Not Just an Adhesion Molecule: LFA-1 Contact Tunes the T Lymphocyte Program

T lymphocytes are the most important component of the immune system and play an indispensible role in the host defense against infection and in the maintenance of immune homeostasis. The timely and targeted migration of T lymphocytes (also called “homing”) through peripheral tissues, secondary lymphoid organs, and into lymphatic vessels or certain vascularized tissues is crucial for immune surveillance. The process of T cell movement continues throughout the lifetime of an immune response, even as it shifts from lymphoid organs to individual target tissues or organs (1). Thus, the migration of T lymphocytes is intrinsically coupled with T cell activation, differentiation, and effector functions (2–4), in addition to its roles in infection control, autoimmunity, allergy, alloreactivity, and tumor eradication. T cells use multiple approaches to maintain self-immune tolerance; such functions are potentially critical for avoiding the pathogenesis of deleterious inflammatory conditions that can result in tissue damage.

T lymphocytes strongly express the α_Lβ₂ integrin LFA-1 (CD11a/CD18), and like other integrins, activated LFA-1 signals bidirectionally: outside-in signaling, in which signals flow from the extracellular stimuli to induce intracellular changes, and inside-out signaling, in which intracellular stimuli can cause extracellular changes and regulate the adhesive state of LFA-1. Depending on its activation status, LFA-1 displays various conformational states, ranging from the low-affinity bent conformation to the high-affinity open conformation (5–7). In its low-affinity conformation or inactive form, the LFA-1 receptor does not bind to its counter ligands and does not signal, which is particularly important for T lymphocytes for circulation through the bloodstream with minimal interactions with ligands normally present in blood, on the vessel wall, or endothelium to avoid inappropriate cell activation that may be life threatening. To leave the circulation, L-selectin on T lymphocytes interacts with its glycosylated ligand present on the endothelium, or ligands on T lymphocytes bind to E- or P-selectin expressed on the stimulated endothelium. Following these initial interactions, T lymphocytes attach loosely to the endothelium and begins to roll along the vascular wall (8, 9). Chemokines secreted by the endothelial cells or the surrounding tissue shift the LFA-1 receptor into a high-affinity conformation (i.e., active state) (5, 6). Activated LFA-1 receptor interacts with ICAMs, primarily ICAM-1, which is highly induced on endothelial surfaces within an inflamed tissue or high endothelial venules. This interaction between LFA-1 and ICAM-1 initiates a plethora of downstream signaling cascades that culminate in dynamic cytoskeletal remodeling (5–15), which is important for T cell motility. In addition, T lymphocytes use several other modes of adhesive interactions to attach and roll along the vascular wall. These include the α₄β₁ integrin VLA-4 (CD49d/CD29) and α₄β₇ integrin on T cells that bind to their corresponding ligands VCAM-1 and mucosal vascular addressin cell adhesion molecule-1 on endothelial cells (14, 16–18). Activated T cells use VLA-4 for capture, rolling on, and firm adhesion to endothelial surfaces, and they use LFA-1 for subsequent crawling and extravasation (19). LFA-1 interacts with VLA-4 in activated T cells and dominates during the late stages of adhesion on the endothelium (19–24). In the spinal cord, Th1 cells preferentially use VLA-4 for infiltration, whereas Th17 cells use LFA-1 to enter the brain parenchyma (25). Binding of the cell surface glycoprotein CD44 to the endothelial hyaluronan is also important for T lymphocyte extravasation into inflamed tissues (26).

At the cellular level, LFA-1 is best known for its key role in T cell trafficking and extravasation, which are essential for the normal functioning of the immune system, as well as some pathological immune responses. The adhesiveness of LFA-1 in circulating T lymphocytes is tightly regulated to ensure that adhesive interactions between LFA-1 and its cognate ligand occur only after stimulation with external stimuli, such as cytokines, chemokines, or specific Ags, through inside-out signaling. The inside-out signaling cascades increase ligand-binding affinity of LFA-1 receptors and cause their redistribution on the T cell surface into highly organized patch-like clusters (6). One of the main components of inside-out signaling cascades that is capable of activating LFA-1 is the small GTPase Rap1, which cycles between the active GTP-bound form and the inactive GDP-bound form (27–30). The GTP-bound active Rap1 in stimulated T lymphocytes recruits the effector proteins Rap1-interacting adaptor molecule (RIAM) and RAPL to Talin1, resulting in the association of Talin with the LFA-1 β₂ cytoplasmic tail and subsequent conformational changes in LFA-1 (31–38). Consequently, the LFA-1 receptor is activated and binds to its ligand, ICAM-1, on the endothelium. Thus, the Rap1–RIAM–Talin inside-out signaling axis plays an essential role in the regulation of LFA-1 activation and adhesion strength between T cell and endothelium and subsequent migration of the T lymphocyte.

