Rap1-GTP is a negative regulator of Th cell function and promotes the generation of CD4+CD103+ regulatory T cells in vivo

The small GTPase Rap1 is transiently activated during TCR ligation and regulates integrin-mediated adhesion. To understand the in vivo functions of Rap1 in regulating T cell immune responses, we generated transgenic (Tg) mice, which express the active GTP-bound mutant Rap1E63 in their T lymphocytes. Although Rap1E63-Tg T cells exhibited increased LFA-1-mediated adhesion, ERK1/2 activation and proliferation of Rap1E63-Tg CD4+ T cells were defective. Rap1E63-Tg T cells primed in vivo and restimulated with specific Ag in vitro, exhibited reduced proliferation and produced reduced levels of IL-2. Rap1E63-Tg mice had severely deficient T cell-dependent B cell responses, as determined by impaired Ig class switching. Rap1E63-Tg mice had an increased fraction of CD4+CD103+ regulatory T cells (Treg), which exhibited enhanced suppressive efficiency as compared with CD4+CD103+ Treg from normal littermate control mice. Depletion of CD103+ Treg significantly restored the impaired responses of Rap1E63-Tg CD4+ T cells. Thus Rap1-GTP is a negative regulator of Th cell responses and one mechanism responsible for this effect involves the increase of CD103+ Treg cell fraction. Our results show that Rap1-GTP promotes the generation of CD103+ Treg and may have significant implications in the development of strategies for in vitro generation of Treg for the purpose of novel immunotherapeutic approaches geared toward tolerance induction.     

SKAP1 protein PH domain determines RapL membrane localization and Rap1 protein complex formation for T cell receptor (TCR) activation of LFA-1

Although essential for T cell function, the identity of the T cell receptor (TCR) "inside-out" pathway for the activation of lymphocyte function-associated antigen 1 (LFA-1) is unclear. SKAP1 (SKAP-55) is the upstream regulator needed for TCR-induced RapL-Rap1 complex formation and LFA-1 activation. In this paper, we show that SKAP1 is needed for RapL binding to membranes in a manner dependent on the PH domain of SKAP1 and the PI3K pathway. A SKAP1 PH domain-inactivating mutation (i.e. R131M) markedly impaired RapL translocation to membranes for Rap1 and LFA-1 binding and the up-regulation of LFA-1-intercellular adhesion molecule 1 (ICAM-1) binding. Further, N-terminal myr-tagged SKAP1 for membrane binding facilitated constitutive RapL membrane and Rap1 binding and effectively substituted for PI3K and TCR ligation in the activation of LFA-1 in T cells.     

Rap1 is a potent activation signal for leukocyte function-associated antigen 1 distinct from protein kinase C and phosphatidylinositol-3-OH kinase

To identify the intracellular signals which increase the adhesiveness of leukocyte function-associated antigen 1 (LFA-1), we established an assay system for activation-dependent adhesion through LFA-1/intercellular adhesion molecule 1 ICAM-1 using mouse lymphoid cells reconstituted with human LFA-1 and then introduced constitutively active forms of signaling molecules. We found that the phorbol myristate acetate (PMA)-responsive protein kinase C (PKC) isotypes (alpha, betaI, betaII, and delta) or phosphatidylinositol-3-OH kinase (PI 3-kinase) itself activated LFA-1 to bind ICAM-1. H-Ras and Rac activated LFA-1 in a PI 3-kinase-dependent manner, whereas Rho and R-Ras had little effect. Unexpectedly, Rap1 was demonstrated to function as the most potent activator of LFA-1. Distinct from H-Ras and Rac, Rap1 increased the adhesiveness independently of PI 3-kinase, indicating that Rap1 is a novel activation signal for the integrins. Rap1 induced changes in the conformation and affinity of LFA-1 and, interestingly, caused marked LFA-1/ICAM-1-mediated cell aggregation. Furthermore, a dominant negative form of Rap1 (Rap1N17) inhibited T-cell receptor-mediated LFA-1 activation in Jurkat T cells and LFA-1/ICAM-1-dependent cell aggregation upon differentiation of HL-60 cells into macrophages, suggesting that Rap1 is critically involved in physiological processes. These unique functions of Rap1 in controlling cellular adhesion through LFA-1 suggest a pivotal role as an immunological regulator.     

