T Cell Lymphopenia in Rhoh^−/− Mice Results from Combined Defects of T Cell Migration and IL-7-Regulated Naïve T Cell Homeostasis.

The Rho family GTPases are increasingly implicated for their important roles in T cell development and function. We have found that RhoH, a hematopoietic-specific member of this family is essential for thymocyte development (Gu et al, Nat Immunol, in press). Further, Rhoh^−/− mice showed T cell lymphopenia. In particular, naïve T cells were reduced in secondary lymphoid organs and blood both in unchallenged and LCMV-challenged Rhoh^−/− mice compared to WT controls. These findings suggest a possible defect in T cell emigration from thymus and peripheral T cell homeostasis in Rhoh^−/− mice. To study the role(s) of RhoH in T cell migration and homeostasis, purified splenic T cells were adoptively transferred into common γ^−/−; Rag2^−/− mice. T cells were then recovered from spleens 3d and 8d post-transplantation. The recovery of Rhoh^−/− T cells was decreased by 60% compared with WT cells. In vitro migration of Rhoh^−/− T cells towards SDF-1α, a homeostatic chemokine for T cells, in a transwell migration assay was significantly reduced compared to WT cells. Flow analysis showed decreased number of CXCR4 expressing and reduced expression levels of CXCR4 on Rhoh^−/− T cells. Furthermore, apoptotic T cells were increased twofold in Rhoh^−/− mice compared to controls. CFSE staining of adoptively transferred T cells demonstrated a comparable proliferation rate between Rhoh^−/− and WT cells in the recipient mice. Our data suggest that RhoH plays an important role in T cell homeostasis via regulating cell survival and SDF-1α-mediated migration. To further investigate how RhoH regulates T cell survival, we focused on IL-7, an essential factor for prolonged survival of naïve T cells. One way IL-7 exerts its effect is through upregulating the Bcl-2 family of antiapoptotic proteins. Our data showed that in vitro survival of Rhoh^−/− T cells in response to IL-7 was impaired, and expression of IL-7Rα and Bcl-2 were both decreased in Rhoh^−/− T cells. To further study a potential role of RhoH in regulation of IL-7R expression, FACsvantage sorted naïve T cells (CD4⁺CD44^low, or CD8⁺CD44^low) were cytokine starved overnight, then cultured with or without the addition of IL-7 for 6 hrs, and analyzed for IL-7Rα expression by flow cytometry. In WT cells, during cytokine starvation CD4 and a subset of CD8 naïve cells upregulated IL-7Rα expression, but this upregulation was reduced followed IL-7 stimulation. In contrast, Rhoh^−/− T cells failed to show either up- or subsequent down-regulation of IL-7Rα in response to cytokine starvation and IL-7 exposure. These data may indicate a role for RhoH in regulating IL-7R expression in naïve CD4 T cells. Taken together, these findings suggest that RhoH is required for IL-7-mediated T cell survival and SDF-1α-mediated homing and/or emigration from thymus. Thus, deficiency of naïve T cells in Rhoh^−/− mice likely results from combined defects in T cell migration and homeostasis.