4). In the male mice the number of DP thymocytes was slightly increased (Fig. 5). These results demonstrate the partial impairment of positive and negative selection in LAR-deficient mice. During selection, the strength of the TCR signal plays a pivotal role in determining cell fate 3–5. In LAR-deficient thymocytes, the

level of TCR stimulation, as measured by the intracellular Ca2+ concentration, 5-Fluoracil mw was reduced compared with control thymocytes (Fig. 6). We think the reduction of Ca2+ responding cells is mainly attributed to DP thymocytes because neither CD4SP nor CD8SP thymocytes express LAR 17, 18 and LAR deficiency might not affect the Ca2+ mobilization in CD4SP as well as CD8SP thymocytes. This reduction in the strength of the TCR signal may result in a decrease in the efficiency of negative and positive selection (Fig. 7). Although LAR deficiency might affect T-cell development in the thymus, the animals do

not appear to be immune-compromised and there has been no report of which in the literature. LAR deficiency might not be reflected in the immunological phenotype; first, because the effect of LAR deficiency in the thymus was subtle and its effect was compensated in the periphery; second, because LAR is a member of receptor type membrane tyrosine phosphatase family including CD45 that is expressed on thymocytes as well as peripheral T cells and CD45 could compensate the effect of LAR deficiency. How does LAR deficiency affect TCR signal transduction? During TCR signal transduction, a receptor-like PTP, CD45, activates two PTK, Lck and Fyn, by dephosphorylating a tyrosine moiety 29,

30. Activated Lck and Fyn then phosphorylate the immunotyrosine-activating Opaganib datasheet motif on CD3ξ, which leads to the activation of thymocytes as well as T cells. Tsujikawa et al. 12 reported that LAR also regulated the activity of Lck and Fyn in CD45-deficient cells. Taken together, our results suggest that LAR is also involved in DCLK1 TCR signal transduction in thymocytes. The effect of LAR deficiency on TCR signal transduction seems subtle since we did not observe significant differences in the proliferation of LAR-deficient thymocytes following TCR stimulation (Supporting Information Fig. 6). Regarding the regulation of Ca2+ mobilization by LAR, Archuleta et al. have demonstrated that activated Lck and Fyn increase tyrosine phosphorylation of phospholipase C-γ1, and activated phospholipase C-γ1 increase formation of IP3, which may be responsible for the rapid increase in intracellular Ca2+ mobilization 31. Taken together, we hypothesize that LAR may regulate Ca2+ mobilization by activating Lck and Fyn, which then leads to tyrosine phosphorylation of phospholipase C-γ1 and increases formation of IP3, which finally regulate intracellular Ca2+ mobilization. Our data suggest that LAR is only expressed during the DN-to-DP transition of T cells in the thymus and that LAR plays a role in pre-TCR- or αβTCR-mediated selection during the differentiation of thymocytes.