Rap1-GTP Promotes the Generation and Effector Function of Cytotoxic CD8+ T Cells.

Rap1, a member of the Ras superfamily, is currently emerging as a small GTPase with a critical role in cell adhesion, junction formation and spreading. In T lymphocytes, transient activation of Rap1 and accumulation of Rap1-GTP at the T cell:APCs interface is one of the physiologic consequences of TCR ligation and regulates b2 integrin activation and LFA-1:ICAM-1 mediated adhesion. To understand the role of Rap1 in the immune responses of the intact host, we generated transgenic (Tg) mice, which express the active, GTP-bound Rap1 mutant Rap1E63 in T cells. Using these mice we observed that Rap1-GTP is a negative regulator of T helper cell function and promotes the generation of CD4+CD103+ Treg cells in vivo. Here we investigated the role of Rap1 in the generation and function of cytotoxic T cells (CTL). First, we examined allogeneic responses of CD8+ T cells. Purified CD8+ T cells from either Rap1E63-Tg or normal littermate control (NLC) mice were cultured with allogeneic Balb/c splenocytes as stimulators. Proliferation of Rap1E63-Tg CD8+ T cells were reduced compared to those of NLC CD8+ cells, but reached the peak within a shorter time interval of culture. Importantly, Rap1E63 transgenic CD8+ T cells produced significantly increased levels of IFN-g compared to NLC cells. Next we examined effector function. CTL differentiation was carried out by a five-day culture of purified Rap1E63-Tg or NLC CD8+ T cells with allogeneic Balb/c splenocytes. Subsequently, effector CTL were isolated and tested for their ability to kill 51 Cr-labeled P815 cells. Rap1E63-Tg effector CTL showed a 3 to 4-fold increased cytolytic activity as compared to NLC effector CTL, at all E:T cell ratios tested. Because Rap1E63 induced high levels of LFA1 activation, we examined whether LFA-1-mediated signals might be responsible for the enhanced cytolytic activity of Rap1E63-Tg CTL and whether such signals affected CTL generation or effector function. Addition of LFA-1 blocking mAb during the effector phase, abrogated CTL function in both Rap1E63 and NLC cells. Addition of LFA-1 mAb during the generation phase, did not alter CTL efficiency of NLC effectors as compared to control cultures without LFA-1. In contrast, addition of LFA1 mAb during the generation phase, dramatically reduced the enhanced killing efficiency of Rap1E63 CTL to levels comparable to NLC CTL, suggesting that Rap1-GTP predominantly regulated the generation phase of CTL effectors. Cell killing by CTL effectors requires the combined actions of the membranolytic protein perforin, and the granule-associated serine proteases, granzymes. After co-release from CTL, perforin mediates transport of granzymes, which initiate the molecular events that culminate cell death. Granzyme B, which cleaves target proteins after aspartate residues is the most efficient mediator of CTL-induced killing. Detailed analysis revealed that Rap1E63-Tg CTL effectors had significantly increased levels of perforin as determined by western blot. Granzyme B enzymatic activity was also higher in Rap1E63-Tg as compared to NLC CTL. These results indicate that Rap1-GTP promotes the generation of highly efficient CTL effectors, and at least one mechanism responsible for this effect involves the enhanced LFA-1-mediated signaling.