Figure 2. The Treg phenotype is dependent upon tissue location and is influenced by IL-2.

(A) CD4⁺ Thy1.1⁺ cells from the spleens of naïve mice were sorted into CD25^hi and CD25^lo populations for adoptive transfer. A fraction of each was stained for intracellular Foxp3 and CD25. Representative dot plots depict the post-sort purity with the mean percentage of CD25^hi (upper left panel) and CD25^lo (lower left panel) populations at the time of adoptive transfer. The bar graphs depict the percentage of CD25^hi (upper graph) or CD25^lo (lower graph) Thy1.1⁺ CD4⁺ Foxp3⁺ donor cells that retained their CD25 phenotype after migration to the spleens or livers of recipient mice at 3 or 7 days following adoptive transfer. The results were equivalent at both time points so the data were pooled and averaged (*p< 0.0052, n=6 recipients/group). (B) CD4⁺ Foxp3⁺ Tregs from the spleens and livers of naïve mice were analyzed for CD25 expression by flow cytometry one day following 3 consecutive days of treatment with anti-IL-2 mAbs or an IgG mAb control. Representative histograms and bar graphs depicting CD25 geometric mean fluorescence intensity (+SEM) for each tissue are shown. The differences in MFI between treated and control groups were significant in both the spleen and liver (*p< 0.0093, n=9–10). (C) Intracellular Ki-67 expression and (D) intracellular Bcl-2 expression of CD4⁺Foxp3⁺ Tregs was analyzed by flow cytometry for the IgG control and the anti-IL-2 treated groups. There was no statistical difference in percentage of Ki-67 positive cells between treatment groups in the spleen or liver. However, Bcl-2 expression was significantly reduced with anti-IL-2 treatment in both tissues (spleen *p= 0.0001, liver *p=0.0329, n=9–10). (E) A bar graph depicting the IL-2 concentration as measured by ELISA from supernatants of purified CD4⁺ Foxp3⁻ T cells from the spleens and livers of naïve and chronically infected mice after 48 hours of in vitro culture. There was a significant difference between naïve spleens and livers as well as chronic spleens and livers (*p<0.0205, n=7–10).