In WT mice, the number of total thymocytes reached its peak between 2 and 8 wk of age (Fig. 1A). The total number of thymocytes from LAR−/− mice at corresponding ages was slightly lower than from WT mice. As shown in Fig. 1B, the average number of total thymocytes in LAR−/− mice was significantly lower than in WT mice. After 11 wk, the number of total thymocytes was similar in both LAR−/− and WT mice (Fig. 1A and B). We then investigated the effect of LAR deficiency on thymocyte differentiation by analyzing CD4 and CD8 expression. The most immature thymocytes

do not express Gefitinib in vitro CD4 or CD8. Immature thymocytes then differentiate into CD4+CD8+ DP thymocytes while passing through a transient CD4−CD8+ (CD8SP) differentiation stage 20. After positive

selection, they lose either CD4 or CD8 expression and differentiate into CD8SP or CD4+CD8− (CD4SP) mature thymocytes. To examine the effects of LAR deficiency on thymocyte differentiation, we analyzed the expression of CD4 and CD8 on thymocytes from WT mice and LAR−/− mice by flow cytometry and calculated the percentage of different thymocyte subpopulations. Of the total thymocytes, 4.0±1.3% and 2.5±0.6% were DN in LAR−/− and WT mice, respectively (Fig. 2), and 84.5±1.2% and 86.3±2.0% were DP, respectively. Furthermore, 8.2±1.4% and 8.5±1.4% of the total thymocytes LY2157299 were CD4SP in LAR−/− and WT mice, respectively, while 3.2±0.4% and 2.7±0.5% were CD8SP in LAR−/− and WT mice, respectively. Taken together, the percentage of DN thymocytes was higher (p=0.0011), that of DP thymocytes was lower (p=0.0022)

and that of CD8SP thymocytes was cAMP higher (p=0.009) in LAR−/− mice compared with WT mice. In CD8SP thymocyte population, the percentage of CD8SP cells that expressed high level of TCRβ was decreased in LAR−/− mice compared with WT mice (p=0.04) (Supporting Information Fig. 3), whereas DP or CD4SP thymocyte population expressing high level of TCRβ was not altered significantly in WT and LAR−/− mice. The results indicate that the percentage of CD8SP cells that expressed no or low level of TCRβ, i.e. immature CD8SP thymocytes, was increased in LAR−/− mice compared with WT mice. Taken together, the differentiation of DN thymocytes to DP thymocytes via intermediate CD8SP thymocytes is partially impaired in LAR−/− mice. The differentiation stages of the DN thymocytes were further subdivided using CD44 and CD25 expression (DN1, CD44+CD25−; DN2, CD44+CD25+; DN3, CD44−CD25+; DN4, CD44−CD25−). We previously showed that IMT-1/LAR was first expressed on DN2 thymocytes and that most DN3 thymocytes continued to express IMT-1/LAR 18. Figure 3 and Supporting Information Fig. 4 show that the proportion and the number of DN subsets defined by the expression of CD44 and CD25 on DN thymocytes was corresponding in LAR−/− and WT mice.