CD98 induces LFA-1-mediated cell adhesion in lymphoid cells via activation of Rap1

CD98 is a multifunctional heterodimeric membrane protein involved in the regulation of cell adhesion as well as amino acid transport. We show that CD98 cross-linking persistently activates Rap1 GTPase in a LFA-1-dependent manner and induces LFA-1/ICAM-1-mediated cell adhesion in lymphocytes. Specific phosphatidylinositol-3-kinase (PI3K) inhibitors suppressed both LFA-1 activation and Rap1GTP generation, and abrogation of Rap1GTP by retroviral over-expression of a specific Rap1 GTPase activating protein, SPA-1, totally inhibited the LFA-1/ICAM-1-mediated cell adhesion. These results suggest that CD98 cross-linking activates LFA-1 via the PI3K signaling pathway and induces accumulation of Rap1GTP in a LFA-1-dependent manner, which in turn mediates the cytoskeleton-dependent cell adhesion process.     

The small GTPase Rap1 is required for Mn(2+)- and antibody-induced LFA-1- and VLA-4-mediated cell adhesion

In T-lymphocytes the Ras-like small GTPase Rap1 plays an essential role in stimulus-induced inside-out activation of integrin LFA-1 (alpha(L)beta(2)) and VLA-4 (alpha(4)beta(1)). Here we show that Rap1 is also involved in the direct activation of these integrins by divalent cations or activating antibodies. Inhibition of Rap1 either by Rap GTPase-activating protein (RapGAP) or the Rap1 binding domain of RalGDS abolished both Mn(2+)- and KIM185 (anti-LFA-1)-induced LFA-1-mediated cell adhesion to intercellular adhesion molecule 1. Mn(2+)- and TS2/16 (anti-VLA-4)-induced VLA-4-mediated adhesion were inhibited as well. Interestingly, both Mn(2+), KIM185 and TS2/16 failed to induce elevated levels of Rap1GTP. These findings indicate that available levels of GTP-bound Rap1 are required for the direct activation of LFA-1 and VLA-4. Pharmacological inhibition studies demonstrated that both Mn(2+)- and KIM185-induced adhesion as well as Rap1-induced adhesion require intracellular calcium but not signaling activity of the MEK-ERK pathway. Moreover, functional calmodulin signaling was shown to be a prerequisite for Rap1-induced adhesion. From these results we conclude that in addition to stimulus-induced inside-out activation of integrins, active Rap1 is required for cell adhesion induced by direct activation of integrins LFA-1 and VLA-4. We suggest that Rap1 determines the functional availability of integrins for productive binding to integrin ligands.     

Rap1-mediated lymphocyte function-associated antigen-1 activation by the T cell antigen receptor is dependent on phospholipase C-gamma1

The small GTPase, Rap1, is a potent activator of leukocyte integrins and enhances the adhesive activity of lymphocyte function-associated antigen-1 (LFA-1) when stimulated by the T cell receptor (TCR) or chemokines. However, the mechanism by which Rap1 is activated remains unclear. Here, we demonstrate that phospholipase C (PLC)-gamma1 plays a critical role in the signaling pathway leading to Rap1 activation triggered by the TCR. In Jurkat T cells, TCR cross-linking triggered persistent Rap1 activation, and SDF-1 (CXCL12) activated Rap1 transiently. A phospholipase C inhibitor, U73122, abrogated Rap1 activation triggered by both the TCR and SDF-1 (CXCL12). PLC-gamma1-deficient Jurkat T cells showed a marked reduction of TCR-triggered Rap1 activation and adhesion to intercellular adhesion molecule-1 (ICAM-1) mediated by LFA-1. In contrast, SDF-1-triggered Rap1 activation and adhesion were not affected in these cells. Transfection of these cells with an expression plasmid encoding PLC-gamma1 restored Rap1 activation by the TCR and the ability to adhere to ICAM-1, accompanied by polarized LFA-1 surface clustering colocalized with regulator of adhesion and polarization enriched in lymphoid tissues (RAPL). Furthermore, when expressed in Jurkat cells, CalDAG-GEFI, a calcium and diacylglycerol-responsive Rap1 exchange factor, associated with Rap1, and resulted in enhanced Rap1 activation and adhesion triggered by the TCR. Our results demonstrate that TCR activation of Rap1 depends on PLC-gamma1. This activity is likely to be mediated by CalDAG-GEFI, which is required to activate LFA-1.     

