Fig 2: 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 3: Phenotype of artARENA-induced CTLs and their dependence on IL-33-ST2 alarmin signaling(A–F) We immunized BALB/c mice with artPICV-P1A and artLCMV-P1A in homologous or heterologous prime-boost vaccination i.v. on day 0 and day 27. On day 34, we analyzed P1A-Tet-binding and CD62L expression by splenic CD8+ T cells (A; gated on CD8+B220– lymphocytes). Unimmunized control mice are shown for comparison in (A) only. Numbers in (A) indicate the percentage of cells in the respective quadrant. Total P1A-Tet+ CTLs (B), P1A-specific effector/effector memory CTLs (CD62Llo; C), and P1A-specific central memory CTLs (CD62Lhi; D) were enumerated in the spleen on day 34. In both subsets of P1A-specific CTLs, CD62Lhi and CD62Llo, we determined the surface expression of KLRG1, CX3CR1, CD27, CD43, and CD127 as well as the master transcription factors Tcf-1, Tbet, and Eomes (E). Total numbers of marker-expressing P1A-specific CTLs were enumerated in (F). (A) shows representative FACS plots from individual mice. Symbols in (B)–(D) and (F) represent individual mice, and bars in (B)–(D) and (F) indicate the mean ± SD. Numbers in (A) and (E) indicate the percentage of gated cells (mean ± SD) or the mean fluorescence intensity (MFI ± SD). Means were calculated from six mice per immunization group (A–F) or from three unimmunized controls (A). N = 2. ∗∗p < 0.01 by unpaired two-tailed Student’s t test.(G and H) We immunized ST2−/− and WT mice with artLCMV-E7E6, artPICV-E7E6 or arCAND-E7E6 i.v. Controls were left unimmunized (“no vaccine” in G). E7-tetramer-binding cells in blood were determined on day 7. Representative FACS plots are shown in (G); values indicate E7-Tet+CD8+ T cells as a percentage of lymphocytes. Symbols in (H) represent individual mice (n = 5 per group) with mean ± SD. N = 2. ∗∗p < 0.01 by two-way ANOVA with Sidak’s post-test.See also Figures S5 and S6.

Fig 4: IL-27 Signaling Intrinsically Modulates CD4 and CD8 T Cell SubsetsLethally irradiated (NOD 3 NOD.Cd45.2)F1 mice were infused with equal numbers of T cell-depleted BM cells from NOD.Cd45.2 and NOD.Il27ra-/- donors. Pre-diabetic recipients were analyzed for wild-type (CD45.2+) and IL-27Ra-deficient (CD45.1+) T cell subsets 10–12 weeks after BM reconstitution.(A) Frequencies of total CD4 and CD8 T cells in the spleens and islets of the mixed BM chimeras. Representative flow cytometry profiles are shown in the left panels, and summarized results are presented in the right panels. Summarized results are representative of at least two independent experiments.(B) T-bet expression in CD3+CD4+Foxp3- T cells in the spleens, PLNs, and islets of mixed BM chimeras. Representative flow cytometry profiles are shown in the left panels, and summarized results are presented in the right panels.(C) IFN? production in CD3+CD4+Foxp3- T cells in the spleens, PLNs, and islets of mixed BM chimeras. Cells were stimulated with PMA and ionomycin. Representative flow cytometry profiles are shown in the left panels, and summarized results are presented in the right panels.(D) Frequencies of CD3+CD4+Foxp3+ Tregs in the spleens, PLNs, and islets of the mixed BM chimeras. Representative flow cytometry profiles are shown in the left panels, and summarized results are presented in the upper middle and right panels. The ratios of IL-27Ra-deficient to wild-type Treg frequencies in spleens versus islets are shown in the lower middle panel. The levels of CD25 expression are summarized in the lower right panel.(E) T-bet expression in CD3+CD8+ T cells in the spleens, PLNs, and islets of mixed BM chimeras. Representative flow cytometry profiles are shown in the left panels, and summarized results are presented in the right panels.(F) IFN? production in CD3+CD8+ T cells in the spleens, PLNs, and islets of mixed BM chimeras. Cells were stimulated with PMA and ionomycin. Representative flow cytometry profiles are shown in the left panels, and summarized results are presented in the right panels.All summarized data in (B)–(F) are from two independent experiments. Statistical significance was determined using Wilcoxon matched-pairs signed rank test (*p < 0.05, **p < 0.01, and ***p < 0.005). NS, not significant. GMFI, geometric mean fluorescence intensity. See also Figure S5.

Fig 5: Increased number, but altered phenotype of PHD2-deficient Treg cells.Lymphoid cells from the thymus, spleen, mesenteric (mLN), and peripheral (pLN) lymph nodes were collected at 12 weeks of age from Foxp3cre and PHD2ΔTreg male and female mice, and the relative frequency and phenotype of Foxp3-expressing cells were established by flow cytometry or qPCR. (a) Frequency of Foxp3-expressing cells among CD4-positive T lymphocytes. (b) Representative flow cytometry expression profiles of Foxp3 and CD25 expression among thymic CD4+ T cells. (c) Frequency of mature-like (CD25+ Foxp3+) and Treg precursors subsets identified respectively as CD25- Foxp3lo and CD25+ Foxp3- cells among thymic CD4+ T cells. (d) Frequency of immature-like, CD24+ Foxp3+ T cells in the thymus of adult mice. (e) Frequency of effector (CD62Llow CD44high), memory (CD62Lhigh CD44high), and naive (CD62Lhigh CD44low) splenic Foxp3-expressing cells. (f) Frequency of splenic Tregs expressing the master transcription factors T-bet, GATA3, and RORγt. (g) Ratio of the Foxp3 MFI of PHD2-KO splenic Tregs to Foxp3cre splenic Tregs. (h) Expression of CD25, CD44, ICOS, PD-1, and CTLA-4 in splenic Treg of Foxp3cre and PHD2ΔTreg mice. Top panel: representative traces of MFI. Bottom panel: ratios of the MFIs of PHD2-KO Treg to Foxp3cre Treg cells are expressed as the mean ± SD. (i) Il10 gene expression relative to RPL32 by ex vivo-purified Tregs was determined by qPCR. (j–l) CD4+ Foxp3- splenic naive T cells were stimulated in vitro with anti-CD3/CD28 (5/1 μg/ml) in the presence of TGF-β (3 μg/ml) and IL-2 (10 μg/ml) for 72 hr to induce Treg polarization. (j) Representative flow cytometry expression profiles of Foxp3 expression at the end of the culture period. The first panel represents a typical profile of cells activated in the absence of polarizing cytokines. (k) Number of Foxp3+ cells generated in the culture conditions. (l) Expression (MFI) of Foxp3 by in vitro-induced Treg cells. Data are representative of at least two independent experiments with n = 15 (a, e–g) n = 9 (h, i) or n = 6 (c, d, k, l) per group. Values are presented as the mean ± SD and were compared by two-tailed unpaired Student’s t-test. Only significant differences are indicated as follows: *p<0.05, **p<0.01, ***p<0.001. Figure 2—source data 1.Increased number, but altered phenotype of PHD2-deficient Treg cells.