Adhesion of T cells after stimulation of the T-cell receptor (TCR) is mediated via signaling processes that have collectively been termed inside-out signaling. The molecular basis for inside-out signalingis not yet completely understood. Here, we show that a signaling module comprising the cytosolic adapter proteins ADAP and SKAP55 is involved in TCR-mediated inside-out signaling and, moreover, that the interaction between ADAP and SKAP55 is mandatory for integrin activation. Disruption of the ADAP/SKAP55 module leads to displacement of the small GTPase Rap1 from the plasma membrane without influencing its GTPase activity. These findings suggest that the ADAP/SKAP55 complex serves to recruit activated Rap1 to the plasma membrane. In line with this hypothesis is the finding that membrane targeting of the ADAP/SKAP55 module induces T-cell adhesion in the absence of TCR-mediated stimuli. However, it appears as if the ADAP/SKAP55 module can exert its signaling function outside of the classical raft fraction of the cell membrane.Within the immune system, integrins play important roles in regulating the interaction of T cells with other cells and with proteins of the extracellular matrix. By mediating T-cell adhesion, integrins control the homing and the trafficking of T cells as well as the interaction between T cells and antigen-presenting cells (34, 41). The major integrins expressed on T cells are the ␤2-integrin LFA-1 (␣L␤2) as well as members of the ␤1-family of integrins (␣4␤1, ␣5␤1, ␣6␤1, and VLA) (25). The physiologic ligands of LFA-1 include the intercellular adhesion molecule 1 (ICAM-1), ICAM-2, and ICAM-3 (25), whereas ligands for ␤1-integrins are vascular cell adhesion molecule 1 (VCAM-1) or proteins of the extracellular matrix, such as fibronectin (13,54).On resting T cells, ␤1-and ␤2-integrins are expressed in an inactive state. However, ligation of the T-cell receptor (TCR) by antigen/major histocompatibility complexes results in a rapid increase in the activity of ␤1-and ␤2-integrins, thereby enhancing ligand binding (15,46,50). Two distinct mechanisms mediate the activation of integrins. First, the affinity of an integrin for its ligand is enhanced, and second, the lateral mobility becomes altered, which results in integrin clustering (avidity regulation) (14). The processes leading to the activation of integrins have collectively been termed inside-out signaling (14, 15, 28).Several molecules have been suggested to play critical roles during TCR-mediated activation of ␤1-and ␤2-integrins (14, 28). Among these is the small GTPase Rap1, whose role for integrin activation has been a matter of intense research during the last few years (4, 29). The mechanisms for how Rap1 becomes activated are not yet completely understood (4). Rap1 activation has been shown to be mediated by particular guanine nucleotide exchange factors (GEFs), such as C3G, and Epac (5,8,11). It has been proposed that Rap1 is associated with CalDAG-GEFI and that TCR-induced Rap1 activation is dependent upon the activation of phosphol...

SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family protein tyrosine kinase Fyn. Previously, several groups have provided experimental evidence that SKAP-HOM (most likely in cooperation with the cytosolic adaptor protein ADAP) is involved in regulating leukocyte adhesion. To further assess the physiological role of SKAP-HOM, we investigated the immune system of SKAP-HOM-deficient mice. Our data show that T-cell responses towards a variety of stimuli are unaffected in the absence of SKAP-HOM. Similarly, B-cell receptor (BCR)-mediated total tyrosine phosphorylation and phosphorylation of Erk, p38, and JNK, as well as immunoreceptor-mediated Ca(2+) responses, are normal in SKAP-HOM(-/-) animals. However, despite apparently normal membrane-proximal signaling events, BCR-mediated proliferation is strongly attenuated in the absence of SKAP-HOM(-/-). In addition, adhesion of activated B cells to fibronectin (a ligand for beta1 integrins) as well as to ICAM-1 (a ligand for beta2 integrins) is strongly reduced. In vivo, the loss of SKAP-HOM results in a less severe clinical course of experimental autoimmune encephalomyelitis following immunization of mice with the encephalitogenic peptide of MOG (myelin oligodendrocyte glycoprotein). This is accompanied by strongly reduced serum levels of MOG-specific antibodies and lower MOG-specific T-cell responses. In summary, our data suggest that SKAP-HOM is required for proper activation of the immune system, likely by regulating the cross-talk between immunoreceptors and integrins.

