Figure 3.
Figure 3. Sequential exposure to IL-15 or antigen and TGFβ induces CD8 “residency” in vitro. (a and b) CD69 expression (%; n = 27; a) and CXCR6 (%; n = 17) or CXCR3 (%; n = 12; b) expressing peripheral CD8 T cells after 3 d culture in the presence of rhIL-15 at indicated doses (red; n = 11). (c and d) Coexpression of CD69 and CD103 on peripheral CD8 T cells (“induced” CD69+CD103+ CD8 T cells; n = 11) after either rhIL-15 alone (red) or rhTGFβ alone (blue; c) at the indicated doses for 6 d, two-step sequential cytokine exposure with 3 d rhIL-15 followed by a further 3 d rhTGFβ (red) at concentrations of both cytokines indicated or vice versa (blue; n = 20; d) or two-step sequential cytokine exposure with 3 d rhIL-15 followed by 3 d rhIL-33 at concentrations of both cytokines indicated (n = 11; e). (f) Frequency of induced CD69+CD103+ CD8 T cells after two-step culture with 3 d 0.25 µg/ml immobilized anti-CD3 followed by 3 d ± addition of rhTGFβ at concentrations indicted (n = 12). (g) Expression of CXCR6 (%; n = 12) and CXCR3 (%; n = 12) on peripheral CD8 T cells after 6 d 0.25 µg/ml immobilized anti-CD3 (n = 10). (h) Frequency of induced CD69+CD103+ within the global or peptide-specific (HBV or CEF) CD8 T cells after culture with either 1 µg/ml of overlapping peptides spanning the core region of HBV genotype D or 0.5 µg/ml CEF (peptide pool against CEF) for 7 d (n = 12). (i) Expression of CD14 (%; n = 17), CD39 (%; n = 20), PD-1 (MFI; n = 17), Blimp-1 (MFI; n = 12), Eomes (MFI; n = 6), and Notch (MFI; n = 6) on CD69−CD103− CD8+ (white) and CD69+CD103+ (“induced resident;” black) CD8 T cells after sequential exposure to 3 d of 50 ng/ml rhIL-15 followed by 3 d of 50 ng/ml rhTGFβ. All figures show summary data from at least four independent experiments. Error bars indicate means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; p-values were determined via a Kruskal-Wallis test (ANOVA) with a Dunn’s post hoc test for pairwise multiple comparisons (a, c, d, e, and f) or a Wilcoxon Signed-rank t test (b, g, h, and i).

Sequential exposure to IL-15 or antigen and TGFβ induces CD8 “residency” in vitro. (a and b) CD69 expression (%; n = 27; a) and CXCR6 (%; n = 17) or CXCR3 (%; n = 12; b) expressing peripheral CD8 T cells after 3 d culture in the presence of rhIL-15 at indicated doses (red; n = 11). (c and d) Coexpression of CD69 and CD103 on peripheral CD8 T cells (“induced” CD69+CD103+ CD8 T cells; n = 11) after either rhIL-15 alone (red) or rhTGFβ alone (blue; c) at the indicated doses for 6 d, two-step sequential cytokine exposure with 3 d rhIL-15 followed by a further 3 d rhTGFβ (red) at concentrations of both cytokines indicated or vice versa (blue; n = 20; d) or two-step sequential cytokine exposure with 3 d rhIL-15 followed by 3 d rhIL-33 at concentrations of both cytokines indicated (n = 11; e). (f) Frequency of induced CD69+CD103+ CD8 T cells after two-step culture with 3 d 0.25 µg/ml immobilized anti-CD3 followed by 3 d ± addition of rhTGFβ at concentrations indicted (n = 12). (g) Expression of CXCR6 (%; n = 12) and CXCR3 (%; n = 12) on peripheral CD8 T cells after 6 d 0.25 µg/ml immobilized anti-CD3 (n = 10). (h) Frequency of induced CD69+CD103+ within the global or peptide-specific (HBV or CEF) CD8 T cells after culture with either 1 µg/ml of overlapping peptides spanning the core region of HBV genotype D or 0.5 µg/ml CEF (peptide pool against CEF) for 7 d (n = 12). (i) Expression of CD14 (%; n = 17), CD39 (%; n = 20), PD-1 (MFI; n = 17), Blimp-1 (MFI; n = 12), Eomes (MFI; n = 6), and Notch (MFI; n = 6) on CD69−CD103− CD8+ (white) and CD69+CD103+ (“induced resident;” black) CD8 T cells after sequential exposure to 3 d of 50 ng/ml rhIL-15 followed by 3 d of 50 ng/ml rhTGFβ. All figures show summary data from at least four independent experiments. Error bars indicate means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; p-values were determined via a Kruskal-Wallis test (ANOVA) with a Dunn’s post hoc test for pairwise multiple comparisons (a, c, d, e, and f) or a Wilcoxon Signed-rank t test (b, g, h, and i).

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