Fig 2: SARS-CoV-2 Spike-Specific CD4+ and CD8+ T Cells Are Diverse and Not Phenotypically Identical to Their CMV-Specific Counterparts(A and B) Shown are t-SNE plots of CyTOF datasets reflecting CD4+ (A) or CD8+ (B) T cells from three representative COVID-19 convalescent donors who had also sustained a previous CMV infection. Cells shown in gray correspond to CD4+ or CD8+ T cells from specimens stimulated with anti-CD49d/CD28 in the absence of any peptides. The top pairs of plots show SARS-CoV-2 spike-specific (red) or CMV pp65-specific (blue) cells as individual dots, with some regions concentrated in antigen-specific cells indicated. The bottom pairs of plots show the same data but with antigen-specific cells shown as contours instead of dots to better visualize regions with the highest densities of antigen-specific cells. Purple ovals outline examples of regions harboring CMV-specific but not SARS-CoV-2-specific cells.See also Figures S3–S6.

Fig 3: Antigen-Specific CD4+ and CD8+ T Cells against SARS-CoV-2 Spike Secrete IFNγ(A and B) Shown on the left are pseudocolor plots of CyTOF datasets reflecting the percentage of CD4+ (A) or CD8+ (B) T cells producing IFNγ in response to the indicated treatment condition for one representative uninfected (COVID−) and recovered convalescent (COVID+) donor. Numbers correspond to the percentage of cells within the gates. The baseline condition corresponds to cells phenotyped by CyTOF immediately following isolation of PBMCs from freshly drawn blood, whereas for all other treatment conditions, cells were cultured for 6 h prior to phenotyping by CyTOF. PMA/ionomycin treatment was used as a positive control. Anti-CD49d/CD28 was used to provide co-stimulation during peptide treatment. Shown on the right are cumulative data from three uninfected individuals and nine recovered convalescent individuals (Table S1). Results are gated on live singlet CD4+ or CD8+ T cells. ∗∗p < 0.01, ∗∗∗∗p < 0.0001, as assessed using Student’s unpaired t test.See also Figures S1 and S2.

Fig 4: SARS-CoV-2-specific T cells elicited by vaccination recognize variants, and in a manner that differs among individuals with prior COVID-19.(A) Identification of vaccine-elicited spike-specific T cells. PBMCs before vaccination (Pre-Vac) or 2 weeks after each dose of vaccination were stimulated with spike peptides and assessed by CyTOF 6 hr later for the presence of spike-specific (IFNγ-producing) CD4+ (left) or CD8+ (right) T cells. The ‘no peptide’ conditions served as negative controls. Shown are longitudinal data from an infection-naïve (PID4101, top) and convalescent (PID4112, bottom) individual. (B) Quantification of the spike-specific CD4+ (left) and CD8+ (right) T cells recognizing the ancestral (squares), B.1.1.7 (triangles), and B.1.351 (circles) spike peptides ininfection-naïve (top) and a convalescent (bottom) individuals before and after vaccination. Note the similar frequencies of T cells responding to all three spike proteins in each donor, the clear boosting of spike-specific CD4+ T cell frequencies in infection-naïve but not convalescent individuals, and the overall higher proportion of responding CD4+ than CD8+ T cells. The dotted line corresponds to the magnitude of the maximal pre-vaccination response in infection-naïve individuals and is considered as background. The y-axes are fitted based upon the maximal post-vaccination response values for each patient group and T cell subset. The p-values shown (**p < 0.01, ***p < 0.001) were calculated by student’s t-test. (C) As expected, nucleocapsid-specific T cell responses are generally low over the course of vaccination, with the exception of convalescent donor PID4112. Shown are the frequencies of nucleocapsid-specific CD4+ (left) and CD8+ (right) T cells, as measured by IFNγ production upon stimulation with ancestral nucleocapsid peptides, in infection-naïve (top) and convalescent (bottom) individuals. The dotted line corresponds to the magnitude of the maximal pre-vaccination response in infection-naïve individuals and is considered as the background signal. Y-axes are labeled to match the corresponding y-axes for spike-specific T cell responses in panel B. (D) The CD4+ T cell response is boosted by the second vaccine dose to a greater extent in infection-naïve than convalescents individuals. Shown are the frequencies of spike-specific CD4+ (left) and CD8+ (right) T cells stimulated by the three spike proteins (squares: ancestral; triangles: B.1.1.7; circles: B.1.351) among the infection-naïve (aqua) and convalescent (coral) donors, after removal of outlier PID4112. ***p < 0.001 comparing the infection-naïve vs. convalescent post-dose two specimens, were calculated using student’s t-test.

Fig 5: SARS-CoV-2-Specific CD8+ T Cells Are Predominantly a Less Differentiated Subset of Temra Cells and Include Long-Lived CD127-Expressing Cells(A) SARS-CoV2-specific CD8+ T cells include CD45RA+CD45RO− and CD45RA−CD45RO+ cells. The phenotypes of total (gray), SARS-CoV-2-specific (red), and CMV-specific (blue) CD8+ T cells are shown as dot plots for three representative donors.(B) SARS-CoV-2-specific CD8+ T cells are predominantly Temra cells. The proportions of SARS-CoV-2-specific and CMV-specific CD8+ T cells belonging to each subset are depicted as pie graphs. Numbers correspond to the percentages of cells belonging to the Temra cell subset. Shown on the right are cumulative data for SARS-CoV-2-specific CD8+ T cells from all 9 convalescent individuals analyzed in this study. ∗p < 0.05, ∗*p < 0.01, as assessed using Student’s paired t test.(C) Compared with CMV-specific and total CD8+ Temra cells, SARS-CoV-2-specific CD8+ Temra cells express high levels of CD27 and CD28. Top: Temra cells were defined as CD45RA+CD45RO− cells expressing low levels of CCR7. Bottom: CD8+ Temra cells were further assessed for expression levels of CD27 and CD28. Numbers correspond to the percentage of total (gray), SARS-CoV-2-specific (red), and CMV-specific (blue) cells within the gate.(D) A subset of SARS-CoV-2-specific CD8+ T cells express CD127. The indicated cell populations were examined for expression levels of CD57 and CD127.See also Figure S7.