The ␤2-integrin lymphocyte functionassociated antigen-1 (LFA-1) plays a crucial role within the immune system. It regulates the interaction between T cells and antigen-presenting cells and facilitates T-cell adhesion to the endothelium, a process that is important for lymphocyte extravasation and homing. Signals mediated via the T-cell receptor and the chemokine receptor CCR7 activate LFA-1 through processes known as inside-out signaling. The molecular mechanisms underlying inside-out signaling are not completely understood. Here, we have assessed the role of the ADAP/SKAP55 module for CCR7-mediated signaling. We show that loss of the module delays homing and reduces intranodal T-cell motility in vivo. This is probably because of a defect in CCR7-mediated adhesion that affects both affinity and avidity regulation of LFA-1. Further analysis of how the ADAP/SKAP55 module regulates CCR7-induced integrin activation revealed that 2 independent pools of the module are expressed in T cells. One pool interacts with a RAPL/Mst1 complex, whereas the other pool is linked to a RIAM/Mst1/Kindlin-3 complex. Importantly, both the RAPL/Mst1 and the RIAM/Mst1/Kindlin-3 complexes require ADAP/SKAP55 for binding to LFA-1 upon CCR7 stimulation. Hence, 2 independent ADAP/SKAP55 modules are essential components of the signaling machinery that regulates affinity and avidity of LFA-1 in response to CCR7. IntroductionWithin the immune system, the ␤2-integrin LFA-1 (lymphocyte function-associated antigen; ␣L␤2) mediates T-cell adhesion to the endothelium, homing of T cells to secondary lymphoid organs, and T-cell activation through the interaction of lymphocytes with antigen-presenting cells (APCs). 1,2 On resting T cells, LFA-1 is expressed in an inactive form and adopts a low-affinity conformation for its ligands, the ICAM molecules (inter cell adhesion molecules). 3,4 Triggering of the T-cell receptor (TCR) by Ag/MHC complexes or of the chemokine receptor CCR7 by CCL21 induces a conformational change of LFA-1 that increases its affinity for ICAM-1 (affinity regulation). Furthermore, these stimuli also facilitate clustering of LFA-1, a process termed avidity modulation. 3,4 The molecular events leading to LFA-1 activation have collectively been termed "inside-out signaling."Research of the last decade has revealed that the small GTPase Rap1, the 2 Rap1 effector proteins RIAM (Rap1 interacting adapter molecule) and RAPL (regulator for cell adhesion and polarization enriched in lymphoid tissues), and the RAPL-interacting mammalian Ste20-like kinase (Mst1) are critically involved in TCR-and CCR7-mediated signaling events regulating T-cell adhesion, LFA-1 affinity and avidity modulation, and T-cell-APC interactions. 3,4 Indeed, RAPL-and Mst1-deficient T cells show defects in adhesion, homing, and intranodal migration in vivo. [5][6][7] In addition to RAPL, RIAM, and Mst1, the cytosolic adapter proteins ADAP (adhesion and degranulating promoting adapter protein), and SKAP55 (Src-kinase associated phosphoprotein of 55 kDa) are crucial for i...

The T cell marker CD26/dipeptidyl peptidase (DP) IV is associated with an effector phenotype and markedly elevated in the human CNS disorder multiple sclerosis. However, little is known about the in vivo role of CD26/DP IV in health and disease, and the underlying mechanism of its function in CNS inflammation. To directly address the role of CD26/DP IV in vivo, we examined Th1 immune responses and susceptibility to experimental autoimmune encephalomyelitis in CD26−/− mice. We show that gene deletion of CD26 in mice leads to deregulation of Th1 immune responses. Although production of IFN-γ and TNF-α by pathogenic T cells in response to myelin Ag was enhanced in CD26−/− mice, production of the immunosuppressive cytokine TGF-β1 was diminished in vivo and in vitro. In contrast to the reduction in TGF-β1 production, responsiveness to external TGF-β1 was normal in T cells from CD26−/− mice, excluding alterations in TGF-β1 sensitivity as a mechanism causing the loss of immune regulation. Natural ligands of CD26/DP IV induced TGF-β1 production in T cells from wild-type mice. However, natural ligands of CD26/DP IV failed to elicit TGF-β1 production in T cells from CD26−/− mice. The striking functional deregulation of Th1 immunity was also seen in vivo. Thus, clinical experimental autoimmune encephalomyelitis scores were significantly increased in CD26−/− mice immunized with peptide from myelin oligodendrocyte glycoprotein. These results identify CD26/DP IV as a nonredundant inhibitory receptor controlling T cell activation and Th1-mediated autoimmunity, and may have important therapeutic implications for the treatment of autoimmune CNS disease.

The adhesion- and degranulation-promoting adaptor protein (ADAP), expressed in T cells, myeloid cells, and platelets, is known to regulate receptor-mediated inside-out signaling leading to integrin activation and adhesion. In this study, we demonstrate that, upon induction of active experimental autoimmune encephalomyelitis (EAE) by immunization with the myelin oligodendrocyte glycoprotein35–55 peptide, ADAP-deficient mice developed a significantly milder clinical course of EAE and showed markedly less inflammatory infiltrates in the CNS than wild-type mice. Moreover, ADAP-deficient recipients failed to induce EAE after adoptive transfer of myelin oligodendrocyte glycoprotein–specific TCR-transgenic T cells (2D2 T cells). In addition, ex vivo fully activated 2D2 T cells induced significantly less severe EAE in ADAP-deficient recipients. The ameliorated disease in the absence of ADAP was not due to expansion or deletion of a particular T cell subset but rather because of a strong reduction of all inflammatory leukocyte populations invading the CNS. Monitoring the adoptively transferred 2D2 T cells over time demonstrated that they accumulated within the lymph nodes of ADAP-deficient hosts. Importantly, transfer of complete wild-type bone marrow or even bone marrow of 2D2 TCR–transgenic mice was unable to reconstitute EAE in the ADAP-deficient animals, indicating that the milder EAE was dependent on (a) radio-resistant nonhematopoietic cell population(s). Two-photon microscopy of lymph node explants revealed that adoptively transferred lymphocytes accumulated at lymphatic vessels in the lymph nodes of ADAP-deficient mice. Thus, our data identify a T cell–independent mechanism of EAE modulation in ADAP-deficient mice.