Fig 2: 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 3: Maternal CD8+ T Cells with Fetal Specificity Expand during Pregnancy and Persist after Parturition(A) Female mice on the C57BL/6 (H-2b) background were mated with Act-OVA male mice on the BALB/c (H-2d) background. For analysis, females were sacrificed either before mating (virgin), at embryonic day 10.5 (E10.5) (pregnant), or 21 days following delivery (postpartum).(B) Number of H-2Kb:OVA257–264-specific CD8+ T cells in the spleen and pooled peripheral lymph nodes of female C57BL/6 virgin or mid-gestation (E10.5) mice compared with postpartum week 1 (PP7), week 3 (PP21), and week 8 (PP56) after allogeneic pregnancy sired by Act-OVA male on the BALB/c background.(C) H-2Kb:OVA257–264 tetramer geometric mean fluorescence intensity (gMFI) and ratio of tetramer gMFI to CD3 gMFI for CD8+ T cells from female C57BL/6 virgin, mid-gestation (E10.5) pregnant, and PP21 after allogeneic pregnancy sired by Act-OVA male mice on the BALB/c background.(D) Percentage of CD44hi, CD62Llo, CCR7, 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 (C).(E) Representative flow cytometry plots showing the identification of H-2Kb:OVA257–264 CD8+ T cells by tetramer staining, and their production of IFN-γ, TNF-α, or IL-2 following PMA-ionomycin stimulation compared with no stimulation controls for cells isolated from the spleen and pooled peripheral lymph nodes for each group of mice described in (C).(F) Frequency of IFN-γ, TNF-α, or IL-2 producing H-2Kb:OVA257–264 CD8+ T cells from female C57BL/6 mice at the indicated time points following allogeneic pregnancies 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: 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 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.