Fig. 1: Allogeneic Tregs match autologous cells in vitro yet protect skin grafts less effectively in vivo. | Nature Communications

Fig. 1: Allogeneic Tregs match autologous cells in vitro yet protect skin grafts less effectively in vivo.

From: HLA matching or CRISPR editing of HLA class I/II enables engraftment and effective function of allogeneic human regulatory T cell therapy in a humanized mouse transplantation model

Fig. 1

CFSE-stained human PBMCs (1 × 105) were stimulated with αCD3/αCD28-coated beads (2 × 104) with and without either autologous or allogeneic Tregs (at 1:2, 1:4, and 1:8 Treg:PBMC ratios) permuted from three donors mismatched at HLA-A, -B, and -DR loci (Suppl. Figure 1A). CFSE dilution after 72 h incubation was measured by flow cytometry, and a division index was calculated. A The percentage suppression of CD4+ proliferation F(1,6) = 0.32, p = 0.59, n2p = 0.015, 95% CI = [−10.23, 16.44], (p > 0.05 for all pairs). B The percentage suppression of CD8+ proliferation F(1,6) = 1.03, p = 0.35, n2p = 0.04, 95% CI = [−8.29, 20.24], (p > 0.05 for all pairs). Both are calculated relative to stimulated PBMCs cultured in the absence of Tregs. C Median fluorescence intensity of CD25 amongst CD4+ responder cells F(1, 6) = 0.19, p = 0.68, n2p = 0.017, 95% CI = [−1482, 2120], (p > 0.05 for all pairs). D Median fluorescence intensity of CD25 amongst CD8+ responder cells F(1,6) = 0.56, p = 0.48, n2p = 0.023, 95% CI = [−2131, 1131], (p > 0.05 for all pairs). A–D Data are plotted as mean ± SEM of 3 autologous and 6 allogeneic responder:Treg donor combinations. All assays were performed in triplicate. Statistical significance for autologous versus allogeneic Tregs, across all Treg:responder ratios, was assessed using two-way repeated measures ANOVA with two-tailed adjusted Bonferroni tests for pairwise comparisons. E Immunodeficient BALB/c Rag2−/− cγc−/− mice grafted with human skin received intraperitoneal human PBMCs (5 × 106) alone (n = 17), with Tregs autologous to the PBMC donor (5 × 106, n = 7) or Tregs allogeneic to the PBMC donor (5 × 106, n = 6). Grafts were monitored for macroscopic signs of rejection over the following 100 days. Data are pooled from two independent experiments with each employing separate skin donors and distinct PBMC/Treg donor pairs. F Percentage of grafts surviving is plotted over time post-adoptive transfer of cells. G Sample size and median survival time for each treatment group is tabulated. Statistical significance was assessed using Mantel-Cox log rank tests: *χ2(1) = 4.28, p = 0.0387; **χ2(1) = 11.69, p = 0.0006, HR = 5.22, 95% CI = [2.17, 12.56]; ***χ2(1) = 18.00, p < 0.0001. Panel E created in BioRender. McCallion, O. (2025) https://BioRender.com/ynhfgt9. Source data are provided as a Source data file.

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