Jason S. Mitchell

WAVE2 Regulates High-Affinity Integrin Binding by Recruiting Vinculin and Talin to the Immunological Synapse

ABSTRACT T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin.

The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL–C3G-mediated activation of Rap1

WAVE2 regulates T cell receptor (TCR)–stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL–C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL–C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.

Lipid Microdomain Clustering Induces a Redistribution of Antigen Recognition and Adhesion Molecules on Human T Lymphocytes

The study of lipid microdomains in the plasma membrane is a topic of recent interest in leukocyte biology. Many T cell activation and signaling molecules are found to be associated with lipid microdomains and have been implicated in normal T cell function. It has been proposed that lipid microdomains with their associated molecules move by lateral diffusion to areas of cellular interactions to initiate signaling pathways. Using sucrose density gradients we have found that human T cell β1 integrins are not normally associated with lipid microdomains. However, cross-linking of GM1 through cholera toxin B-subunit (CTB) causes an enrichment of β1 integrins in microdomain fractions, suggesting that cross-linking lipid microdomains causes a reorganization of molecular associations. Fluorescent microscopy was used to examine the localization of various lymphocyte surface molecules before and after lipid microdomain cross-linking. Lymphocytes treated with FITC-CTB reveal an endocytic vesicle that is enriched in TCR and CD59, while β1 integrin, CD43, and LFA-3 were not localized in the vesicle. However, when anti-CTB Abs are used to cross-link lipid microdomains, the microdomains are not internalized but are clustered on the cell surface. In this study, CD59, CD43, and β1 integrin are all seen to colocalize in a new lipid microdomain from which LFA-3 remains excluded and the TCR is now dissociated. These findings show that cross-linking lipid microdomains can cause a dynamic rearrangement of the normal order of T lymphocyte microdomains into an organization where novel associations are created and signaling pathways may be initiated.

Control of α4β7 Integrin Expression and CD4 T Cell Homing by the β1 Integrin Subunit

The α4β7 integrin promotes homing of T cells to intestinal sites. The α4 integrin subunit that pairs with β7 integrin can also pair with β1 integrin. In this paper, we show that the preferential pairing of β1 integrin with α4 integrin regulates the expression of α4β7 on T cells. In the absence of β1 integrin, naive mouse CD4 T cells have increased α4β7 expression, resulting in increased adhesion to mucosal addressin cell adhesion molecule-1 and enhanced homing to Peyer’s patches (PP). In a reciprocal manner, overexpression of β1 integrin causes the loss of α4β7 expression and decreased homing to PP. A similar upregulation of β1 integrin and suppression of α4β7 expression occurs rapidly after CD4 T cell activation. β1 integrin thus dominates β7 integrin for α4 integrin pairing, thereby controlling the abundance of unpaired α4 integrin. Increasing the abundance of α4 integrin relative to β1 integrin is critical to retinoic acid-mediated expression of α4β7 integrin during T cell activation. In the absence of β1 integrin, endogenous Ag-specific CD4 T cells uniformly express high levels of α4β7 after Listeria monocytogenes infection. The resulting β1-deficient early memory T cells have decreased localization to the bone marrow and enhanced localization to PP after infection. Thus, the preferential association of β1 integrin with α4 integrin suppresses α4β7 integrin expression and regulates the localization of memory CD4 T cells.

The p110γ isoform of phosphatidylinositol 3-kinase regulates migration of effector CD4 T lymphocytes into peripheral inflammatory sites

The role of PI‐3K in leukocyte function has been studied extensively. However, the specific role of the p110γ isoform of PI‐ 3K in CD4 T lymphocyte function has yet to be defined explicitly. In this study, we report that although p110γ does not regulate antigen‐dependent CD4 T cell activation and proliferation, it plays a crucial role in regulating CD4 effector T cell migration. Naïve p110γ−/− CD4 lymphocytes are phenotypically identical to their wild‐type (WT) counterparts and do not exhibit any defects in TCR‐mediated calcium mobilization or Erk activation. In addition, p110γ‐deficient CD4 OT.II T cells become activated and proliferate comparably with WT cells in response to antigen in vivo. Interestingly, however, antigen‐experienced, p110γ‐deficient CD4 OT.II lymphocytes exhibit dramatic defects in their ability to traffic to peripheral inflammatory sites in vivo. Although antigen‐activated, p110γ‐deficient CD4 T cells express P‐selectin ligand, β2 integrin, β1 integrin, CCR4, CXCR5, and CCR7 comparably with WT cells, they exhibit impaired F‐actin polarization and migration in response to stimulation ex vivo with the CCR4 ligand CCL22. These findings suggest that p110γ regulates the migration of antigen‐experienced effector CD4 T lymphocytes into inflammatory sites during adaptive immune responses in vivo.

Physical association and functional interaction between β1 integrin and CD98 on human T lymphocytes

CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.

