Figure 6.

Abrogation of GVHD in Nrf2^−/−allo-T cell recipients is associated with a greater proportion of donor-derived CD4⁺Helios⁺Tregs. (A-G) Lethally irradiated BALB/c recipients were transplanted with WT B6 TCD BM and 0.5 × 10⁶ B6 WT or Nrf2^−/− T cells. Donor CD4⁺ T-cell subsets in recipient spleens were analyzed on day 7 after HCT. Data represent the mean ± standard deviation. (A) Absolute numbers of WT and Nrf2^−/− donor-derived CD4⁺ Th1 (T-bet⁺) cells, Th2 (GATA-3⁺) cells, Th17 (RORγt⁺) cells, and Tregs (FoxP3⁺CD25⁺) (n = 15 per group, combined from 3 independent experiments). (B) Representative flow cytometric dot plots showing the frequency of Tregs within donor CD4⁺ populations. (C) Representative flow cytometric analysis and bar graphs showing the mean fluorescence intensity (MFI) of intracellular Helios expression within donor-derived Tregs (n = 5 per group, 1 of 2 reproducible experiments). (D) The proportion of donor-derived Tregs expressing Helios (n = 15 per group, combined from 2 independent experiments). (E) Representative flow cytometric analysis and bar graph showing the MFI of intracellular FoxP3 expression within donor-derived Tregs (n = 5 per group, 1 of 3 reproducible experiments). (F) IL-2 levels in the recipient sera were analyzed using Luminex (n = 19 or 20 per group combined from 4 independent experiments). (G) An IL-2 secretion assay was performed to detect IL-2 levels on the donor T-cell surface, and MFI was analyzed using flow cytometry (n = 5 per group, 1 of 2 reproducible experiments). (H) WT CD45.1⁺ B6 TCD BM plus CD45.2⁺ WT vs Nrf2^−/− T cells were transplanted into lethally irradiated CD45.1⁺ B6 (syngeneic HCT [syn-HCT]) or BALB/c (Allo-HCT) recipients. Bar graphs show the MFI of CD25 receptor and intracellular Helios expression within donor-derived Tregs. Data represent the mean + standard deviation from 1 of 2 reproducible experiments (n = 5 per group). (I) Lethally irradiated BALB/c recipients transplanted with WT B6 TCD BM alone or with a total of 0.5 × 10⁶ B6 putative Treg (CD4⁺CD25⁺)-depleted B6 WT or Nrf2^−/− conventional CD4⁺ and CD8⁺ T cells (Tcon) were monitored daily for survival. Kaplan-Meier analysis showing combined data from 2 independent experiments. (J) Schematic model of the proposed mechanism by which Nrf2 regulates CD4⁺ T cells in GVHD. *P < .05, **P < .01, ***P < .005, ****P < .0001.