S2a,b). A previous report suggested increased apoptosis in Ts65Dn thymuses

in situ[10] and analysis of the thymocytes ex vivo by Annexin V staining also indicated increased apoptosis of thymocytes from Ts65Dn mice. Consistent with the role of IL-7Rα, increased apoptosis was only observed in the DN thymocyte populations in the Ts65Dn mice (Fig. 3e), whereas there were no differences in Annexin V staining in mature DP and SP thymocytes (Fig. S2c). One find more mechanism by which IL-7Rα regulates thymocyte survival, is through induction of the expression of the anti-apoptotic protein Bcl-2.[17] However, no significant differences in Bcl2 expression were detected in all the thymocyte populations by intracellular staining (Fig. 3f, Fig. S2d). Alvelestat manufacturer Because of the role(s) of IL-7Rα in survival of peripheral T cells, especially CD8+ memory T cells, surface expression of IL-7Rα was also measured in the splenic T-cell subsets. A small decrease in IL-7Rα-positive cells was observed in both CD4+ (see Supplementary material, Fig. S3a) and CD8+ (Fig. S3b) subsets, although the magnitude of decrease was not commensurate with that observed in DN thymocytes. Hence, alterations in IL-7Rα expression appear to be limited to immature lymphocyte progenitors and not the

more committed mature cells. B-cell proliferative responses were also assessed in the Ts65Dn mice to determine whether immune dysfunction was limited to T cells. Total splenocytes were stimulated with varying concentrations of anti-IgM, anti-IgM in combination

with IL-4, and Escherichia coli lipopolysaccharide, a known B-cell activator. Proliferation was then assessed in CD19+ B-cells by flow cytometry using CFSE dilution as in Fig. 2. Compared with cells from euploid control mice, there was a significant decrease in the percentage of Ts65Dn CD19+ cells that had undergone at least one division after 48 hr (Fig. 4a) and 72 hr (Fig. 4b) in response to stimulation by anti-IgM, and anti-IgM in combination with IL-4. In contrast, no significant difference was observed when cells were stimulated with various concentrations Nintedanib (BIBF 1120) of lipopolysaccharide either at 48 hr (Fig. 4c) or 72 hr (Fig. 4d). To determine whether changes in B-cell development in the Ts65Dn mice reflected the changes in B-cell function, peripheral B-cell subsets were defined by flow cytometry.[26] Consistent with decreased proliferation of spleen B cells, there were small but significant decreases in the presence of both follicular (Fol I) and transitional (T1 and T3) B-cell subsets in the splenic B cells from Ts65Dn mice (Fig. 5a). Furthermore, there was an increased percentage of CD19+ cells expressing high levels of both MHC II and CD80, which has been proposed as markers of memory B cells[29] (Fig. 5b).