Stimulation of the LFA-1 receptor following contact with ICAM-1 on endothelial surfaces, in the absence of immune synapse formation, triggers a vast array of context-specific downstream signaling cascades, leading to a variety of outcomes that are dependent on the local cytokine milieu and the cell of contact. Because the intracellular domain of LFA-1 receptor has no catalytic function (39, 40), its spatiotemporal interaction with other transducing molecules is crucial for downstream signaling. The molecular signals initiated following LFA-1 contact are propagated to the T cell interior via the activation/deactivation of kinases and phosphatases and the formation of signaling complexes, which mediate intracellular signal transduction and cellular responses. The “immediate to short-term” intracellular changes arising from LFA-1/ICAM-1 ligation in T lymphocytes are activation of kinases, phosphatases, and other enzymes, phosphorylation or dephosphorylation of specific substrates, redistribution of certain signaling proteins leading to dynamic rearrangements of cytoskeletal systems, and the initiation of migration. In the “medium-term,” LFA-1 contact induces changes in gene expression that could influence T cell differentiation and effector functions. In the “long-term,” LFA-1 signal can potentially dictate the development and maintenance of immunological memory, particularly in the context of TCR engagement at the immune synapse (41). A dual role for LFA-1/ICAM-1 in tuning the effector response and programming the sensitivity of memory CD8 T cells to secondary stimuli has been reported (42). LFA-1 costimulation with TCR also plays a crucial role in cell fate choices between differentiating αβ and γδ T cells. On TCR engagement and subsequent activation via the Rap1–RIAM–Talin signaling axis, LFA-1 can program mature T cells for growth promotion, growth inhibition, and cell death. For example, LFA-1 signal has been found to be a key factor in the apoptosis of peripheral γδ T cells upon TCR cross-linking (43) (Fig. 1).

Following stimulation and ligand binding, LFA-1 triggers an array of interconnected signaling pathways involving kinases, phosphatases, adaptor proteins, and metabolic mediators that coordinate remodeling of the cytoskeletal systems and T cell locomotion (5–14). Major early players in the outside-in signaling include proteins that are bound to the LFA-1 cytoplasmic tails, such as VAV1, Talin1, kindlin3, RAP1, Lck, Syk family tyrosine kinase ζ-chain–associated protein kinase of 70 kDa (ZAP70), and a coactivator of the c-Jun transcription factor Jun-activation domain-binding protein 1 (JAB1) (7, 15, 39, 44–46). Engagement of LFA-1 with ICAM-1 causes these early mediators to become phosphorylated (or autophosphorylated), activated, or dissociated to form discrete signaling complexes, thereby initiating a cascade of further downstream signal transduction. In a polarized migrating T cell, ZAP70 colocates with activated LFA-1 and regulates the speed of migration in response to the LFA-1 signal (13). Blocking ZAP70 function prevents the conversion of LFA-1 into the high-affinity conformation (13), which indicates that active phosphorylation of ZAP70 is required for the inside-out signaling and conformational shift that regulates the ligand-binding affinity of LFA-1 from intermediate to high. Of note, unlike platelets and myeloid cells, “phosphorylation-primed” Lck (47) and ZAP70 kinases are directly associated with LFA-1 in T cells (6). The direct association of Lck/ZAP70 with LFA-1 helps to control the high-affinity LFA-1 conformation at the time of T cell arrest on the vasculature that happens very quickly (in milliseconds). The LFA-1/Lck/ZAP70 complex recruits other proteins, such as Talin1, kindlin3, Arp2/3, vinculin, and WASP, to the lamella of the polarized T cell (44). This multiprotein clustering ultimately regulates dynamic cytoskeletal remodeling in migrating T cells and other downstream signals, such as PLC-γ. PLC-γ catalyzes the generation of second messengers, which trigger calcium release and the subsequent activation of PKC, Akt, and MAPK signaling pathways (6). In addition, several adaptor proteins that facilitate intracellular protein–protein interactions have been implicated in LFA-1 outside-in signaling (44). These adaptor proteins include SLP-76, ADAP, cytohesin-1, c-Cbl, and centrosome and Golgi localized protein kinase N-associated protein (also called AKAP450) (44, 48–55). This “ready to fire” protein complex and the subsequent activation of signaling facilitate rapid motility of T cells.

