Fig 2: Percentage of CD4+ and CD8+ T cells in skin. C57BL/6J mice were injected intradermally in the ear with vehicle (Veh), proteinase K-digested porins (PorK), porins (PorID), or intraperitoneally with porins (PorIP) or heat-inactivated S. Typhi (HIS) at day 0. All groups were boosted at day 14 with porins injected intradermally in the ear. Count of the percentage of CD4+ (A) and CD8+ (B) T cells found in skin at day 28. (n = 7; 2 independent experiments; significant difference for a Kruskal–Wallis test ** p < 0.05 * p < 0.1 with Dunn’s multiple comparison).

Fig 3: NIX Is Critical for Formation of Effector Memory in Ova-Specific CD8+ T CellsSpleens from OT-I mice (A–D) or wild-type (WT) and T/NIX-/- mice (E–K) were collected at designated time points.(A) Kinetics of Nix expression in Ova-specific CD8+ T cells (Ova-CD8+) after VSV-Ova immunization.(B) Gene expression of Nix in Ova-CD8+ 24 h after addition of IL-15. CD8+ T cells from naive OT-I mice were activated with anti-CD3 and anti-CD28 for 72 h, followed by IL-15 addition.(C) Kinetics of Nix expression in Ova-CD8+ after CD3-stimulation, followed by IL-15 addition.(D) Kinetics of Il15ra expression in Ova-CD8+ after VSV-Ova immunization. Ova-CD8+ from mice within the same experimental group in (A)–(D) were pooled before analysis.(E) Representative dot plot showing percentage of Ova-EM in WT or T/NIX-/- spleens on day 30 p.i. with 104 plaque-forming units (PFU) of VSV-Ova.(F) Mean frequencies of Ova-EM from (E).(G) Experimental model for adoptive transfer experiment performed in (H).(H) Left: representative plot showing percentages of CD45.1+ WT and CD45.2+ T/NIX-/- Ova-EM in CD45.2+ T/NIX-/- mice 30 days after VSV-Ova immunization. Right: mean frequencies of CD45.1+ WT and CD45.2+ T/NIX-/- Ova-EM from experiment performed in the left panel.(I) Kinetics of effector memory formation in Ova-CD8+ in vivo in WT or T/NIX-/- mice after VSV-Ova immunization.(J) Gene expression of Nix in day 0 naive, day 6 Ova-activated, day 10 Ova-CD8+ MPECs, day 30 Ova-EM, and day 30 Ova-CM in WT mice after VSV-Ova immunization.(K and L) Gene expression of Foxo1 (left panel) and Tcf7 (right panel) in day 10 Ova-CD8+ MPECs (K) or day 30 Ova-EM (L) harvested from WT or T/NIX-/- spleens after VSV-Ova immunization.(M) In vitro differentiation of Ova-EM. Left: representative plot for percentage of WT or T/NIX-/- Ova-EM on day 8. Right: mean frequency of Ova-EM from left panel.In (E) and (M), CD8+Ova_tetramer+ population (Ova-EM) was gated on CD3+CD8+CD43-CD62L-CD44+ population. Data are representative of two or more independent experiments (n = 3–10). Data were analyzed using one-way ANOVA with Bonferroni’s posttest (mean ± SEM) in (A)–(D); two-tailed Student’s t test (mean ± SEM) in (F), (H, right), (J)–(L), and (M, right); and two-way ANOVA with Bonferroni’s posttests (mean ± SEM) in (I). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. See also Figures S1 and S2.

Fig 4: TSC1-deficient CD8+ T cells accelerate the acute allograft rejection. We isolated and infused TSC-/- (WT?TSC-/-) or WT CD8+ (WT?WT) T cells into B6 recipient mice (2 × 106 cells per mouse) within 24 h pre-transplantation (A) and analyzed the survival of cardiac allografts (B). Log-rank test; * P < 0.05, n = 6 per group.

Fig 5: MVA5/MVA1, vNY1/MVA1 and vNY5/MVA1 vaccination regimens induce TH1-biased immune responses.(A). End-point titers of SARS-CoV-2 spike-specific IgG2C and IgG1 antibodies in mouse sera collected 2 weeks after vaccination regimens. ns-not significant; **p<0.01; ****p<0.0001. (B). End-point titer IgG2C/IgG1 ratio calculated based on data from (A) (n = 5 for each group). (C). ELISpot analyses of mouse splenocytes collected 4 weeks after vaccination regimens for their expression of IL-2, IFN-?, TNF-a, IL4 and IL6 cytokines (n = 5 for each group). Data represented as mean ± SEM. SFC–spot-forming cells. *p<0.05; **p<0.01; ***p<0.001. (D & E). SARS-CoV-2 spike-specific CD8+ (in D) and CD4+ (in E) T effector memory cells (CD44+CD62L-) in splenocytes, as detected by flow cytometry (n = 5 for each group). Data represented as mean ± SD. ns–not significant; ***p<0.001; ****p<0.0001.