Contrasting Roles for Domain 4 of VCAM-1 in the Regulation of Cell Adhesion and Soluble VCAM-1 Binding to Integrin α4β1

Cell adhesion mediated by the interaction between integrin α4β1 and VCAM-1 is important in normal physiologic processes and in inflammatory and autoimmune disease. Numerous studies have mapped the α4β1 binding sites in VCAM-1 that mediate cell adhesion; however, little is known about the regions in VCAM-1 important for regulating soluble binding. In the present study, we demonstrate that 6D VCAM-1 (an alternatively spliced isoform of VCAM-1 lacking Ig-like domain 4) binds α4β1 with a higher relative affinity than does the full-length form of VCAM-1 containing 7 Ig-like extracellular domains (7D VCAM-1). In indirect binding assays, the EC50 of soluble 6D VCAM-1 binding to α4β1 on Jurkat cells (in 1 mM MnCl2) was 2 × 10−9 M, compared with 7D VCAM-1 at 11 × 10−9 M. When used in solution to inhibit α4β1 mediated cell adhesion, the IC50 of 6D VCAM-1 was 13 × 10−9 M, compared with 7D VCAM-1 measured at 150 × 10−9 M. Removal of Ig-like domains 4, 5, or 6, or simply substituting Asp328 in domain 4 of 7D VCAM-1 with alanine, caused increased binding of soluble 7D VCAM-1 to α4β1. In contrast, cells adhered more avidly to 7D VCAM-1 under shear force, as it induced cell spreading at lower concentrations than did 6D VCAM-1. Finally, soluble 6D VCAM-1 acts as an agonist through α4β1 by augmenting cell migration and inducing cell aggregation. These results indicate that the domain 4 of VCAM-1 plays a contrasting role when VCAM-1 is presented in solution or as a cell surface-expressed adhesive substrate.

Clustering T-cell GM1 lipid rafts increases cellular resistance to shear on fibronectin through changes in integrin affinity and cytoskeletal dynamics

Lipid rafts are small laterally mobile microdomains that are highly enriched in lymphocyte signaling molecules. GM1 gangliosides are a common lipid raft component and have been shown to be important in many T‐cell functions. The aggregation of specific GM1 lipid rafts can control many T‐cell activation events, including their novel association with T‐cell integrins. We found that clustering GM1 lipid rafts can regulate β1 integrin function. This was apparent through increased resistance to shear flow‐dependent detachment of T cells adherent to the α4β1 and α5β1 integrin ligand fibronectin (FN). Adhesion strengthening as a result of clustering GM1 enriched lipid rafts correlated with increased cellular rigidity and morphology through the localization of cortical F‐actin, the resistance to shear‐induced cell stretching, and an increase in the surface area and symmetry of the contact area between the cell surface and adhesive substrate. Furthermore, clustering GM1 lipid rafts could initiate integrin ‘inside‐out’ signaling mechanisms. This was seen through increased integrin–cytoskeleton associations and enhanced soluble binding of FN and VCAM‐1, suggesting the induction of high‐affinity integrin conformations. The activation of these adhesion‐strengthening characteristics appears to be specific for the aggregation of GM1 lipid rafts as the aggregation of the heterogeneous raft‐associated molecule CD59 failed to activate these functions. These findings indicate a novel mechanism to signal to β1 integrins and to activate adhesion‐strengthening processes.

Intravital mucosal imaging of CD8 + resident memory T cells shows tissue-autonomous recall responses that amplify secondary memory

CD8+ T cell immunosurveillance dynamics influence the outcome of intracellular infections and cancer. Here we used two-photon intravital microscopy to visualize the responses of CD8+ resident memory T cells (TRM cells) within the reproductive tracts of live female mice. We found that mucosal TRM cells were highly motile, but paused and underwent in situ division after local antigen challenge. TRM cell reactivation triggered the recruitment of recirculating memory T cells that underwent antigen-independent TRM cell differentiation in situ. However, the proliferation of pre-existing TRM cells dominated the local mucosal recall response and contributed most substantially to the boosted secondary TRM cell population. We observed similar results in skin. Thus, TRM cells can autonomously regulate the expansion of local immunosurveillance independently of central memory or proliferation in lymphoid tissue.Masopust and colleagues show that mucosal tissue-resident memory T cells proliferate in situ in response to local antigen and dominate the local recall response.

Diverse roles of integrins in human T lymphocyte biology

T lymphocytes are the primary cells responsible for maintaining the immune system. There are many intricate mechanisms involved in the regulation of T cells and the integrin family of adhesive surface proteins plays a pivitol role in the control of T lymphocyte activation and functions. Integrins are heterodimeric transmembrane proteins that are not merely adhesion molecules but also function in T cell coactivation by providing a scaffold for signaling and cytoskeletal proteins that are adept at transmitting signals from the inside of the cell to the outside (“inside-out signaling”) or from the outside of the cell to the inside (“outside-in signaling”). The signaling property of integrins allows for rapid responses to changes T cell needs to adhere or detach, integrins can quickly accommodate either state of the cell. Once cells are guided to sites of infection, inflammation, or antigen presentation, integrins can also participate in the initiation, maintenance, or termination of the response. This review will focus on the aspects of integrin-mediated T cell coactivation, affinity and avidity control of integrins, signaling molecules involved with integrins, association of integrins in lipid microdomains, and negative regulation of integrins.