Talin1 regulates TCR-mediated LFA-1 function

The leukocyte integrin LFA-1 plays a critical role in T cell trafficking and T cell adhesion to APCs. It is known that integrin-mediated adhesion is regulated by changes in integrin ligand-binding affinity and valency through inside-out signaling. However, the molecular mechanisms involved in TCR-mediated LFA-1 regulation are not well understood. In this study, we show that the cytoskeletal protein talin1 is required for TCR-mediated activation of LFA-1 through regulation of LFA-1 affinity and clustering. Depletion of talin1 from human T cells by small interfering RNAs impairs TCR-induced adhesion to ICAM-1 and T cell-APC conjugation. TCR-induced LFA-1 polarization, but not actin polarization, is defective in talin1-deficient T cells. Although LFA-1 affinity is also reduced in talin1-deficient T cells, rescue of LFA-1 affinity alone is not sufficient to restore LFA-1 adhesive function. Together, our findings indicate that TCR-induced up-regulation of LFA-1-dependent adhesiveness and resulting T cell-APC conjugation require talin1.     

The small GTPase, Rap1, mediates CD31-induced integrin adhesion

Integrin-mediated leukocyte adhesion is a critical aspect of leukocyte function that is tightly regulated by diverse stimuli, including chemokines, antigen receptors, and adhesion receptors. How cellular signals from CD31 and other adhesion amplifiers are integrated with those from classical mitogenic stimuli to regulate leukocyte function remains poorly understood. Here, we show that the cytoplasmic tail of CD31, an important integrin adhesion amplifier, propagates signals that induce T cell adhesion via beta1 (VLA-4) and beta2 (LFA-1) integrins. We identify the small GTPase, Rap1, as a critical mediator of this effect. Importantly, CD31 selectively activated the small Ras-related GTPase, Rap1, but not Ras, R-Ras, or Rap2. An activated Rap1 mutant stimulated T lymphocyte adhesion to intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), as did the Rap1 guanine nucleotide exchange factor C3G and a catalytically inactive mutant of RapGAP. Conversely, negative regulators of Rap1 signaling blocked CD31-dependent adhesion. These findings identify a novel important role for Rap1 in regulating ligand-induced cell adhesion and suggest that Rap1 may play a more general role in coordinating adhesion-dependent signals during leukocyte migration and extravasation. Our findings also suggest an alternative mechanism, distinct from interference with Ras-proximal signaling, by which Rap1 might mediate transformation reversion.     

2E8 Binds to the High Affinity I-domain in a Metal Ion-dependent Manner: A SECOND GENERATION MONOCLONAL ANTIBODY SELECTIVELY TARGETING ACTIVATED LFA-1*

The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α_L subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K_D) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn²⁺ was replaced sequentially by Mg²⁺ and Ca²⁺. Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4⁺ and CD8⁺ T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.     

Ly6C induces clustering of LFA-1 (CD11a/CD18) and is involved in subtype-specific adhesion of CD8 T cells

Ly6C is a hemopoietic cell differentiation Ag found on a subset of CD8 T cells in the periphery. It is involved in target cell killing by CTLs, augments TCR-mediated activation of IL-2 and IFN-gamma production in CD8 T cells, and regulates CD8 T cell homing in vivo. In this study, we show that cross-linking of Ly6C causes clustering of LFA-1 (CD11a/CD18) on the surface of CD8 T cells via a mechanism dependent on reorganization of actin cytoskeleton and intracellular protease, calpain, but not the phosphatidylinositol 3-kinase pathway. In the capillary flow-adhesion assay, Ly6C cross-linking significantly augments lymphocyte adhesion to endothelium, and this is inhibited by an Ab that blocks LFA-1 function. Furthermore, upon in vitro cross-linking and during in vivo homing into lymph nodes, Ly6C is transiently lost from cell surface but becomes re-expressed on lymph node-resident CD8 T cells. The abilities of Ly6C to induce LFA-1 clustering and to be re-expressed after signaling-associated down-regulation may be important in regulating the homing of CD8 T cells into lymph nodes and in subsequent steps of CD8 T cell activation and effector function that again involve LFA-1.     

