Daan Pieren

123 Regulatory KIR+RA+ CD8+ T cells accumulate with age after the initial report. Respiratory viruses included in our selection were real time PCR-based Multiplex Ligation-dependent Probe Amplification (MPLA)- confirmed cases of Influenza A virus (n=15), Influenza B virus (n=5), Respiratory Syncytial Virus (RSV) (n=2), Human Metapneumovirus (hMPV) (n=4), seasonal Coronavirus (n=5), or Rhinovirus (n=5) infection (RespiFinder® Smart 22 kit, Pathofinder) ( Supplementary Table 1 ). Of these participants, the self-reported occurrence and duration of ILI-associated symptoms were used to calculate an overall symptom score by totaling the duration of all symptoms per individual divided by the number of symptoms of the individual. Participants who were asymptomatic and laboratory-confirmed respiratory infection negative were sampled and used as healthy controls in this ILI-cohort. Healthy individuals Blood samples obtained from healthy (asymptomatic) individuals (n=50, 21-82 years) investigated in the current study were either derived from the aforementioned ILI-cohort, or from healthy blood donors (Sanquin Blood Supply Foundation) or from healthy participants of a trial performed at the RIVM (Netherlands Trial Register NTR2070) [29]. Overall, the samples investigated in this study were relatively evenly distributed over age and sex ( Supplementary Table 2 ). All healthy and symptomatic participants from the aforementioned cohorts that were included in this study were tested for cytomegalovirus (CMV) antibodies by an in-house Multiplex Immunoassay [30]. Only participants who tested negative were included in the study, to prevent bias towards CMV- induced T-cell alterations. SARS-CoV-2 infected adults with COVID-19 With the recent COVID-19 pandemic caused by the novel severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), we expanded our analyses of KIR + RA + T cells by investigating the presence and phenotype of these cells in the blood of adults with COVID-19. Whole blood of PCR-confirmed SARS- CoV-2 infected participants (32-52 years of age, n=9) and that of asymptomatic household members who tested negative for SARS-CoV2 (indicated as healthy controls) (16-51 years of age, n=9) was obtained as part of the First Few X (FFX) cases of SARS-CoV-2 infection in the Netherlands ( Supplementary Table 3 ) [31]. Blood samples were drawn within the first two weeks after COVID-19-like symptoms were reported (acute phase of infection), as well as two and four weeks thereafter. The study was approved by the Utrecht ethical committee (METC Utrecht; NL13529.041.06). 5