Likewise, LFA-1 engagement activates signaling pathways during the formation of the immunological synapse, the specialized structure that forms between T lymphocytes and APCs during T cell activation (55). LFA-1 forms the peripheral ring of the immunological synapse (56). LFA-1 stimulation in T cells within the immune synapse activates Src family tyrosine kinase Lck, leading to the phosphorylation of ITAMs in the TCR/CD3 complex (13). This phosphorylation event leads to the recruitment and activation of ZAP70 to the immune synapse (13). Lck activation also activates PI3K, which catalyzes the generation of phosphatidylinositol (3-5)-trisphosphate lipids that interact with and recruit ITK onto the plasma membrane (46). ITK then interacts with and phosphorylates several adaptor proteins, including LAT and SLP-76, which are critical for efficient signal transduction and activation of TCR signaling (44).

Although the pathways and processes involved in LFA-1–mediated lymphocyte migration and in the immune synapse are increasingly well understood, a number of studies have expanded the realm of LFA-1 signaling in the modulation of gene expression and T cell effector functions, including development and differentiation and, in particular, the modulation of cytokine signaling (3, 4, 56–62). For example, work from our laboratories has demonstrated that the LFA-1 signal plays a potent role in gene induction, with the generation of a pattern of gene expression creating a functional profile characterized by refractoriness to TGF-β (4). In addition, an inhibitory cross-talk between LFA-1 and other receptors, such as VLA-4, has been reported (20, 21). In particular, inside-out signaling following the activation of TCR causes phosphorylation of LFA-1 β₂-chain at Thr⁷⁸⁸, reducing binding between VLA-4 and 14-3-3 protein and, consequently, inhibiting VLA-4–mediated adhesion and migration (21). Furthermore, the LFA-1/ICAM-1 signal also induces Notch-mediated polarization of the T cell functional phenotype toward a Th1 profile (3).

A crucial role for LFA-1 in regulating the development, immune homeostasis, and functions of regulatory T cells (Tregs) has been demonstrated (63–65). For example, in LFA^−/− mice, LFA-deficient CD4⁺CD25⁺ Tregs failed to mediate suppression in vitro and protection from colitis induced by the transfer of CD4⁺CD25⁻ T cells into lymphopenic hosts in vivo (63, 64). LFA-1 deficient (CD18^−/−) mice showed a propensity for autoimmunity (63). Additionally, a mechanism involving upregulation of the intestinal-specific trafficking molecules α4β7 and CCR9, LFA-1 has been found to play a role in the optimal proliferation of CD4⁺ T cells upon oral Ag immunization in mice (66). LFA-1 engagement provided sufficient costimulatory signals to T cells and induced IL-2 gene expression and T cell activation (67). Furthermore, LFA-1 decreases the Ag dose for T cell activation, lowers the threshold of TCR signals for low-affinity ligands, as well as signaling through cytohesin-1 and JAB1, and cooperates with TCR in sustaining Erk1/2 phosphorylation, thus delivering costimulatory signals in T lymphocytes (62, 68, 69). Earlier studies using TCR-transgenic mice demonstrated that simultaneous blockade of ICAM-1 and ICAM-2 resulted in DO11.10-TCR mice with a 100–1000-fold increase in Th2 cytokine production (61), although this may reflect exaggerated changes in a murine transgenic model. A more recent study demonstrated that LFA-1/ICAM-1 interactions are crucial for liver-resident T cells to patrol and remain in the hepatic sinusoids (70). Such findings reveal limitations in the understanding of earlier paradigms of LFA-1’s role in T cell motility and strongly support the notion that, on contact with ICAM-1, the LFA-1 signal is not just a receptor for T lymphocyte locomotion but is also a rheostat that can fine-tune cellular responses, leading to a variety of outcomes, potentially during the process of cell migration.

