Signaling by small GTPases in the immune system.

The Ras superfamily consists of over 50 low-molecular-weight proteins that cycle between an inactive guanosine diphosphate-bound state and an active guanosine triphosphate (GTP)-bound state. They are involved in a variety of signal transduction pathways that regulate cell growth, intracellular trafficking, cell migration, and apoptosis. Several methods have been devised to measure the activation state of Ras proteins, defined as the percent of Ras molecules in the active GTP-bound state. We have previously developed a quantitative biochemical method that can be applied to animal and human tissues and have used it to measure the activation state of Ras, Rap1, Rheb, and Rho proteins in cultured cells and in animal and human tumors. Ras, Rac, and Rho all play roles in regulating the functions of T and B lymphocytes and dendritic cells, and these proteins are clearly important in maintaining normal immune system function.

Farnesyltransferase inhibitors inhibit T-cell cytokine production at the posttranscriptional level.

Several cytoplasmic proteins, such as GTPases of the Ras family, containing a C-terminal CAAX motif are prenylated by farnesyltransferase to facilitate localization to cellular membranes where activation occurs. Farnesyltransferase inhibitors (FTIs) interfere with this farnesylation process, thereby preventing proper membrane localization and rendering the proteins unavailable for activation. Currently, FTIs are being explored as antineoplastic agents for the treatment of several malignancies. However, since farnesylated proteins like Ras are also involved in intracellular signaling in lymphocytes, FTIs might interfere with T-cell activation. Based on this hypothesis we examined the effect of several FTIs on cytokine production in response to anti-CD3 + anti-CD28 monoclonal antibodies or PMA + ionomycin. Murine Th1 and Th2 clones, stimulated in the presence of FTIs, showed a dose-dependent reduction of lineage-specific cytokine secretion (IFN-gamma, IL-2, IL-4, IL-5). However, no inhibition of ERK or JNK MAP kinases was observed, nor was induction of cytokine mRNA affected. Rather, intracellular cytokine protein synthesis was blocked. Inhibition of human T-cell INF-gamma production also was observed, correlating with reduced phosphorylation of p70S6K. These results indicate that FTIs inhibit T-cell activation at the posttranscriptional level and also suggest that they may have potential as novel immunosuppressive agents.

Differential Ras signaling via the antigen receptor and IL-2 receptor in primary T lymphocytes.

Ras can become activated via multiple distinct receptors in T lymphocytes. However, mechanistic studies of Ras signaling in normal T cells have been hampered by the lack of an efficient technology for gene transfer into resting post-thymic cells. We have overcome this limitation by utilizing adenoviral transduction of T cells from Coxsackie/adenovirus receptor transgenic mice. Unexpectedly, dominant negative Ras17N blocked activation of Ras and ERK in response to IL-2R engagement but not TCR/CD3 ligation. However, TCR-induced ERK activation was suppressed by inhibitors of PKC and PLC-gamma. This first biochemical study of DN Ras in normal quiescent T cells reveals a striking contrast in Ras signaling via two receptors, and suggests that the principal mechanism of TCR-induced Ras activation in normal T cells may be distinct from that utilized in T-lineage tumor cell lines.

T cell development and function in CrkL-deficient mice.

The adapter protein CrkL has been implicated in multiple signal transduction pathways in hematopoietic cells. In T lymphocytes, the recruitment of CrkL-C3G complexes has been correlated with hyporesponsiveness, implicating CrkL as a potential negative regulator. To test this hypothesis we examined T cell activation in CrkL-deficient mice. The CrkL(-/-) genotype was partially embryonic lethal. In viable CrkL(-/-) mice, peripheral blood counts were normal. The thymus from CrkL(-/-) mice had 40% fewer cells compared to littermates, but the proportion of thymocyte subsets was comparable. There was no discernable alteration in T cell function as reflected by T cell numbers, expression of memory markers, IL-2 production, proliferation, and differentiation into Th1/Th2 phenotypes. Immunization induced comparable levels of IgG2a and IgG1 antibodies. Chimeric mice, generated by transfer of CrkL(-/-) fetal liver cells into irradiated RAG2(-/-) recipients, also showed normal T cell function, arguing against selection via partial embryonic lethality. Our results indicate that CrkL is not absolutely required for T cell development or function, and argue against it being an essential component of a negative regulatory pathway in TCR signaling.