The

population of CD3-positive T cells in the spleen or mesenteric lymph node was reduced by ~ 35–45% in T-cell-specific Stat3-deficient mice (Fig. 2a). Absolute total splenocyte numbers were counted using a haemocytometer, and T-cell and non-T-cell numbers were drug discovery calculated according to flow cytometry results. The total number of splenocytes was significantly reduced in T-cell-specific Stat3-deficient mice (Fig. 2b). The number of CD3-positive T cells was reduced to a greater degree than that of splenocytes in T-cell-specific Stat3-deficient mice; non-T-cell numbers in the spleen were similar in both groups (Fig. 2c). This implies that the reduced volume, weight and cell number in spleens of T-cell-specific Stat3-deleted mice was a result of the T-cell deficiency. Because it has been reported that Lck-driven Cre expression is toxic for developing T cells,[23] we also compared the splenic volumes, the proportion and the absolute number of T cells in spleens

from Stat3WT/WT Lck-CRE−/− and Stat3WT/WT Lck-CRE+/− to exclude the possibility that our results were attributable to the off-target effect of Cre-recombinase to developing T cells. Both the volume of spleens and the absolute number of T cells showed only minimal decrease in Stat3WT/WT Lck-CRE+/− mice compared with Stat3WT/WT Lck-CRE−/− mice at 8 weeks (see Supplementary material, Fig. S2a–c), while the significant T-cell depletion was observed in spleens from Stat3fl/fl Lck-CRE+/− mice compared with Ulixertinib nmr those from Stat3WT/fl Lck-CRE+/− mice (Fig. S2d,e). Furthermore, 2-hydroxyphytanoyl-CoA lyase we analysed the subpopulation of thymocytes in Stat3WT/WT Lck-CRE−/− and Stat3WT/WT Lck-CRE+/− mice (Fig. S2f–h). Both the population and the absolute number of double-positive, CD4 and CD8 SP cells were unvarying between CRE−/− and CRE+/− mice at 6 months (Fig. S2f–h). These results indicate that the T-cell deficiency in Stat3fl/fl Lck-CRE+/− mice largely

resulted from Stat3 deletion, rather than from the off-target toxicity of Cre-recombinase. We next investigated the proportion of CD4- or CD8-positive T cells in spleen and lymph node. Both the CD4 and CD8 populations were considerably decreased in the Stat3-deleted group (Fig. 2d–f). Also, the population and the absolute number of CD4+ Foxp3+ T cells, which are regarded as regulatory T cells, were notably decreased in spleens from Stat3-deficient mice when compared with the control group (Fig. 2g,h). To observe the variation of naive or effector/memory T-cell population in peripheral T cells from wild type or Stat3 knockout mice, we performed flow cytometry analyses with CD4, CD8, CD62L and CD44 staining (Fig. 3). The CD62Lhigh and CD44low population in both CD4- and CD8-positive T lymphocytes, which has been identified as naive T cells, was considerably reduced in splenocytes and lymph node cells from the Stat3-deficient group (Fig.