In this review, we argue that, in addition to regulating T lymphocyte migration, LFA-1 contact defines T cell functions that are dependent on the local cytokine milieu and the cell of contact. Within this context, we discuss the progression of our knowledge of LFA-1 signaling beyond its role in T cell migration and outline important considerations. We also draw attention to several gaps in the current understanding of how the temporal nature of LFA-1 contact shapes T cell functioning.

Previous studies on various integrins highlight that, in addition to adhesion, these receptors regulate cell migration, proliferation, cell-fate determination, cell cycle progression, apoptosis prevention, and cell survival and differentiation in diverse cell types (71–77). Binding of activated integrins to their ligands results in a number of signaling, structural, and phenotypic changes within the cell. Several multiprotein complexes assemble onto integrins’ cytoplasmic tails to engage and reorganize the cytoskeletal systems, as well as to facilitate the transmission of signaling cascades that ultimately lead to changes in the expression of certain genes. For example, VLA-4 regulates G9a activity that controls epigenetic changes and the nuclear properties that are required for lymphocyte migration (73). In mammary epithelium, β₁ integrin plays a major role in controlling differentiation and development, as well as in the cellular programming of epithelial functions (74, 75). In addition, in breast carcinoma cells, integrin α₆β₄ regulates a coordinated genetic program, including the expression of genes, such as metastasis-associated S100A4, which predispose the cell to migratory and invasive phenotypes (76). β₄ integrin regulates expression of secreted protein acidic and rich in cysteine protein that promotes cell invasion (77). In transformed keratinocytes, integrin α₃β₁ regulates the expression of fibulin-2, contributing to the invasive phenotypes (78). Integrins play a crucial role in the expression of endothelin-1 in endothelial cells (79). These receptors also regulate gene expression in luminal cells (72). In polymorphonuclear neutrophils, β₂ integrin family receptors control cytokine gene expression during the inflammatory response (60). Another study identified a set of gene transcripts that were differentially expressed on β7⁺ versus β7⁻ blood memory CD4⁺ T cell subsets, including adhesion receptors (α₄ integrin and L-selectin) and chemokine receptors (CCR9 and CCR10) (80). Of note, a low level of α₄β₇ integrin is expressed on naive T lymphocytes and plays an important role in mediating rolling adhesion via interaction with mucosal vascular addressin cell adhesion molecule-1 on the high endothelial venules of gut-associated secondary lymphoid organ (79). A number of β₇⁺-integrin–associated gene transcripts contribute to the complex multistep signaling processes that regulate the trafficking of CD4⁺ memory T cells with various homing behaviors, such as organ-specific lymphocyte homing (81). In addition, signaling pathways triggered by the activated integrin receptor interconnect with growth factor–mediated signaling at multiple levels of cross-talk (81–83).

In most studies, α_Lβ₂ LFA-1 integrin has typically been considered a cell surface receptor involved in T lymphocyte adhesion and migration. But, as we are now aware, LFA-1 contact also influences the gene-expression program in the medium-term and modulates T cell effector functions that depend on distinct combinations of signals and the cytokine milieu. Thus, LFA-1 is clearly more than just a simple adhesion receptor mediating physical attachment of T cells to their cognate ligands on the endothelium or target tissues.

Compelling evidence from several studies clearly indicates that LFA-1 engagement in migrating T cells transmits signals that play a crucial role in complex cell functions, such as proliferation, activation, cytokine production, differentiation, and survival, that require the regulation of gene expression (4, 39, 56, 59). LFA-1–specific blocking Abs prevent interactions between T lymphocytes and APCs and, thus, disrupt T cell activation and transendothelial migration. In conjunction with CD3/CD28 signaling, LFA-1–mediated signals accelerate the production of IL-2 and IFN-γ, reduce the late-stage responsiveness of T cells to IL-4, and promote the entry of naive T lymphocytes into the cell cycle (3, 39, 67, 70, 84). A number of signaling proteins, transcription factors, and RNA-binding factors that directly interact with the LFA-1 cytoplasmic domains or are indirectly activated following LFA-1 stimulation and regulate gene transcription have been identified. These include cytohesin-1, JAB1 (a transcriptional coactivator that interacts with the nuclear proteins c-Jun and JunD), STAT3, and AP-1, as well as the RNA-binding protein HuR (3, 10, 15, 39). Furthermore, we determined that LFA-1/ICAM-1 cross-linking in human T lymphocytes caused significant modulation of the genetic program (4), in particular gene signatures that are involved in TGF-β and Notch pathways (Fig. 2); this is described in more detail in the following sections.

