency of CD69 + cells in KIR + RA + T cells at the three time points in influenza-A infected older adults (n=15) and healthy asymptomatic controls (n=8). ( C ) The frequency of CD69 + cells in six different CD8 + T-cell subsets. The frequency of CD69 + in KIR + RA + T cells in the blood of older adults infected with ( D ) a seasonal coronavirus (n=5), ( E ) influenza B virus (n=5), ( F ) Rhinovirus (n=5), ( G ) human metapneumovirus (hMPV) (n=4), or ( H ) respiratory syncytial virus (RSV) (n=2). ( I ) The number of CD8 + T cells specific for the immunodominant Influenza A matrix protein epitope GILGFVFTL detected by flow cytometry over time in the indicated CD8 + T-cell subsets of HLA-A*02 + influenza-A infected individuals (n=4). ( J ) The frequency of CD69 + cells in KIR + RA + T cells in the blood of adults infected with SARS-CoV-2 and suffering from COVID-19 (n=9). Relationship between the frequency of CD69 + cells within the KIR + RA + T-cell subset and ( K ) the calculated symptom score, ( L ) duration of cough, and ( M ) duration of fever in older adults suffering from influenza A virus infection (n=15). Correlations ( r values) were assessed by Spearman test. Statistical significance of data presented in bar graphs (mean ± s.d.) was determined using row-matched one-way ANOVA (with Geisser-Greenhouse correction and Dunnett’s post-test) for the difference between the time points and Mann-Whitney U test was used to determine the difference between infected and asymptomatic healthy individuals ( A, B, D-G, J ). Row-matched one-way ANOVA (with Geisser-Greenhouse correction and Dunnett’s post-test) was used in ( C ). (* p <0.05, ** p <0.01, *** p <0.001, ns=not significant, or the exact p -value is shown.) DISCUSSION Here we identify a novel human CD8 + T-cell subset with regulatory properties, which we refer to as KIR + RA + T cells based on their unique expression of KIR and CD45RA. We show that KIR + RA + T cells accumulate during healthy aging and may contribute to diminished CD8 + T-cell responses observed in older adults. Furthermore, our findings show that KIR + RA + T cells become activated in older adults suffering from viral respiratory diseases and may negatively affect resolution of disease. Human CD8 + CD45RA + T cells that express KIR and/or NKG2A have been described as “virtual memory T cells” and these cells have been reported to increase with age [21-23]. We now show that the virtual memory T-cell subset comprises two distinct cell subsets: KIR + RA + T cells that accumulate with age and NKG2A + RA + T cells that decline with age. Our data show that KIR + RA + T cells can be considered a distinct CD8 + T-cell subset that increases with aging. Moreover, KIR + RA + T cells constitute 30% of the T-cell subset that has been designated T EMRA cells [48-50]. Since KIR + RA + T cells have remained hidden among the T EMRA subset, our findings indicate that part of previously reported age-related increase of the conventional T EMRA subset and expression of TIGIT 5