Essential role of the adhesion receptor LFA-1 for T cell-dependent fulminant hepatitis

Viral hepatitis affects more than 2 billion people worldwide. In particular, no effective treatment exists to abrogate death and liver damage in fulminant hepatitis. Activation of T cells is an initial and critical event in the pathogenesis of liver damage in autoimmune and viral hepatitis. The precise molecular mechanisms that induce T cell-mediated hepatocyte injury remain largely unclear. In mice, T cell-dependent hepatitis and acute liver damage can be modeled using ConA. In this study, we examined the role of the adhesion receptor LFA-1 in ConA-induced acute hepatic damage using LFA-1(-/-) (CD11a) mice. Massive liver cell apoptosis and metabolic liver damage were observed in LFA-1(+/+) mice following ConA injection. By contrast, LFA-1(-/-) mice were completely resistant to ConA-induced hepatitis and none of the LFA-1(-/-) mice showed any hepatic damage. Whereas activated hepatic T cells remained in the liver in LFA-1(+/+) mice, activated T cells were rapidly cleared from the livers of LFA-1(-/-) mice. Mechanistically, T cells from LFA-1(-/-) mice showed markedly reduced cytotoxicity toward liver cells as a result of impaired, activation-dependent adhesion. Importantly, adoptive transfer of hepatic T cells from LFA-1(+/+) mice, but not from LFA-1(-/-) mice, sensitized LFA-1(-/-) mice to ConA-induced hepatitis. Thus, LFA-1 expression on T cells is necessary and sufficient for T cell-mediated liver damage in vivo. These results provide the first genetic evidence on an adhesion receptor, LFA-1, that has a crucial role in fulminant hepatitis. These genetic data identify LFA-1 as a potential key target for the treatment of T cell-mediated hepatitis and the prevention of liver damage.     

HIV envelope gp120 activates LFA-1 on CD4 T-lymphocytes and increases cell susceptibility to LFA-1-targeting leukotoxin (LtxA)

The cellular adhesion molecule LFA-1 and its ICAM-1 ligand play an important role in promoting HIV-1 infectivity and transmission. These molecules are present on the envelope of HIV-1 virions and are integral components of the HIV virological synapse. However, cellular activation is required to convert LFA-1 to the active conformation that has high affinity binding for ICAM-1. This study evaluates whether such activation can be induced by HIV itself. The data show that HIV-1 gp120 was sufficient to trigger LFA-1 activation in fully quiescent naïve CD4 T cells in a CD4-dependent manner, and these CD4 T cells became more susceptible to killing by LtxA, a bacterial leukotoxin that preferentially targets leukocytes expressing high levels of the active LFA-1. Moreover, virus p24-expressing CD4 T cells in the peripheral blood of HIV-infected subjects were found to have higher levels of surface LFA-1, and LtxA treatment led to significant reduction of the viral DNA burden. These results demonstrate for the first time the ability of HIV to directly induce LFA-1 activation on CD4 T cells. Although LFA-1 activation may enhance HIV infectivity and transmission, it also renders the cells more susceptible to an LFA-1-targeting bacterial toxin, which may be harnessed as a novel therapeutic strategy to deplete virus reservoir in HIV-infected individuals.     

Cytoplasmic tails of beta 1, beta 2, and beta 7 integrins differentially regulate LFA-1 function in K562 cells.

