Fig 2: High Enrichment of CD39 Expression on Tumor-Infiltrating MAIT Cells(A) Left: CD39 expression shown by UMAP plot. Center: representative staining of CD39 expression on MAIT cells from PBMC, adjacent tissue, and tumor of CRC, gated on CD45+ live, DNA+, CD14– CD16– CD3+ T cells. Right: frequencies of CD39+ MAIT cells in different compartments. PBMC = 10, colon = 19, tumor = 24. Data are mean with SD from at least 7 experiments. Mann-Whitney U test.(B). Expression intensities of selected markers on CD39+ and CD39– MAIT cells in 4 different tumors (left). The numbers are patient IDs. Also shown is analysis of selected markers expression between the CD39+ and CD39– subset; n = 22 (right). Data are from at least 7 experiments. Two-tailed paired t test.(C) CD39 co-expression with PD-1 or CTLA-4 gated on total MAIT cells from one representative tumor sample.(D) Representative staining of CD39 and Ki-67 expression on tumor-infiltrating MAIT cells (left) and Ki67 expression on CD39+ and CD39– tumor-infiltrating MAIT cells (right); n = 18. Data are from at least 3 experiments. Two-tailed paired t test.(E) Representative example of IFN?, TNF-a, IL-17, and CD39 expression on tumor-infiltrating MAIT cells upon 4 h of PMA/ionomycin stimulation (left) and polyfunctionality profile of CD39+ and CD39– tumor-infiltrating MAIT cells (right); n = 8. Data are mean with SD from 2 experiments. Two-tailed paired t test.(F) Flow cytometry data of CD39 and IFN? expression after different stimulation conditions, assessed on CD3+ tet-MR1+ CD161+ cells; n = 12. Data are mean with SD from at least 3 experiments. Two-tailed paired t test.See also Figure S3.

Fig 3: Functional characterization following stimulation to peptide cocktails leads to multiclonal expansion of Ag-specific CD4+ and CD8+ T cells possessing cytolytic capabilities at the end of the culture period (D19).Dot-plot showing expression of perforin and granzyme B on (A) CD8+ T cells in HD1 (top panel) and MM1 (bottom panel) following restimulation with each peptide from CT3. T cell receptor (TCR) Vß repertoire based on flow analysis of (B) CD3+CD4+ and (C) CD3+CD8+ of HD1 or MM1 harvested on day 19. Representative data are shown and statistical analysis indicated no significant differences among the ten samples analyzed (Student’s t-test).

Fig 4: Stimulation with peptide cocktails enriches T cells equivalently regardless of the disease status.(A) Depiction of percentages of CD4+ (white) and CD8+ (grey) T cells at the end of culture period for 5 HDs (left panel) and 5 MM patients (right panel). (B) Pie charts showing percentages of immune cell subsets on D0 or D19 at end of culture period of PBMCs of HD (left panel) and MM patient (right panel) with MUC1 cocktail and cocktails 1, 3, and 4. CD19 (orange), CD56 (green), CD33 (blue) and CD3 (purple) are shown. CD3+ population on D19 was always greater than 85% positive. (C) CD3+ T cells were further analyzed for CD4+ (blue), CD8+(orange) and CD56+ (green) for HD (left panel) and MM patient (right panel). Percents of CD4+ and CD8+ T cells depended upon the particular cocktail used for primary stimulation and the HLA genotype of the individual. Data were similar for all ten samples. No statistically significant differences were observed (Student’s t-test). Representative data are shown.

Fig 5: Mild COVID-19 Induces a Sustained Enrichment of RBD-Specific IgG+ Memory B Cells(A) Representative gating of live CD3–CD14–CD16– cells for SARS-CoV-2 RBD-specific cells (RBD tetramer+decoy tetramer–) from CoV2+ and HC PBMCs at V1 and V2.(B) Number of RBD-specific B cells (RBD tetramer+decoy tetramer–CD20+) per 1 × 106 PBMCs.(C) Representative flow cytometry plots and number of RBD-specific PBs (RBD tetramer+decoy tetramer-CD20-CD138hi) (na = could not be calculated because all values 0).(D) Representative gating of RBD-specific B cells for naive B cells (CD21+CD27-) and MBCs (CD21+CD27+/CD21–CD27+/CD21–CD27– populations outlined in green).(E) Proportion of RBD-specific B cells that are naive (CD21+CD27-), classical MBCs (CD21+CD27+), or activated MBCs (CD21-CD27+/-), statistics for the proportion that are MBCs.(F) Number of RBD-specific MBCs (classical and activated).(G) Representative gating of RBD-specific MBCs for BCR isotype (IgD, IgM, IgA, and IgG) expression.(H) Proportion of RBD-specific MBCs expressing the BCR isotypes IgD, IgM, IgA, and IgG. Statistics are for the proportion that are IgG+.(I) Number of RBD-specific IgG+ MBCs.(J) Representative gating of RBD-specific MBCs for T-bet expression and number of RBD-specific T-bet+ MBCs.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 < 0.04. Error bars represent mean and SD. Data from 2 experiments per visit. See also Figure S5.