Fig 2: Cellular screening of SARS-CoV-2-specific response.a Memory CD8+ T cells (CD45RO+) from peripheral blood mononuclear cells (PBMC) of COVID-19 convalescents were seeded at 2000 cells/well in multiple wells containing irradiated allogenic PBMC, PHA, IL-2, IL-7, and IL-15 as described in Methods. The individual cultures were expanded to make up the libraries of polyclonally amplified CD8+ T cells. Then the libraries were washed and examined as to their antigen specificity by stimulating with a series of viral protein-expressing aAPC. Once being judged as positive by IFNγlevel (measured by ELISA), they were further expanded with relevant aAPC and cytokines. These libraries (cell lines) were subjected to several downstream analyses including epitope screening, cytokine profiling, and/or cytotoxicity assays, etc. b A representative result of the CD8+ T cell library assay. Libraries from a healthy donor (HC-010) or COVID-19 convalescent (CV-004) were divided into 7 groups, and each group was co-cultured with indicated aAPCs: S (n = 15 for HC-010; n = 47 for CV-004), M (n = 15 for HC-010; n = 47 for CV-004), N (n = 15 for HC-010; n = 47 for CV-004), ORF3a (n = 14 for HC-010; n = 41 for CV-004), or ORF1ab NSP6 (n = 14 for HC-010; n = 53 for CV-004) -expressing aAPCs, influenza virus and cytomegalovirus (Flu/CMV) derived peptides (n = 15 for HC-010; n = 47 for CV-004)-pulsed aAPC or aAPCs (None) (n = 15 for HC-010; n = 47 for CV-004). Each dot represents the IFNγ level of each library. The threshold of positivity was determined by mean + 3 SD of IFNγ level in the group co-cultures with aAPCs (indicated as “None”). c Heatmap representation of results from all participants (healthy controls (HC), convalescents from mild, moderate (mod), and severe (sev) disease) subjected to the library assay. The frequency of positive library in response to aAPCs (None), indicated viral protein-expressing, or influenza virus and cytomegalovirus (Flu/CMV) derived peptides-pulsed aAPC are shown. d Comparison of positive library frequency between healthy donors (HC) (open circles, n = 8) and COVID-19 convalescents (COVID-19) (filled circles, n = 20) in each group. p values were calculated by two-sided Mann–Whitney test. Data represent mean ± SD. **p = 0.0064. e Comparison of positive library frequency in response to SARS-CoV-2-M protein-expressing aAPC among indicated groups (healthy donors (HC) (n = 8), convalescents from mild severity COVID-19 (mild COVID-19) (n = 8), those from moderate severity COVID-19 (moderate COVID-19) (n = 8) and those from severe severity COVID-19 (severe COVID-19) (n = 4)). p values were calculated by two-sided Mann–Whitney test. Data represent mean ± SD. ***p = 0.0002; *p = 0.024.

Fig 3: Correlation between Intratumoral CD8+ T cell subsets and tumor burden in NMIBC. (A) Scatter plots represent the abundance of various subsets of intratumoral CD8+ T cells in NMIBC patients with either Ta or T1 tumor stage. (B,C) Pearson correlation and Spearman correlation between different subsets of intratumoral CD8+ T cells with either (B) tumor size, or (C) tumor multifocality. (A–C) Analysis done with 27 NMIBC patient samples.

Fig 4: Intratumoral PD1+CD38+Tim3+ CD8+ T cells in pre-BCG tumor tissues predict the post-BCG recurrence in NMIBC. (A) Pie chart representation of average frequency of 7 different subsets of intratumoral CD8+ T cells in NMIBC patients with either BCG responsive tumor or BCG non-responsive tumor. (B) Pearson correlation and Spearman correlation analysis between EORTC recurrence-risk and frequency of different intratumoral CD8+ T cell subsets. (C) Scatter plots represents frequency of various intratumoral CD8+ T cell subsets obtained from pre-BCG tumor tissue of NMIBC patients who eventually received BCG and either responded or recurred. Data from 27 NMIBC patients are presented. ** p < 0.01. Mann-Whitney U Test was applied to evaluate statistical significance between the groups.

Fig 5: Heterogeneity of CD8+ T cells in Bladder Tumor tissue. (A,B) Flow cytometry-based analysis of: (A) frequency of CD3+ T cells in peripheral blood and tumor tissues, and (B) frequency of intratumoral CD4+ and CD8+ T cells. Scatter plots represent the cumulative data from 27 NMIBC patient samples. (C–F) cell surface expression of (C) PD1, (D) CD38, (E) Tim3, and (F) Lag3 on CD8+ T cells. Adjacent plots represent the cumulative data from 27 NMIBC patient samples. (G,H) Scatter plots represent the percentage of CD8+ T cells positive for intracellular cytokine (G) IFNγ, and (H) GzmB. (I) Gating strategy based on expression of different inhibitory immune checkpoint molecules on CD8+ T cells. (J) Heat map of CD8+ T cell subsets co-expressing various immune checkpoint molecules. **** p < 0.0001. The Mann–Whitney U Test was applied to evaluate statistical significance between the groups.