Modulation of the strength of signals from the TCR determines the outcome of positive and negative selection in thymocyte development. Previous studies have demonstrated that SHP-1 plays a role in determining signal strength from the TCR. Here, we have taken a genetic approach to test whether SHP-1 plays a role in T cell selection in the thymus. Experiments in which a dominant negative mutant of SHP-1 was expressed in the BYDP hybridoma cell line confirmed that SHP-1 regulated TCR signaling in a cell-autonomous manner and suggested that Lck is one of its targets. To examine the role of SHP-1 in T cell development, we crossed the ovalbumin-specific DO11.10 TCR transgene onto the motheaten background, which lacks SHP-1 expression. Analysis of the progeny of these crosses provided evidence that SHP-1 regulates thymocyte selection: (i) flow cytometric analyses revealed alterations in the percentages of thymocyte subpopulations in the me/me background; (ii) ex vivo deletion experiments demonstrated that me/me:Tg thymocytes undergo negative selection at lower concentrations of OVA peptide compared to +/+:Tg thymocytes; and (iii) ex vivo proliferation analyses indicated that me/me:Tg thymocytes were hyper-sensitive to stimulation by the specific OVA peptide. Our observation that the absence of SHP-1 leads to altered selection of TCR transgenic thymocytes demonstrates that SHP-1 regulates the strength of TCR-mediated signals in vivo and, in turn, helps to set the threshold for thymocyte selection.

A subpopulation of T cells, named regulatory T cells (Treg cells), has been shown to play a key role in tolerance and the prevention of autoimmunity. It is not known how changes in TCR signal strength during thymic T cell development affect the generation of a Treg population. In this study, we took two different strategies to modulate the TCR signal strength: an intrinsic approach, where signaling was enhanced by the loss of a negative regulator, and an extrinsic approach, where signaling strength was altered through variations in the concentrations of the selecting peptide. The tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a known negative regulator of TCR-mediated signaling. motheaten mice, lacking expression of SHP-1, showed a 2- to 3-fold increase in the percentage of CD4+CD25+ Treg cells within the CD4+ T cells. Similarly, the percentage of Treg cells was heightened in fetal thymic organ cultures (FTOCs) derived from motheaten mice compared with wild-type FTOCs, thus establishing the thymic origin of these Treg cells. Using FTOCs derived from DO11.10 TCR transgenic mice, we demonstrated that exposure to increasing concentrations of the cognate OVA peptide favored the appearance of Treg cells. Our data suggest that the development of CD4+CD25+ Treg cells is intrinsically different from non-Treg cells and that Treg cells are selectively enriched under conditions of enhanced negative selection. Our data also reveal a key role for the SHP-1-mediated regulation of TCR signal strength in influencing the ratio of Treg vs non-Treg cells.