Extended Data Fig. 4: FOXP3⁺ T_regs play an indispensable role in TLI/ATS-induced cDC1 EPOR-dependent immune tolerance.

a, Representative pseudocolor plots showing FOXP3⁺ T_reg depletion efficiency in recipient mice on day 6 after DT treatment (DT injections on days 0, 2, and 4). b,c, Representative pseudocolor plots of C57BL/6, Batf3^−/−, or Epor^ΔXcr1 recipient conventional CD4⁺ T cell percentages and FOXP3⁺ T_reg percentages in CD4⁺ T cells. d,e, Absolute cell number of indicated cell populations. b,d, Day 0 (UNT, n = 5; n = 5; n = 5 and TLI/ATS, n = 6; n = 5; n = 4) and c,e, Day 14 of UNT or TLI/ATS-treated groups post allo-BM infusion (UNT, n = 11; n = 6; n = 9 and TLI/ATS, n = 3; n = 11; n = 10). f, MHCII expression on cDC1s and cDC2s from MHCII^flox/flox and MHCII^ΔXcr1 spleens. g,h,i, MHCII^flox/flox (n = 6) and MHCII^ΔXcr1 (n = 6) recipients were given TLI/ATS and i.v. infused with BALB/c donor BM cells. 14 days post BM infusion, the percentages of donor type (H2K^d+) cells among leukocyte populations were determined in the peripheral blood of hosts. g,i, Recipient MHCI (H-2K^b)⁺TCRβ⁺CD4⁺ T cell frequency among total live cells and FOXP3⁺ frequency among CD4⁺ T cells were analyzed on day 14. j, CD45.2⁺FOXP3^WTEpor^flox/flox (+ PBS/without DT, n = 8; +DT, n = 8) or Epor^ΔXcr1 (+ PBS/ without DT, n = 8; +DT, n = 8) mice were injected with 30 million CD45.1⁺FOXP3^DTR CD4⁺ T cells isolated by MACS. Two consecutive doses of DT or PBS were given on each of the following 2 days. Subsequently, the mice were treated with TLI/ATS, and 2W1S-BALB/c donor BM cells were infused i.v., and 14 days later, 2W1S-tetramer⁺CD44⁺H-2K^b+TCRβ⁺CD4⁺ T cells from the spleens were analyzed for FOXP3 expression by flow cytometry. FOXP3 expression in CD45.1⁺ or CD45.2⁺2W1S-tetramer⁺ CD4⁺ T cells is shown. One experiment (j) or one of two independent experiments with similar results are shown (a-i). Statistical analysis was performed using unpaired two-tailed Student’s t-test (g,i), two-way ANOVA with Tukey’s multiple-comparison test (d,e,j). Data are mean ± s.e.m. (d,e,g,i,j). The diagram in j was created in BioRender. Zhang, X. (2025) https://BioRender.com/cx0n3vn.

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