Fig 2: 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.

Fig 3: 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 4: 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 5: Expanded TFR Cells Express an Abnormal Phenotype and Altered GC Distribution after Immunization of Prdm1fl/flFoxP3Cre Mice(A and B) Flow cytometry (left) and numbers (right) of splenic (A) or mLN (B) Treg (FoxP3+CD4+) (upper), TFR (CD4+CD3+PD-1+CXCR5+FoxP3+), and non-TFR (CD4+CD3+CXCR5−FoxP3+) (bottom) from WT and KO mice (7–9 weeks old) 10 days post-immunization with NP-KLH in CFA.(C) Histogram of Ki67, annexin V, and CD69 expression in mLN TFR cells in (B) (upper) and quantification of mean fluorescence intensity (MFI) in mLN TFR and CXCR5− Treg cells (bottom).(D) Expression of TFR-associated gene products by splenic TFR from (A) and quantification of MFI (right).(E) IL-17A, IFNγ, and IL-4 expression (left) and frequency (right) by splenic TFR (CD4+CD3+PD-1+BTLA+FoxP3+) cells from WT or KO mice (6–8 weeks old) at day 7 post-immunization with NP-OVA in CFA.(F) Frequency of CD25lo (left), CD25int (center), and CD25hi (right) splenic TFR from WT and KO mice (6–8 weeks old) 10 days post-immunization with NP-OVA in CFA. (Bottom) FoxP3 and CTLA-4 expression in the CD25-expressing TFR subsets.(G) MFI of FoxP3, CTLA-4, GITR, and RORγt expression and frequencies of IL-17A+ cells in YFP+ Blimp1-deficient (KO) compared to YFP− Blimp1-sufficient (WT) TFR cells from the spleens of female Prdm1fl/flFoxP3Cre/+ mice (6–8 weeks old) 10 days post-immunization with NP-OVA in CFA.(H and I) CXCR5 (H) and CCR7 (I) expression in the splenic TFR cells in (A) and mLN TFR cells in (B).(J) Histology of 7-week-old WT and KO mice 14 days post-immunization with NP-OVA in CFA. TFR cells (CD3+FoxP3+, white arrows) and TFH cells (CD3+FoxP3−) in the GC area (Ki67+) within the B cell follicle (IgD+). Insets indicate the B cell follicles. (Bottom) Numbers of TFR and TFH cells in the GC.In (A)–(I), the data are representative of four independent experiments (A–F, n = 4/group; G, n = 5/group; H and I, n = 4–5/group). In (J), the data are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (unpaired two-tailed Student’s t test). Error bars indicate means ± SEMs.See also Figure S2.