Fig 2: DNA methylation status of TSDRs in WT and Tet2/3fl/flFoxp3Cre DKO Treg cells. a–f DNA methylation status of TSDRs (Treg-specific demethylated regions). Graphs depict the percentage of 5mC + 5hmC determined by BS-seq in peripheral Tregs in: (a) 4 CpGs in Foxp3 CNS1, and (b) 11 CpGs in Foxp3 CNS2, (c) 4 CpGs in Il2ra intron 1a, (d) 7 CpGs in Tnfrsf18 exon 5, (e) 6 CpGs in Ikzf4 intron 1b and (f) 5 CpGs in Ctla4 exon 2. Error bars show mean ± s.d. of thousands of sequencing reads from two independent experiments. Wild type (WT) Treg cells (8–10 weeks-old) are shown in black, CD4Cre mediated Tet2/3 DKO Treg cells (3–4 weeks-old) are shown in red12 and Foxp3Cre mediated Tet2/3 DKO Treg cells (8–10 weeks-old) are shown in blue

Fig 3: Maternal CD8+ T Cell-Blunted Responsiveness to Fetal Antigen Re-stimulation during Secondary Pregnancy(A) C57BL/6 (H-2b) females 21 days following allogeneic mating with Act-OVA male mice on the BALB/c background were compared to pregnant mice mid-gestation during secondary pregnancy sired by Act-OVA or non-transgenic male mice on the BALB/c background.(B) Number of H-2Kb:OVA257–264 CD8+ T cells in the spleen and pooled peripheral lymph nodes of mice described in (A).(C) Percentage of IFN-?-, TNF-a-, or IL-2-producing H-2Kb:OVA257–264 CD8+ T cells following PMA-ionomycin stimulation for each group of mice described in (A).(D) H-2Kb:OVA257_264 tetramer staining intensity and CD44 expression levels by H-2Kb:OVA257_264 CD8+ T cells for each group of mice described in (A).(E) PD-1, LAG-3, CTLA-4, and TIM-3 expression levels by H-2Kb:OVA257_264 CD8+ T cells for each group of mice described in (A).(F) Relative expression of PD-1, LAG-3, CTLA-4, and TIM-3 among H-2Kb:OVA257–264 CD8+ T cells mid-gestation during primary or secondary pregnancy, each sired by Act-OVA male mice on the BALB/c background.Data are from at least 3 independent experiments, each with similar results, with each point representing data from an individual mouse.Bar, mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001.

Fig 4: 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 5: 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.