Figure 4. Lack of protection is attributable to reversion of iTregs to a proinflammatory phenotype in vivo.

(A-E) Lethally irradiated Balb/c mice (n=12) were transplanted with 8 × 10⁶ B6.PL (Thy1.1) BM and spleen cells (adjusted to yield a dose of 0.6 × 10⁶ αβ T cells) with 0.6 × 10⁶ iTregs (Thy1.2). Spleen, liver, lung, and pooled colon samples were analyzed for Thy1.2⁺ cells to determine EGFP expression. (A) Representative dot plots gated on CD4⁺ T cells depicting the percentage of Thy1.2⁺ H-2K^b+ cells that expressed EGFP 10 days after transplantation. (B,C) The relative percentage of CD4⁺ Thy1.2⁺ EGFP⁻ cells is shown in B, and the absolute number of EGFP⁺ and EGFP⁻ cells in the specified tissue sites is depicted in C. Data are presented as the mean ± SEM and are the cumulative results of three independent experiments. (D) Representative dot plots and (E) percentages of CD4⁺Thy1.2⁺EGFP⁻ cells that secreted IFN-γ or IL-17A as determined by intracellular cytokine staining. (F) Lethally irradiated Balb/c mice were transplanted with B6.PL BM, B6.PL spleen cells (adjusted to yield a dose of 0.6 × 10⁶ αβ T cells) and 1.8 × 10⁴ B6 Thy1.2⁺ CD4⁺ EGFP⁻ cells. Representative dot plots gated on CD4⁺ T cells showing Thy1.2⁺ expressing cells in the spleen, liver, lung, and colon from animals 10 days after transplantation. Statistics: * p<0.05, **p<0.01.