The effector phenotypes of T lymphocytes are determined by complex interactions of naive T cells with APCs. This physiological process also involves a multitude of factors, including the dominant cytokine environment, costimulatory molecules, and a plethora of signaling cascades. One of the key factors that defines these protective and pathogenic immune responses is TGF-β, which regulates a wide variety of biological functions in the immune system (85). TGF-β is also highly expressed in the vicinity of mucosal tissues, at sites of inflammation, and by many epithelial cells (86–88); hence, the lymphocytes migrating toward an epithelial surface may be exposed to significant exogenous TGF-β. TGF-β prevents the maturation of dendritic cells, counteracts macrophage activation, and inhibits B cell Ab production. Dysfunction of TGF-β–mediated regulation of immunosuppression is associated with many autoimmune and inflammatory diseases (85). In addition, the molecular principles of TGF-β regulation of T cell activation are important in a range of clinical settings. Depending on the immunological environment and its context, TGF-β guides T cells toward specific differentiation programs, including the development of inflammatory T cells that secrete IL-17 (Th17) and induced Tregs (iTregs) (89–91). A more recent study demonstrates that TGF-β regulates the formation of kidney-resident T cells via promoting the tissue entry of effector T cells (92). FOXP3⁺ iTreg cells also produce TGF-β that suppresses endothelial cell activation in vitro and in vivo (93). Tregs are immunosuppressive cells that possess the ability to suppress the proliferation and functions of other immune cells, including T effector cells, B cells, NK cells, macrophages, and dendritic cells. In addition, TGF-β reprograms the differentiation of Th2 cells, promotes an IL-9–producing subset, enhances effector Th1 cell activation, and converts Th1 cells into Th17 cells through stimulation of Runx1 expression (94–96). In contrast, the proinflammatory cytokines, such as IL-6, inhibit TGF-β–mediated induction of FOXP3 in CD4⁺ T cells and promote the differentiation of T cells toward a Th17 effector cell lineage (89, 97). The immunological imbalance among various T cell lineages, such as skewed Tregs and Th17 balance, has been observed in various diseases, including cancers (98).

In a previous study using comprehensive gene-expression analysis and in vitro experiments, we showed that, in addition to participating in T cell migration, LFA-1/ICAM-1 cross-linking contributed to the genetic program by upregulating molecules, including SKI, SKIL, SMURF2, and SMAD7 (4), all of which can induce refractoriness to TGF-β (Fig. 2). TGF-β responsiveness of T cells is critical for their commitment to the FOXP3⁺ iTreg lineage and maintenance of peripheral T cell tolerance (89, 90). Notably, LFA-1–stimulated T cells were refractory to TGF-β–mediated induction of FOXP3⁺ Tregs and RORγt⁺ Th17 differentiation (4). Furthermore, LFA-1 stimulation modulated the expression of several other genes, including transmembrane prostate androgen-induced RNA and chromosome 18 open reading frame 1, which have known effects on TGF-β signaling pathways (99, 100). Earlier studies using LFA-1–knockout mice showed that development of the optimal number of natural thymic and peripheral CD4⁺CD25⁺ Tregs was impaired in the absence of LFA-1 protein expression (63, 64). Nonetheless LFA-1–knockout mice cannot easily recapitulate the multiple individual stages involved in migration and immune synapse formation with respect to antigenic or other inflammatory signals. However, Tregs in LFA-1^−/− mice were defective in suppressive functions in vitro and in vivo (63, 64). A subsequent study demonstrated an active involvement of Rap1-GTP, but not LFA-1, in the development of natural Tregs in the mouse thymus (65). However, the development and suppressive functions of murine peripheral Tregs were dependent on Rap1-GTP and LFA-1 (65). The discrepancy in these findings could be due to the different model systems used. Previous studies using LFA-1–knockout mice primarily examined the development and functions of Tregs in the absence of LFA-1 expression or LFA-1–mediated signals, whereas we demonstrated that prolonged LFA-1/ICAM-1 contact caused human T cells’ refractoriness to TGF-β (4). The nonresponse to TGF-β may influence the T cell program as cells migrate to an inflammatory site to take up an effector role. In the latter case, it is possible that LFA-1 contact–mediated T cell TGF-β unresponsiveness serves as a regulatory check to control T cell effector functions. Hence, there is significant complexity in the analysis of the multiple components of signaling as a cell proceeds through a migratory phase to a site of inflammation and, thus, to engagement with APCs in a tissue-resident site. Of note, human and mouse T lymphocytes may also differ in terms of certain phenotypes and functions (101).

