Fig 2: Increased activation and proliferation of CD8+ and CD4+ lymphocytes after therapy. Flow cytometry analysis of CD8+ and CD4+ lymphocytes collected from all patients. Fold change in the proportion of Ki67+ (a) and ICOS+ (b) CD8+ T cells, and Ki67+ (c) and ICOS+ (d) CD4+ T cells between baseline and day 21, comparing patients treated with pembrolizumab (pembro; n = 9), nivolumab (nivo; n = 6), or combination nivolumab/ipilimumab (combo; n = 10). Fold change in the proportion of (e) Ki67+ and (f) ICOS+ CD8+ and CD4+ T cells between baseline and day 21, in responding (n = 20) vs non-responding (n = 4) patients. Proportion of CD8+ (g) and CD4+ (h) lymphocytes expressing Ki67 and ICOS at baseline (BL; n = 20), day 21 (D21; n = 20), week 10 (Wk10; n = 18) and time of discontinuation or progression (Prog.; n = 10), for responding patients only, colour-coded by treatment group (i) Proportion of CD3+ lymphocytes expressing PD-L1 at baseline (BL), day 21 (D21), week 10 (Wk10) and time of discontinuation or progression (Prog.), for responders (n = 20, 20, 18, 10) and non-responders (n = 4, 4, 2, 2), colour-coded by treatment group. p values calculated by Kruskal–Wallis test with multiple comparisons (a–d), Mann–Whitney test (e,f) or mixed effects analysis (g–i); *p = 0.05, ** p = 0.005; error bars, SEM.

Fig 3: SARS-CoV-2 Infection Induces Durable, Functional Spike-Reactive CD4+ T Cells(A) Representative flow cytometry plots of ICOS and CD40L expression on antigen-experienced (non-CD45RA+CCR7+) CD4+ T cells 20 h after incubation of PBMCs from HC and CoV2+ individuals at V1 and V2 with vehicle or SARS-CoV-2 spike.(B) Number of antigen-experienced ICOS+CD40L+CD4+ T cells per 1x106 CD4+ T cells from HC and CoV2+ samples after incubation with vehicle (Veh.) or spike (S) at both time points (right) and calculated number of spike-responsive CD4+ T cells (number after incubation with spike minus number after incubation with vehicle) compared across time points (left).(C) Number of antigen-experienced CXCR5+ICOS+CD40L+CD4+ T cells (cTfh) per 1 × 106 CD4+ T cells from HC and CoV2+ samples after incubation with Veh. or S at both time points (right) and calculated number of spike-responsive cTfh cells (number after incubation with spike minus number after incubation with vehicle) compared across time points (left).(D) Representative flow cytometry plots of sorted CD4+ naive (CD45RA+CCR7+), TCM (CD45RA-CCR7+), or TEM (CD45RA-CCR7-) T cells from HC and CoV2+ PBMCs after 5–6 days of co-culture with SARS-CoV-2 spike-protein-pulsed autologous monocytes and measuring proliferation by CPD dilution.(E) SARS-CoV-2 spike-specific expansion of sorted CD4+ naive T, TCM, and TEM cells from V1 (circles) and V2 (squares) reported as frequency of CXCR3+CPDlo cells after incubation with spike minus frequency after incubation with vehicle.(F) Number of cytokine-producing, antigen-experienced CD69+CD4+ T cells per 1 × 106 CD4+ T cells after incubation with Veh. or S (right) and calculated number of spike-responsive, cytokine-producing CD4+ T cells (number after incubation with spike minus number after incubation with vehicle) (left).(G) Frequency of antigen-experienced CD69+CD4+ T cell subsets, CCR6+/- Teff (CXCR5-), and CCR6+/- cTfh (CXCR5+) producing IL-2, IFN-?, and IL-17A effector cytokines after incubation with spike for 20 h.(H) Representative flow cytometry plots of CD69 and IFN-? expression on antigen-experienced CD8+ T cells from HC and CoV2+ PBMCs at V2 after 20 h of incubation with vehicle or SARS-CoV-2 spike.(I) Number of antigen-experienced IFN-?+CD69+CD8+ T cells per 1 × 106 CD8+ T cells after 20 h of incubation with Veh. or S (right) and calculated number of spike-responsive CD8+ T cells (number after incubation with spike minus number after incubation with vehicle) (left).Statistics for unpaired data determined by 2-tailed Mann-Whitney tests and, for paired data, by 2-tailed Wilcoxon signed-rank tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value < 0.05. Error bars represent mean and SD. Data from 2 experiments per visit. See also Figure S6.