2a). Previous reports on DS have suggested that limited thymic output would lead to decreased function and immunosenescence in peripheral immune cells. To assess lymphocyte functional capacity in Ts65Dn mice, whole splenocytes were stimulated with immobilized anti-CD3 antibody (at 0.5 or 5 μg/ml) for 48 and 72 hr in vitro. Proliferation of CFSE-labelled splenocytes was measured in TCR+ T cells by flow cytometry (representative flow data shown in Fig. 2b,c) as described in the ‘Materials and methods’. There was a significant decrease in the percentage of TCR+ cells that had undergone
at least one division in cells from Ts65Dn mice compared with euploid Navitoclax in vitro controls after 48 hr (Fig. 2d) and 72 hr (Fig. 2e). There was also a significant decrease in the percentage of TCR+ cells that had undergone more than three divisions after 72 hr (Fig. 2f) in cells from Ts65Dn mice. Changes in proliferation were not the result of cell death because the percentage of viable cells was not different between euploid and Ts65Dn cells at both time-points (not shown). Hence, although the proportions
of peripheral immune cells in Ts65Dn mice are relatively unchanged, there are significant defects in the function of peripheral T cells that suggest a senescent phenotype in this mouse model of DS. As a possible mechanism for thymic alterations in Ts65Dn mice, IL-7Rα was assessed because it plays a non-redundant role in thymic development,
promoting proliferation and survival of immature, DN thymocytes. Consistent with our previous observations selleck in bone marrow lymphoid progenitors, the percentage of specific thymocyte subsets that were IL-7Rα+ was decreased in the lineage-negative (total DN thymocytes), DN2 and DN3 populations (Fig. 3a). The absolute number DAPT cell line of cells expressing the IL-7Rα chain was significantly decreased in the Lin− and all the DN thymocyte populations (Fig. 3b). Interleukin-7Rα is normally down-regulated in DP thymocytes and re-expressed in positively selected CD4 and CD8 SP thymocytes. In contrast to the data in DN thymocytes, when mature DP and SP thymocytes were analysed neither the percentage of IL-7Rα+ cells (Fig. S1c) nor the number of positive cells (not shown) was decreased. To measure whether changes in IL-7Rα were associated with altered cell proliferation in the thymus, mice were injected with BrdU for 2 days and incorporation was assessed ex vivo. Consistent with those populations having decreased IL-7Rα expression, lower percentages of BrdU+ cells were found in the DN2 and DN3 populations (Fig. 3c), and significantly fewer BrdU+ cells were detected in the DN2, DN3 and DN4 populations of Ts65Dn mice in comparison to euploid mice (Fig. 3d), indicating defects in thymocyte proliferation.