Fig. 2: Allogeneic Treg survival is impaired in vivo and reversed by depletion of host CD8⁺ cells.

A Human ex vivo-expanded Tregs were cultured with VPD-stained autologous or allogeneic PBMCs (1 × 10⁵) in the presence of αCD3/αCD28-coated beads at Treg:PBMC ratios of 1:2, 1:4, and 1:8. After 3 days, live Tregs were quantified by flow cytometry and plotted as a fraction of the input number. Data are presented as mean ± SEM. Six Treg donors were used across conditions. Statistical analyses used a two-way repeated measures ANOVA with Bonferroni post-tests: F(1,15) = 2.00, p = 0.18; all pairwise comparisons p > 0.05. B Immunodeficient BALB/c Rag2^−/−cγc^−/− mice received intraperitoneal CFSE-labeled human Tregs (5 × 10⁶) with 5 × 10⁶ autologous (n = 5) or allogeneic (n = 4) unstained PBMCs from one (autologous) or two (allogeneic) donors. After 7 days, peritoneal cells were recovered by lavage, and CD4⁺CFSE⁺ Tregs enumerated by flow cytometry. C Treg numbers/mouse plotted with mean ± SD for each treatment group. Mann–Whitney U = 0, p = 0.0159 (two-tailed). D–F Immunodeficient BALB/c Rag2^−/−cγc^−/− mice received intraperitoneal CFSE-labeled human Tregs (5 × 10⁶) with 5 × 10⁶autologous (n = 3) or allogeneic (n = 11) PBMCs from one (autologous) or two (allogeneic) donors (Supplementary Fig. 2A). Allogeneic PBMCs were either whole (n = 4), depleted of CD8⁺cells (n = 3), or depleted of CD56⁺ cells (n = 4). Depletions were performed using magnetic beads. Cells were analysed on day 7. Treg numbers are shown/mouse with group mean ± SD. D Absolute Treg numbers. Statistical significance was calculated using two-tailed t-tests: autologous versus allogeneic PBMCs t(5) = 10.50, p = 0.0001, n² = 0.96, 95% CI = [−67700, −41000]; whole versus CD8⁺-depleted PBMCs t(5) = 7.20, p = 0.0008, n² = 0.91, 95% CI = [43000, 91000]; and whole versus CD56⁺-depleted allogeneic PBMCs t(6) = 0.2393, p = 0.8188, n² = 0.009, 95% CI = [−3674, 4470]. E Absolute number of undivided Tregs. Statistical significance was calculated using two-tailed t-tests: autologous versus allogeneic PBMCs t(5) = 9.60, p = 0.0002, n² = 0.95, 95% CI = [−13000, −7000]; whole versus CD8⁺-depleted PBMCs t(5) = 7.20, p = 0.0008, n² = 0.91, 95%CI = [4700, 10000]; and whole versus CD56⁺-depleted allogeneic PBMCs t(6) = 1.38, p = 0.22, n² = 0.24, 95% CI = [−550, 2000]. F Percentage of CFSE^hi (representing undivided) Tregs. Statistical significance was calculated using two-tailed t-tests: autologous versus allogeneic PBMCs t(5) = 2.89, p = 0.034, n² = 0.63, 95% CI = [−17, −1]; whole versus CD8⁺-depleted PBMCs t(6) = 5.9, p = 0.0011, n² = 0.85, 95% CI = [9.21, 22.4]; and whole versus CD56⁺-depleted allogeneic PBMCs t(6) = 2, p = 0.0983, n² = 0.39, 95% CI = [−13.5, 1.51]. Panel B created in BioRender. McCallion, O. (2025) https://BioRender.com/35ntrd5. Source data are provided.