Gene Targeting of Cdc42 Reveals Its Essential Role in T Cell Development and Homeostasis.

Cdc42 of the Rho GTPase family is known to play an essential role in diverse cell functions. Previous studies by using dominant mutants or transgenic mice suggest that Cdc42 is involved in T cell polarization, immune synapse formation, migration, and development. Because the dominant mutant overexpressing approach imposes significant experimental limitations, we have characterized the T cell-specific, conditional gene targeted mice with the Lck-Cre;Cdc42^loxP/loxP genotype in an effort to define the physiological role of Cdc42. Firstly, Cdc42 gene disruption in T cells caused an increase of CD4⁺CD8⁺ double positive T cells by ∼10% whereas a reduction of both CD4⁺CD8⁻ and CD4⁻CD8⁺ single positive T cells by >50% in the Cdc42^{−/ −} thymus. The thymus of Cdc42-deleted mice showed small and inconspicuous thymic medulla and the thymic cortex of Cdc42-deficient mice appeared prominent. Examination of CD69 expression in Cdc42^{−/ −} CD4⁺CD8⁺ T cells revealed a defective positive selection. In peripheral organs, loss of Cdc42 caused a drastic reduction of mature T cell populations in lymph nodes, blood and spleens. Spleens of the Cdc42 null mice contained ∼1/7 of CD4⁺ T cells and ∼1/8 of CD8⁺ T cells compared with that of wild type (WT) mice. These phenotypic observations indicate that Cdc42 regulates T cell development and homeostasis. Secondly, the perturbed T cell homeostasis in Cdc42 null mice is associated with defective T cell survival characterized by an increase in apoptosis and a gain of resistance to IL-7-mediated cell survival. The apoptotic phenotype of Cdc42^{−/ −} T cells correlates with an increased expression of pro-apoptotic Fas and decreased expression of anti-apoptotic BCL-2. Concomitantly, Cdc42 deficiency resulted in an increase in homeostatic proliferation as manifested by increased in vivo BrdU incorporation in Cdc42-deficient T cells and accelerated division of Cdc42 null cells upon adoptive transfer into Rag2^{−/ −} mice, possibly due to a compensatory effect of lymphopenia. Thus, a combined effect on survival and proliferation by Cdc42 deficiency may contribute to the defective T cell homeostasis. Thirdly, F-actin assembly, T cell receptor (TCR) capping, and cell migration were impaired in T cells lacking Cdc42. Cdc42 deficiency caused a ∼50% reduction in the percentage of CD4⁺ T cells with capped TCR in response to TCR cross-linking. Chemotaxis of Cdc42^{−/ −} CD4⁺ T cells toward SDF-1a was reduced by 40% in comparison with that of WT cells, suggesting that Cdc42 regulates T cell migration and polarization, which may also be involved in the defective homeostatic distribution of Cdc42^{−/ −} T cells. Fourthly, Cdc42 deficiency appears to promote the activation of mature T cells with an elevated expression of a T cell activation marker, CD69. Cdc42^{−/ −} T cells proliferated faster than WT cells and showed increased BrdU incorporation upon in vitro culture with CD3 antibody. This activation phenotype may be attributed to a constitutively elevated ERK activity found in the Cdc42^{−/ −} T cells. Finally, loss of Cdc42 led to an increase of naturally-occurring and the lymphocytic choriomeningitis virus-specific effector and memory T cells. Autoimmune-protective CD4⁺CD25⁺ regulatory T cells were markedly reduced and the production of T helper cell-dependent IgG2a increased by ∼6 folds in the absence of Cdc42. Taken together, our results suggest that Cdc42 plays a critical role in T cell homeostasis by regulating survival and proliferation. Further, Cdc42 is important for T cell actin cytoskeleton rearrangement, polarization and migration and for effector and memory T cell response.