Fig 2: Mechanism of Blimp1-Dependent Control of TFR Differentiation: Contribution of the IL-23R-STAT3 Axis and CXCR5-CCR7 Expression(A) WT and KO mice (6 weeks old) were immunized with NP-OVA in CFA. Seven days later, TFR cells (CD4+CD3+PD-1+CXCR5+YFP+) were sorted for microarray analysis. Differential gene expression in TFR from WT and KO mice (>1.5-fold) is shown.(B) Pathway analysis revealed control of genes associated with TH cell differentiation by Blimp1.(C) Genes related to cytokine-cytokine receptor interaction by DAVID.(D) IL-23R expression by donor TFR cells in Tcra-/- hosts after tamoxifen-induced deletion of Blimp1, as in Figure 4.(E) IL-23R expression in the CD25-expressing TFR subsets from WT and KO mice (6–8 weeks old) 10 days post-immunization.(F) FoxP3-GFP reporter mice were immunized with NP-OVA in CFA. Seven days later, Treg (CD4+CD3+GFP+CD25+) and CD4+ naive T cells (CD4+CD3+GFP-CD44-) were sorted, chromatin prepared, and ChIP-PCR analyses performed for Blimp1, acetylated H3 (AcH3), and H3K27me3 at Blimp1-binding sites at the 3rd intron of Il23r, the 1st intron of Cxcr5, the 5' distal element of CTLA-4, the 3rd intron of Ccr7, the FoxP3 CNS2, the 1st intron of Il2ra, and a non-specific region (C, control) of Il2ra. Data are shown as the percentage of input. Naive cells were used as controls for the anti-Blimp1 assay. Rabbit IgG isotype (R-iso) and mouse IgG isotype (M-iso) served as controls for the anti-AcH3 assay and anti-H3K27 assay, respectively.(G) Prdm1fl/fliCre- (WT) and Prdm1fl/fliCre+ (Del) mice (8 weeks old) were treated with tamoxifen at day 0, followed by immunization with NP-OVA in CFA at day 1 and injection of tamoxifen daily for 4 days. Expression of pSTAT3 and pSTAT5 in TFR cells at day 7 post-immunization. At right, pSTAT3:pSTAT5 ratios in TFR cells are shown (n = 4/group).In (D), (E), and (G), the data represent one of two experiments. In (F), the data represent one of three independent experiments. **p < 0.01 (unpaired two-tailed Student’s t test). Error bars indicate means ± SEMs.See also Figure S4.

Fig 3: AMPKa1-deficient Treg cells present a ‘fragile’ phenotype in TME(A) Gating strategy of CD4+YFP+ Treg cells from tumor tissues.(B) Representative FACS images of ICOS, CD25, CTLA-4, GITR, PD1, and Nrp1 expression on CD4+Foxp3-YFP+ Treg cells from day 13 tumor tissues of AMPKa1Treg+/+ and AMPKa1Treg-/- mice.(C) Relative MFI of cell surface ICOS (n = 5), CD25 (n = 6), CTLA-4 (n = 6), GITR (n = 6), PD1 (n = 6) and Nrp1 (n = 5) on CD4+Foxp3-YFP+ Treg cells from day 13 tumor tissues of AMPKa1Treg+/+and AMPKa1Treg-/- mice. (Data are presented as individual values and mean ± SD and analyzed by Student's t test).(D) Representative FACS images and frequency of YFP+IL-10+ Treg cells in CD4+Foxp3-YFP+ Treg cells from day 13 tumor tissues of AMPKa1Treg+/+and AMPKa1Treg-/- mice. (n = 6 in each group, data are presented as individual values and mean ± SD and analyzed by Student's t test).(E) Relative MFI of IL-10 in CD4+Foxp3-YFP+ Treg cells from day 13 tumor tissues of AMPKa1Treg+/+and AMPKa1Treg-/- mice. (n = 6 in each group, data are presented as individual values and mean ± SD).(F) Representative FACS images and frequency of YFP+IFN-?+ Treg cells in CD4+Foxp3-YFP+ Treg cells from day 13 tumor tissues of AMPKa1Treg+/+and AMPKa1Treg-/- mice. (n = 6 in each group, data are presented as individual values and mean ± SD and analyzed by Student's t test).(G) Relative MFI of IFN-? in CD4+Foxp3-YFP+ Treg cells from day 13 tumor tissues of AMPKa1Treg+/+and AMPKa1Treg-/- mice. (n = 6 in each group, data are presented as individual values and mean ± SD and analyzed by Student's t test).

Fig 4: No difference in grades of rejection between mock-infected mice and mice that received all three infections. Donor Balb/c hearts were excised from C57BL/6 recipients at the time of sacrifice. H&E staining was performed, and slides were sent to a cardiac pathologist for blinded analysis. Grades of rejection were scored using the ISHLT-2004 acute cellular rejection grading scheme. A score of 0 indicates no rejection, 1 indicates mild rejection, 2 indicates moderate rejection, and 3 indicates severe rejection. (A) Representative histology slides from mock-infected mice with PBS or infected with all three viruses that received fully mismatched heterotopic heart transplants, treated with chronic CTLA4-Ig. Summary data show no significant difference in grades of rejection between groups at day 80 post-transplant (p = 0.7333). (B) Representative slides from mock-infected or infected mice treated with chronic CTLA4-Ig and rapamycin. Summary data show no significant difference in grades of rejection between groups at day 75 post-transplant (p = 3333). (C) Representative slides from mock-infected or infected mice treated with CTLA4-Ig on days 0, 2, 4, and 7. Summary data show no significant difference in grades of rejection between groups at day 14 post-transplant (p = 0.2381). Statistical analysis of grades of rejection between mock-infected and infected mice was performed using nonparametric Mann–Whitney test.

Fig 5: Latent and persistent viral infection does not impact allograft rejection in the setting of costimulation blockade (CoB). (A) Mice infected with polyomavirus, murine cytomegalovirus, and HV68 (n = 12) under chronic CoB with CTLA4-Ig fail to reject their allograft, while mock-infected mice (n = 10) do go on to reject their allograft (p = 0.0258). (B) No difference in allograft survival was observed between infected (n = 4) and mock-infected (n = 4) mice under chronic immunosuppression with CTLA4-Ig and rapamycin (p > 0.9999). (C) When treated with short course CTLA4-Ig, survival curves were not significantly different between infected (n = 7) and mock-infected (n = 8) mice (p = 0.1174). Decrement survival indicates immunosuppressive death with a functioning allograft. Allograft survival data were assessed using the Log-rank (Mantel–Cox) test for statistical significance.