Fig 2: TSC1 deficiency enhances the proliferation of CD8+T cells in vivo after transplantation. We transplanted the MHC full-mismatched cardiac allografts from BALB/c mice into WT or TSC1-/- mice and determined the percentage and number of CD4+ (A, B, and C) and CD8+ (D, E and F) T cells on day 4 post-transplantation. Student’s t-test; ***P < 0.005, n = 4 per group. In mixed lymphocyte reaction (MLR), we labeled the splenocytes from primary WT or TSC1-/- mice with CFSE and stimulated them with mitomycin C-treated splenocytes from primary BALB/c mice for 72 h in vitro. We then analyzed the proliferation of CD4+ and CD8+ T cells (G, H, and I). Student’s t-test; * P < 0.05, n = 3 per group.

Fig 3: Impacts of vaccine adjuvants on T cell distribution of splenocytes post in vitro live cancer cell stimulation. Mice were vaccinated once with NAP and UVA treated H22 vaccine plus PBS, poly IC, CpG or alum as an adjuvant or controls (n = 5 per group). (a) Scheme of the adjuvant study; (b) representative flow cytometry analysis diagrams of CD4+ and CD8+ T cell subpopulations of the isolated splenocytes with in vitro live H22 cell stimulation and controls; (c) - (d) percent bar graphs of CD4+, CD8+ and CD4-CD8- cells in isolated CD3+ splenocytes without or with live untreated H22 cells (*P < 0.05 as compared with corresponding PBS control within the panel); (e) scatter plot of percentage of CD4+ and CD8+ subpopulations of individual mouse of all adjuvant groups and controls post in vitro H22 cell stimulation; (f) scatter plot of individual coordinates from principal component analysis of subpopulations of CD3+ splenocytes post in vitro H22 cell stimulation. Confidence ellipse of each group was overlaid as indicated by colors.

Fig 4: TSC1 deficiency upregulates the functional molecule production by CD8+ T cells. We implanted allografts into WT or TSC1-/- mice, sacrificed them on day 4 post-transplantation, and determined the mRNA levels of Ifn-? (A), Il-2 (B), perforin (C), and granzyme B (D) in allografts via real-time PCR. (A) and (B) were determined using the Student’s t-test; (C) and (D) were determined using the Mann-Whitney U test; *P < 0.05, **P < 0.01, ***P < 0.005, n = 3 per group. On day 4 post-transplantation, IFN-? production by splenic CD4+ and CD8+ T cells was tested after PMA, ionomycin, and Golgistop treatment for 4-5 h ex vivo (E and F). Student’s t-test; * P < 0.05, n = 4 per group. Serum levels of IFN-? in recipient B6 mice that were implanted with TSC-/- or WT CD8+ T cells before cardiac allograft transplantation were tested on day 3 post-transplantation (G). Student’s t-test; * P < 0.05, n = 4 per group. We also determined T-bet expression levels of splenic CD4+ (H) and CD8+ (I) T cells in the allografts from WT or TSC1-/- mice on day 4 post-transplantation. (G) and (I) were analyzed using the Student’s t-test. (H) was analyzed using the Mann-Whitney U test; *P < 0.05, ***P < 0.005, n = 4 per group.

Fig 5: TSC1-deficiency increases CXCR3 expression and inner-graft proliferation of CD8+ T cells. MHC full-mismatched cardiac allografts from BALB/c mice were transplanted into WT or TSC1-/- mice, and these recipient mice were sacrificed on day 4 post-transplantation. The mRNA levels of Cxcr3 in the allografts were measured using real-time PCR (A). Mann-Whitney U test; ***P < 0.005, n =3 per group. CXCR3 expression levels in splenic CD8+ T cells were determined (B, C, D, and E). Student’s t-test; *P < 0.05, ***P < 0.005, n = 4 per group. We also tested t Ki67 expression levels in CD4+ (F) and CD8+ (G) T cells in allografts. Student’s t-test; **P < 0.01, n = 4 per group.