The beta 2 integrin lymphocyte function-associated antigen 1 (LFA-1) mediates activation-dependent adhesion of lymphocytes. To investigate whether lymphocyte-specific elements are essential for LFA-1 function, we expressed LFA-1 in the erythroleukemic cell line K562, which expresses only the integrin very late antigen 5. We observed that LFA-1-expressing K562 cannot bind to intercellular adhesion molecule 1-coated surfaces when stimulated by phorbol 12-myristate 13-acetate (PMA), whereas the LFA-1-activating antibody KIM185 markedly enhanced adhesion. Because the endogenously expressed beta 1 integrin very late antigen 5 is readily activated by PMA, we investigated the role of the cytoplasmic domain of distinct beta subunits in regulating LFA-1 function. Transfection of chimeric LFA-1 receptors in K562 cells reveals that replacement of the beta 2 cytoplasmic tail with the beta 1 but not the beta 7 cytoplasmic tail completely restores PMA responsiveness of LFA-1, whereas a beta 2 cytoplasmic deletion mutant of LFA-1 is constitutively active. Both deletion of the beta 2 cytoplasmic tail or replacement by the beta 1 cytoplasmic tail alters the localization of LFA-1 into clusters, thereby regulating LFA-1 activation and LFA-1-mediated adhesion to intercellular adhesion molecule 1. These data demonstrate that distinct signaling routes activate beta 1 and beta 2 integrins through the beta-chain and hint at the involvement of lymphocyte-specific signal transduction elements in beta 2 and beta 7 integrin activation that are absent in the nonlymphocytic cell line K562.     

LFA-1 Affinity Regulation Is Necessary for the Activation and Proliferation of Naive T Cells

