Rap1-GTP Augments TGF-b-Mediated Signaling in T Lymphocytes Via a Mechanism Dependent on the b Chain of LFA-1 Integrin

Integrin-mediated adhesion of lymphocytes to antigen presenting cells (APCs) is a critical event linking innate and adaptive immunity. Integrins function as bidirectional receptors and can transmit signals from both sides of the plasma membrane, a property referred to as inside-out and outside-in signaling. Lymphocyte adhesion to APC is mainly accomplished through the principle adhesion molecule on the lymphocyte surface, the lymphocyte functional antigen 1 (LFA-1), which binds to intercellular adhesion molecule 1 (ICAM-1) on the surface of APCs. In order to mediate its function, LFA-1 must be activated via a process, which results in conformation changes of the receptor that extends the ectodomains of the α and β chains leading to a high affinity state. Among the few signaling molecules that have been implicated in integrin activation in hematopoietic cells are the small GTPase Rap1A (thereafter named Rap1) and its downstream effectors RapL and RIAM. In response to Rap1-GTP, RapL regulates LFA-1 activation by interacting with the integrin α chain, whereas RIAM mediates recruitment of talin to the cytoplasmic tail of the β chain leading to its conformational change to the high affinity state. To understand the role of Rap1 in T cell responses we generated transgenic (Tg) mice that selectively express the active, GTP-bound Rap1 mutant Rap1E63 in mature T cells. Rap1E63-Tg and littermate control mice had no statistically significant difference in absolute thymocyte numbers and differentiation profiles. In contrast, in peripheral lymphoid organs, Rap1E63-Tg mice had a significant reduction in total T cells but a 4-fold increase in the CD4⁺CD103⁺ T cell fraction. CD103 (also known as α_Eβ₇ integrin) defines a subset of peripherally generated Treg with potent suppressive function. TGF-β is the strongest stimulus for induction of CD103 (α_Eβ₇). To examine whether Rap1-GTP can affect TGF-β-mediated signaling in T cells, we used stable Jurkat T cell lines expressing GTP-bound Rap1 mutant, Rap1E63, or Jurkat T cell lines in which the endogenous Rap1 was depleted by shRNA (Rap1-KD), and also primary T cells from Rap1E63-Tg mice and Rap1-KO mice. After interaction with two membrane-bound receptors, TGF-βRI and II, TGF-β propagates downstream signaling via the Smad family transcription factors. Incubation of Rap1E63 Jurkat T cells with TGF-β resulted in enhanced and sustained phosphorylation of Smad2 and Smad3, which was observed with very low concentrations of TGF-β that were incapable of inducing detectable phosphorylation of Smads in control Jurkat T cells. In contrast, diminished level and duration of Smad2 and Smad3 phosphorylation was observed in Rap1-KD Jurkat T cells. Similar patterns of responses to those observed in Rap1E63 Jurkat T cells and in Rap1-KD Jurkat T cells were observed in primary mouse T cells isolated from Rap1E63-Tg mice and Rap1-KO mice, respectively. To investigate whether the LFA-1 integrin α and/or β chain had an active role in the enhanced TGF-β-mediated signaling in the presence of Rap1-GTP, we used Rap1E63 Jurkat T cells in which RapL or RIAM were depleted by shRNA (Rap1E63/RapL-KD and Rap1E63/RIAM-KD) because these adaptors selectively regulate the LFA-1 α and the LFA-1 β chain, respectively, downstream of Rap1-GTP. Although in Rap1E63/RapL-KD cells the enhanced TGF-β-induced Smad3 phosphorylation remained unaffected, in Rap1E63/RIAM-KD cells the enhanced TGF-β-induced Smad3 phosphorylation was abrogated. To investigate the biological relevance of these observations, we used T cells from Rap1E63-Tg mice crossed with mice deficient for the LFA-1 α chain. TGF-β resulted in enhanced Smad3 phosphorylation in T cells from Rap1E63-Tg/LFA1-a KO mice similarly to T cells from Rap1E63-Tg mice, indicating that this effect was not dependent on the activation of LFA-1 α chain. In contrast, T cells from RIAM^flox/flox-Lck-Cre mice, in which activation of the LFA-1 β chain is impaired, displayed abrogated activation of Smad3 in response to TGF-β. Our data reveal a novel mechanism by which Rap1 regulates T cell responses via outside-in integrin signaling and may have important implications on TGF-β-mediated T cell homeostasis, differentiation and immune quiescence.