Fig 2: Magnitude, dynamics, and cross-reactivity of CD8+ epitope-specific responses after diverse SARS-CoV-2 exposures.a. Antigen specificity of each T cell inferred from dextramer-barcode UMI counts. Representative distribution of the number of UMIs in cells called dextramer-positive (pink) and dextramer-negative (yellow). b. T cells within a clone have largely consistent specificity assignments, except T cells that cross-react with common cold coronavirus epitopes (B15_NQK_A/B15_NQK_Q pair). Each bar shows a fraction of cells of a given clonotype attributed to different dextramers. The 43 most abundant clones (more than 20 cells) are shown. c. The correlation between the number of UMIs for B15_NQK_Q (SARS-CoV-2) and B15_NQK_A (OC43 and HKU1) dextramers (Spearman ?=0.8, p<0.001). d. Cross-reactivity between HLA-B*15:01-NQK epitope variants confirmed in vitro. Jurkat cell line expressing aßTCR identified from scTCRseq data binds pMHC multimers loaded with both SARS-CoV-2 and CCCoV variants of the epitope. e. The magnitude of epitope-specific CD8+ T cell responses. Each point depicts an estimated frequency of epitope-specific T cells in a sample. Estimated frequency was calculated as a fraction of dextramer-specific T cells in scRNAseq results multiplied by bulk frequency of dextramer-stained CD8+ cells of all CD8+ cells measured by flow cytometry. Central line on boxplot shows the median. Epitopes from spike protein are in bold font. f. Composition of HLA-A*01-restricted T cell response in HLA-A*01 positive donors. Increasing proportion of spike-targeting T cells (pink) is observed after vaccination of infected individuals. g. Boosting of spike-specific epitope fraction after vaccination (donor R6). h. Previously infected individuals have a higher proportion of spike-specific T cells after vaccination than before vaccination (p=0.025, one-sided Wilcoxon signed-rank test). Spike T cell proportion (shown on a log10-scale) was calculated as a fraction of spike-specific T cells out of all CD8+ epitope-specific T cells of a donor in scRNAseq data. Central line on the violin shows the median.

Fig 3: F5111 IC expands functional Tregs without compromising infection immunity(A–C) C57BL/6 mice (n = 3 per group) were treated daily for 4 days (intraperitoneally) with PBS or 8.2 μg F5111 IC (1.5 μg IL-2 equivalence) and spleens were harvested at the indicated times.(A) Representative flow plots showing Treg percentage and IL-2Rα versus FOXP3 levels in CD4+ T cells at the indicated times. (B) Percent Tregs within CD4+ T cells at the indicated times. (C) Treg:CD8+ T (left), Treg:TConv (middle), and Treg:NK (right) at the indicated times. Dashed lines show baseline values for the PBS-treated cohort harvested 1 day after the last dose.(D) Serum half-life of F5111 IC in C57BL/6 mice (n = 5) treated retro-orbitally with 2 mg/kg F5111 IC (~0.4 mg/kg IL-2 equivalence).(E) C57BL/6 CD45.1 RFP-FOXP3 mice were treated daily for 4 days with PBS (n = 3) or 6.2 μg F5111 IC (1.125 μg IL-2 equivalence, n = 2). Percent proliferation suppression of TConv cells from untreated C57BL/6 CD45.2 mice (n = 5) by titrating ratios of Tregs from spleens of treated mice is shown.(F) C57BL/6 mice (n = 5 per group) were dosed on days 0, 2, and 4 with PBS or 8.2 μg (1.5 μg IL-2 equivalence) control IC or F5111 IC. Spleens were harvested on day 6. Number of cTregs (CD4+FOXP3+IL-2RαHighBCL-2High) and effector (eTregs) (CD4+FOXP3+IL-2RαLowBCL-2Low) are shown.(G–I) C57BL/6 mice were administered 25 cysts of T. gondii on day 0. The control group was not given cysts. Starting on day 1, mice were treated daily for 5 days with PBS (control, n = 5; PBS, n = 4) or 8.2 μg (1.5 μg IL-2 equivalence) control IC (n = 5) or F5111 IC (n = 5). Mice were sacrificed on day 10. (G) Mouse weight. (H) Parasite burden. (I) Treg:T-bet+Tetramer (Tet)+ CD8+ T cell ratio in the spleen. (G–I) Mean ± SEM. All other data are mean ± SD. Statistical significance in percent initial weight on day 10 compared with F5111 IC is shown. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. See also Figure S5.

Fig 4: Relationships between SARS-CoV-2-specific T cell responses, persistent viral shedding, and PASC(A) The percentage of SARS-CoV-2-specific CD4+ and CD8+ T cells at T1 as measured by the AIM and ICS assays in study participants with detectable SARS-CoV-2 RNA in saliva at T1.(B) SARS-CoV-2-specific T cell frequency at T1 in study participants with any COVID-19-related symptoms at T1.(C and D) SARS-CoV-2-specific T cell frequency at T1 (C) and T4 (D) in study participants with post-acute sequelae of SARS-CoV-2 infection (PASC; persistent symptoms) at T4.(E) Longitudinal frequency of N-specific IFNγ+ and IFNγ−/CD107a+ memory (mem) CD8+ T cells in those with PASC. For (A)–(D), dots represent individual participant values, and bars and lines in cross-sectional data represent median values and interquartile ranges: ∗p < 0.05 by non-parametric Kruskal-Wallis tests (T1 N and S AIM data available for 67 participants, ICS IFNγ N CD8 and CD4 n = 61, T1 ICS IFNγ S CD8 and CD4 n = 60, T4 IFNγ N and S CD4 and CD8 n = 46.) For (E), solid line and shaded region represent the median model prediction (including individual effects) and 50% prediction interval, respectively (n = 61 participants: 32 with PASC, 29 without PASC). T mem, T memory cells.

Fig 5: Long-term durability of SARS-CoV-2-specific CD4 and CD8 cell responsesGating strategy for identifying SARS-CoV-2-specific memory T cell responses in the activation-induced marker (AIM) (A) and intracellular cytokine staining (ICS) (B) assays. Percentage of AIM+ Nucleocapsid (N)-specific CD4+ (C) and CD8+ (D) T cell responses over time (Spike [S]-specific responses were similar and are shown in Figure S3; n = 68 participants). Percentage of IFNγ+ Nucleocapsid (N)- or S-specific CD4 (E and F) and CD8 (G and H) T cell responses over time (n = 64 participants). Points and connecting lines represent raw data for each individual. Solid line and shaded region represent the median model prediction and 95% prediction interval, respectively, from linear mixed effects modeling including individual effects. Dashed lines represent assay limits of detection. T mem, T memory cells (i.e., excluding naive CCR7+CD45RA+ T cells). Longitudinal responses as measured by IFNγ+/TNF-α dual expression and IFNγ+/CD107a expressing lymphocytes are show in Figure S3.