The activation of LFA-1 (lymphocyte function-associated antigen) is a critical event for T cell co-stimulation. The mechanism of LFA-1 activation involves both affinity and avidity regulation, but the role of each in T cell activation remains unclear. We have identified antibodies that recognize and block different affinity states of the mouse LFA-1 I-domain. Monoclonal antibody 2D7 preferentially binds to the low affinity conformation, and this specific binding is abolished when LFA-1 is locked in the high affinity conformation. In contrast, M17/4 can bind both the locked high and low affinity forms of LFA-1. Although both 2D7 and M17/4 are blocking antibodies, 2D7 is significantly less potent than M17/4 in blocking LFA-1-mediated adhesion; thus, blocking high affinity LFA-1 is critical for preventing LFA-1-mediated adhesion. Using these reagents, we investigated whether LFA-1 affinity regulation affects T cell activation. We found that blocking high affinity LFA-1 prevents interleukin-2 production and T cell proliferation, demonstrated by TCR cross-linking and antigen-specific stimulation. Furthermore, there is a differential requirement of high affinity LFA-1 in the activation of CD4⁺ and CD8⁺ T cells. Although CD4⁺ T cell activation depends on both high and low affinity LFA-1, only high affinity LFA-1 provides co-stimulation for CD8⁺ T cell activation. Together, our data demonstrated that the I-domain of LFA-1 changes to the high affinity state in primary T cells, and high affinity LFA-1 is critical for facilitating T cell activation. This implicates LFA-1 activation as a novel regulatory mechanism for the modulation of T cell activation and proliferation.LFA-1 (lymphocyte function-associated antigen), an integrin family member, is important in regulating leukocyte adhesion and T cell activation (1, 2). LFA-1 consists of the α_L (CD11a) and β₂ (CD18) heterodimer. The ligands for LFA-1, including intercellular adhesion molecule ICAM³-1, ICAM-2, and ICAM-3, are expressed on antigen-presenting cells (APCs), endothelial cells, and lymphocytes (1). Mice that are deficient in LFA-1 have defects in leukocyte adhesion, lymphocyte proliferation, and tumor rejection (3–5). Blocking LFA-1 with antibodies can prevent inflammation, autoimmunity, organ graft rejection, and graft versus host disease in human and murine models (6–10).LFA-1 is constitutively expressed on the surface of leukocytes in an inactive state. Activation of LFA-1 is mediated by inside-out signals from the cytoplasm (1, 11). Subsequently, activated LFA-1 binds to the ligands and transduces outside-in signals back into the cytoplasm that result in cell adhesion and activation (12, 13). The activation of LFA-1 is a critical event in the formation of the immunological synapse, which is important for T cell activation (2, 14, 15). The active state of LFA-1 is regulated by chemokines and the T cell receptor (TCR) through Rap1 signaling (16). LFA-1 ligation lowers the activation threshold and affects polarization in CD4⁺ T cells (17). Moreover, productive LFA-1 engagement facilitates efficient activation of cytotoxic T lymphocytes and initiates a distinct signal essential for the effector function (18–20). Thus, LFA-1 activation is essential for the optimal activation of T cells.The mechanism of LFA-1 activation involves both affinity (conformational changes within the molecule) and avidity (receptor clustering) regulation (21–23). The I-domain of the LFA-1 α_L subunit is the primary ligand-binding site and has been proposed to change conformation, leading to an increased affinity for ligands (24–26). The structural basis of the conformational changes in the I-domain of LFA-1 has been extensively characterized (27). Previously, we have demonstrated that the conformation of the LFA-1 I-domain changes from the low affinity to the high affinity state upon activation. By introducing disulfide bonds into the I-domain, LFA-1 can be locked in either the closed or open conformation, which represents the “low affinity” or “high affinity” state, respectively (28, 29). In addition, we identified antibodies that are sensitive to the affinity changes in the I-domain of human LFA-1 and showed that the activation-dependent epitopes are exposed upon activation (30). This study supports the presence of the high affinity conformation upon LFA-1 activation in cell lines. It has been demonstrated recently that therapeutic antagonists, such as statins, inhibit LFA-1 activation and immune responses by locking LFA-1 in the low affinity state (31–34). Furthermore, high affinity LFA-1 has been shown to be important for mediating the adhesion of human T cells (35, 36). Thus, the affinity regulation is a critical step in LFA-1 activation.LFA-1 is a molecule of great importance in the immune system, and its activation state influences the outcome of T cell activation. Our previous data using the activating LFA-1 I-domain-specific antibody MEM83 indicate that avidity and affinity of the integrin can be coupled during activation (37). However, whether affinity or avidity regulation of LFA-1 contributes to T cell activation remains controversial (23, 38, 39). Despite the recent progress suggesting that conformational changes represent a key step in the activation of LFA-1, there are considerable gaps to be filled. When LFA-1 is activated, the subsequent outside-in signaling contributes to T cell activation via immunological synapse and LFA-1-dependent signaling. It is critical to determine whether high affinity LFA-1 participates in the outside-in signaling and affects the cellular activation of T cells. Nevertheless, the rapid and dynamic process of LFA-1 activation has hampered further understanding of the role of high affinity LFA-1 in primary T cell activation. The affinity of LFA-1 for ICAM-1 increases up to 10,000-fold within seconds and involves multiple reversible steps (23). In addition, the activation of LFA-1 regulates both adhesion and activation of T cells, two separate yet closely associated cellular functions. When LFA-1 is constitutively expressed in the active state in mice, immune responses are broadly impaired rather than hyperactivated, suggesting the complexity of affinity regulation (40). Therefore, it is difficult to dissect the mechanisms by which high affinity LFA-1 regulates stepwise activation of T cells in the whole animal system.In the present study, we identified antibodies recognizing and blocking different affinity states of mouse LFA-1. These reagents allowed us to determine the role of affinity regulation in T cell activation. We found that blocking high affinity LFA-1 inhibited IL-2 production and proliferation in T cells. Furthermore, there is a differential requirement of high affinity LFA-1 in antigen-specific activation of CD4⁺ and CD8⁺ T cells. The activation of CD4⁺ T cells depends on both high and low affinity LFA-1. For CD8⁺ T cell activation, only high affinity LFA-1 provides co-stimulation. Thus, affinity regulation of LFA-1 is critical for the activation and proliferation of naive T cells.     

Rap1 Functions as a Key Regulator of T-Cell and Antigen-Presenting Cell Interactions and Modulates T-Cell Responses

