The provisions of the Animal Welfare Acts (P
June 29, 2021
The provisions of the Animal Welfare Acts (P.L. titers (replicating virus) in the spleen within the two groups: WT control and Foxp3DTR, 8 months post MCMV infection. Titers were quantified by plaque assay 7 days after Treg depletion, indicated here as Day7. 0/number of mice in each group indicates absence of actively replicating virus and confirms the establishment of latency. (N = 9/group).(PDF) ppat.1006507.s002.pdf (158K) GUID:?A2F814A4-40FC-4973-AB9E-964F33F4AFB0 S1 Fig: Clofarabine Treg in the spleen during latent MCMV. Splenocytes were isolated from na?ve (9.5months old), or aged matched MCMV-latently infected mice (8months p.i.). Cells were stained for CD4 and Foxp3 and analyzed by flow cytometry. Graph shows the number of Foxp3+ cells in total CD4+ cells. Na?ve (N = 4), WT MCMV infected (N = 6).(PDF) ppat.1006507.s003.pdf (6.4K) GUID:?F221FF33-E8C7-481B-B1E1-D09B1E165375 S2 Fig: Highly activated, proliferating MCMV-specific CD8+ T cells in the spleen post Treg depletion. 5C6 week old WT C57BL/6 and Foxp3DTR mice were inoculated with 1 106 pfu of MCMV. 8 months post-MCMV infection, splenocytes were isolated from infected mice. Cells were stained for IE3, m139, M38 (CD8 T cell) tetramers day 0 (-DT) and analyzed with flow cytometry. A) Graph shows the total number of IE3-, m139- and M38-specific CD8 T from WT C57BL/6 (white bars, N = 3) and Foxp3DTR (black bars, N = 6) mice. 5C6 week old C57BL/6 and Foxp3DTRmice were inoculated with1 106 pfu of MCMV. 8 months post-MCMV infection, both groups were injected with Diphtheria toxin (DT) on day 0, 3, 6 and sacrificed on day 7. Spleen cells were analyzed by flow cytometry. B) Bar graphs show the average frequency and absolute number of CD4+ Foxp3+Treg in the spleen (mean+SEM). WT C57BL/6 (N = 11). Foxp3DTR (N = Clofarabine 10). Spleen cells isolated from the two groups were stained with MCMV CD8-specific tetramers and then surface stained for expression of KLRG-1 and CD127 and intra-cellular expression of Ki67. C) Bar graph shows the total numbers of effector subpopulations within Rabbit Polyclonal to IL11RA gated m139-specific CD8 T cells (mean+SEM). WT C57BL/6 (N = 6), Foxp3DTR (N = 6). D) Bar graphs show the frequency and absolute number of Ki67+ cells within M45-, IE3-, m139- and M38-specific CD8 T (mean+SEM). WT C57BL/6 (N = 6), Foxp3DTR(N = 6). Statistical analysis, 0.05, ** 0.01 (Students test).(PDF) ppat.1006507.s008.pdf (245K) GUID:?4945832B-3281-4839-8ED0-E8BD25D7AEC3 S7 Fig: Treg promote MCMV replication in the spleen. 5C6 week old WT C57BL/6 (white) and Foxp3DTR (black) mice were inoculated with 1 106 pfu of MCMV (N = 8/group). 5 months post-MCMV infection, both groups were injected with Diphtheria toxin (DT) on day 0, 3, 6, 9,12 and sacrificed on day 14. A) Bar graph shows the percentage of mice positive for virus replication in the spleen day14 quantified by plaque assay post Treg depletion with the numbers of mice in each group shown above the bars. Viral titers were 18.6 pfu/ml +/- 15.5 in WT C57BL/6 mice and 2.4 pfu/ml +/- 2.24 in FoxP3-DTR mice; p = 0.31. B) Genomic DNA was isolated from the spleens of WT C57BL/6 and DTR mice at day 14 post Treg depletion. MCMV E1 was detected by quantitative PCR, and data expressed as genome copy number per 100 ng genomic DNA as Clofarabine described in Materials and Methods (mean+SEM); p = 0.36.(PDF) ppat.1006507.s009.pdf (214K) GUID:?BECD0FDF-5F1D-471E-9781-B8AE4B8CC118 S8 Fig: IFN- production upon Treg depletion in the SG. Single cell suspensions were generated from the SGs of MCMV infected mice (day7 post Treg depletion). Cells were stained for CD4, Foxp3 and IFN- following stimulation with or without PMA and ionomycin for 5 hours, in the presence of brefeldinA. Bar graph shows the average of frequency of IFN-+ in Foxp3-.