Daan Pieren

139 Regulatory KIR+RA+ CD8+ T cells accumulate with age T NAIVE T CM T EM T EMRA KIR + RA + NKG2A + RA + 0 20 40 60 80 100 % CD122 + cells of subset *** *** *** *** *** T NAIVE T CM T EM T EMRA KIR + RA + NKG2A + RA + 0 20 40 60 80 100 % TIGIT + Helios + cells of subset *** ** ****** *** T NAIVE T CM T EM T EMRA KIR + RA + NKG2A + RA + 0 20 40 60 80 100 % Helios + cells of subset ************ *** A B C D CellTrace (BV421) Responder only (10,000cells) 1:4 (Suppressor: Responder) (2,000+ 8,000cells) % of total Responder only (8,000cells) 81.0% 66.2% 80.1% E 1:1 1:2 1:4 1:8 1:16 0 20 40 60 80 Suppressor : Effector ratio % Suppression Figure 5. KIR + RA + T cells show a distinct regulatory CD8 + T-cell phenotype and suppress proliferation of KIR - NKG2A - CD8 + T cells. Frequency of ( A ) Helios + , ( B ) CD122 + , and ( C ) TIGIT + Helios + cells within the indicated CD8 + T-cell subsets. For suppression assays, suppressor cells (KIR + RA + T cells) and responder cells (KIR - NKG2A - T cells) were sorted, added in different dose-range ratios ranging from 1:1 to 1:16 (suppressor:responder), and cultured with stimulatory anti-CD3 for three days. ( D ) Representative flow cytometry histograms show the frequency of proliferated KIR - NKG2A - CD8 + T cells in responder only conditions (10,000 and 8,000 cells/well) and in a 1:4 suppressor:responder ratio. ( E ) Percentage of suppression derived from suppression assays performed in n=3-6 individual assays per indicated suppressor:responder ratio. Statistical significance of data presented in bar graphs (means ± s.d.) was determined using Friedman test (with Dunn’s post-test) ( A-C ). (** p <0.01, *** p <0.001). Activation of KIR + RA + T cells correlates with prolonged influenza A-induced symptoms We reasoned that accumulation of suppressive KIR + RA + ­ T cells that become activated during respiratory viral infectious disease may suppress protective CD8 + T-cell responses and thereby prolong virus-induced respiratory disease in older adults. Therefore, we monitored the occurrence and duration of ILI- associated symptoms (cough, fever, myalgia, nasal congestion, sore throat, difficulty breathing, and headache) fromwhich we calculated an overall symptom score in the influenza A-infected older individuals ( Supplementary Figure 7D ). The frequency of CD69 + KIR + RA + T cells during ILI positively correlated with the overall symptom score, hinting towards a link between the average duration of symptoms and the presence of activated KIR + RA + T cells ( Figure 6K ). Moreover, the presence of these activated cells positively correlated with the duration of 5