To detect possible enzymatic activity-independent Danshensu effects of the phosphatase, we also generated animals transgenic for the inactive C227S mutants of LYP-W620. In the presence of LYP-W620, we observed increased phosphatase activity of LYPW620 in thymocytes, associated with decreased TCR signaling in total thymocytes and DP thymocytes. No anomalies in thymic TCR signaling were detected in animals carrying the inactive form of LYP-W620, despite supraphysiological expression of LYP directed by the transgene in these animals. Results with LYPW620 C227S variant-bearing mice thus suggest that effects of phosphatase overexpression on thymocyte TCR signaling critically depend upon enzymatic function of LYP. Overexpression of active LYP-W620 did not lead to impaired positive or Dexrazoxane hydrochloride negative selection of HY-TCR transgenic thymocytes in male mice or in OT-1 TCR-transgenic thymocytes crossed to Rip-mOva mice. Indeed, the finding of modestly-reduced autoreactive thymocyte numbers in BACLYP transgenic H-Y males suggests that LYP-W620 may actually promote negative selection of CD8 + T cells. Such a phenotype is in line with the report by Dai et al., in which PepW619-knockin autoreactive thymocytes exhibit augmented clonal deletion. We speculate that such subtle positive effects of LYP-W620 on thymocyte selection are obliterated in our TgLYP models by the combination of supraphysiological expression levels and the use of an exogenous promoter. Next, we considered the hypothesis that overexpression of the LYP-W620 variant would only impact negative selection at an intermediate TCR affinity range and thus might particularly impact those clones bearing TCR with affinity for self-antigen close to the threshold of negative selection. We addressed this hypothesis by comparing the affinity of the bulk population of Kb/ Ova-reactive T cells that survive negative selection in Rip-mOva mice. Normally, in such mice only T cells with low affinity for Kb/ Ova can be detected in the periphery. We reasoned that if LYP-W620 selectively reduces negative selection of T cells bearing antigen receptors with affinity at or just above the threshold of negative selection, then expression of TgLYPW would confer increased in the overall functional avidity of Kb/Ova-reactive T cells in Rip-mOva or Rip-mOvaxVb5 mice. Our negative data clearly argue against this hypothesis. We also considered whether LYP-W620 might selectively impact CD4 + T cell selection. The pathogenesis of disease in Skg mice depends on reduced TCR signaling in thymocytes, which results in reduced positive and negative selection of CD4 + thymocytes, but the repertoire of Skg thymic emigrants contains T cells with significant auto-reactive potential. When we overexpressed LYP-W620 in the thymus of Skg mice, we did not observe alterations of CD4 + thymocyte repertoire or a gain in disease severity. Higher affinity interactions with thymocyte TCR by autoantigens are thought to be required for the differentiation of Treg in the thymus, thus suggesting that alterations of TCR signaling in the thymus might shape the amount and repertoire of Treg. However, we did not see changes in the output of regulatory T cells from the TgLYPW Skg thymus, further arguing against the notion that LYP-W620 selectively affects high-affinity TCR interactions important for development. Importantly, in the TgLYPW model, expression of the transgene is controlled by the Lck proximal promoter and is only expressed in the thymus but not in the periphery. Therefore, we can exclude that we created a balanced situation such that we might have augmented the thymic threshold but, at the same time, caused similar increases in the peripheral T cell activation threshold.