Activation of T cells by antigen requires adhesive interactions with antigen-presenting cells (APC) in which leukocyte function-associated antigen 1 (LFA-1) and intercellular adhesion molecules (ICAMs) are important. However, it is not well understood what signaling molecules regulate this process and how the modulation of adhesive events influences T-cell activation. Here we show that Rap1 is activated in T cells in an antigen-dependent manner and accumulated at the contact site of T-cell and antigen-loaded APC. Inhibition of Rap1 activation by a dominant-negative Rap1 or SPA-1, a Rap1 GTPase-activating protein, abrogates LFA-1-ICAM-1-mediated adhesive interactions with antigen-pulsed APC and the subsequent T-cell-receptor triggering and interleukin-2 production. Conversely, augmented antigen-dependent Rap1 activation by the expression of wild-type Rap1 enhances these responses but culminates in apoptosis by Fas and FasL. Thus, Rap1 functions as a key regulator of T-cell and APC interactions and modulates T-cell responses from productive activation to activation-induced cell death by regulating the strength of adhesive interactions. Moreover, constitutive Rap1 activation rendered T cells unresponsive with accumulation of p27(Kip1). Our study indicates that the activation state of Rap1 has a decisive effect on the T-cell response to antigen.     

Activation of LFA-1 by ionomycin is independent of calpain-mediated talin cleavage

Activation of calpains by calcium flux leading to talin cleavage is thought to be an important process of LFA-1 activation by inside-out signalling. Here, we tested the effects of the calcium ionophore ionomycin and calpain inhibitor calpeptin on LFA-1-mediated adhesion of a T cell hybridoma line, cytotoxic T cells and primary resting T cells. Ionomycin activated LFA-1-mediated adhesion of all three types of T cells, and calpeptin inhibited the effects of ionomycin. However, calpeptin also inhibited activation of LFA-1 by PMA, which did not induce calcium flux. Cleavage of talin was undetectable in ionomycin-treated T cells. Furthermore, treatment with ionomycin and calpeptin induced apoptosis of T cells. Inhibitors of phosphatidyl Inositol-3 kinase inhibited activation of LFA-1 by ionomycin, but not by PMA, whereas the protein kinase C inhibitor inhibited the effects of PMA, but not ionomycin. Thus, activation of LFA-1 by ionomycin is independent of calpain-mediated talin cleavage.     

CD18 is required for optimal development and function of CD4+CD25+ T regulatory cells

CD4+CD25+ T regulatory (Treg) cells inhibit immunopathology and autoimmune disease in vivo. CD4+CD25+ Treg cells' capacity to inhibit conventional T cells in vitro is dependent upon cell-cell contact; however, the cell surface molecules mediating this cell:cell contact have not yet been identified. LFA-1 (CD11a/CD18) is an adhesion molecule that plays an established role in T cell-mediated cell contact and in T cell activation. Although expressed at high levels on murine CD4+CD25+ Treg cells, the role of LFA-1 in these cells has not been defined previously. We hypothesized that LFA-1 may play a role in murine CD4+CD25+ Treg function. To evaluate this, we analyzed LFA-1-deficient (CD18-/-) CD4+CD25+ T cells. We show that CD18-/- mice demonstrate a propensity to autoimmunity. Absence of CD18 led to diminished CD4+CD25+ T cell numbers and affected both thymic and peripheral development of these cells. LFA-1-deficient CD4+CD25+ T cells were deficient in mediating suppression in vitro and in mediating protection from colitis induced by the transfer of CD4+CD25- T cells into lymphopenic hosts. Therefore, we define a crucial role for CD18 in optimal CD4+CD25+ Treg development and function.     

F-actin flow drives affinity maturation and spatial organization of LFA-1 at the immunological synapse

Integrin-dependent interactions between T cells and antigen-presenting cells are vital for proper T cell activation, effector function, and memory. Regulation of integrin function occurs via conformational change, which modulates ligand affinity, and receptor clustering, which modulates valency. Here, we show that conformational intermediates of leukocyte functional antigen 1 (LFA-1) form a concentric array at the immunological synapse. Using an inhibitor cocktail to arrest F-actin dynamics, we show that organization of this array depends on F-actin flow and ligand mobility. Furthermore, F-actin flow is critical for maintaining the high affinity conformation of LFA-1, for increasing valency by recruiting LFA-1 to the immunological synapse, and ultimately for promoting intracellular cell adhesion molecule 1 (ICAM-1) binding. Finally, we show that F-actin forces are opposed by immobilized ICAM-1, which triggers LFA-1 activation through a combination of induced fit and tension-based mechanisms. Our data provide direct support for a model in which the T cell actin network generates mechanical forces that regulate LFA-1 activity at the immunological synapse.