The in vitro generation of iTregs for their potential application as new therapies for chronic inflammation, autoimmune diseases, allergies, and graft rejection are increasingly being investigated in several laboratories. The in vitro experimental approach for the generation of iTregs requires T lymphocytes to be responsive to TGF-β (102). Despite studies demonstrating potent immunosuppressive activity of iTregs in vitro and the envisaged therapeutic applications of TGF-β–induced iTregs, there are some unresolved issues regarding the ability of these cells to suppress immune functions in vivo. In vitro–generated iTregs could potently inhibit autoimmune diseases in mice, whereas the same number of TCR-activated natural Tregs was ineffective in suppressing the same disease (91). The exact reason for this discrepancy in immunosuppressive function is not clear. It is anticipated that a subpopulation of lymphocytes may be refractory to TGF-β, possibly due to LFA-1 encounter at a local level; thus, these cells are unable to perform suppressive functions. The corollary of this would be that T lymphocytes maintain a nonprimed functional phenotype prior to their migration into the tissue site of danger, injury, or inflammation. Failure of such pathways could play a role in unwanted tissue damage and/or immune-mediated damage at tissue sites of inflammation. In this context, LFA-1–stimulated T cells becoming refractory to TGF-β raises important questions concerning their functioning and suggests that LFA-1 contact also primes the moving lymphocytes toward a functional effector phenotype that is capable of executing its intended function at a specific tissue site. Moreover, our data demonstrate that LFA signaling inhibits FOXP3-induced iTreg generation and the generation of RORγt-induced Th17 cells (4). In the context of a migratory signal delivered by LFA-1, these data could suggest that this signal also primes the moving lymphocyte toward a functional effector phenotype that is capable of executing its intended function at a specific tissue site.

A more recent study has demonstrated a novel molecular link between LFA-1 contact and Notch pathway activation (3). LFA-1/ICAM-1 cross-linking in human T cells resulted in Ser⁹ phosphorylation of GSK-3β, nuclear translocation of the cleaved Notch intracellular domain, and upregulation of the expression of Notch target genes Hey1 and Hes1 (3) (Fig. 2). Notch is a cell surface receptor, and the expression of this protein is widely conserved in numerous species. There is mounting evidence that Notch signaling is an important pathway for mediating cell fate decisions in developing thymocytes and peripheral T cells (103). In particular, Notch directs the differentiation of Th cell subsets (104, 105).

In addition to Th17 cells/Tregs, a dynamic balance between Th1 and Th2 effector cells is important in the maintenance of immune homeostasis in healthy individuals; if this balance is lost, the human body becomes vulnerable to infections, chronic inflammation, and tumorigenesis (98). Studies from our group (3) and other investigators (59) have demonstrated that the LFA-1 signal, concomitant with TCR stimulation, upregulated T-bet expression in human T cells and enhanced the secretion of Th1 cytokines IL-2 and IFN-γ. These LFA-1/ICAM-1–mediated effects in human T cells were dependent on ERK/GSK-3β signaling and the Notch pathway (3). In contrast, LFA-1 contact inhibited Th2 differentiation by downmodulating IL-4 responsiveness (3, 59). LFA-1 costimulation shifted the degree of IL-4 responsiveness and increased the threshold required to drive Th2 differentiation above the levels of IL-4 produced during T cell priming (59). Although LFA-1 costimulation did not inhibit early IL-4 production, it reduced the sensitivity of T cells to IL-4–induced Th2 differentiation 2–4 d poststimulation (59). A similar role for VLA-4 activation in regulating the Th1/Th2 balance has been reported (106). Ab engagement of VLA-4 promoted in vitro polarization of human CD4⁺ T cells toward Th1 by upregulating IFN-γ secretion (106). In another study using Brown Norway rats, in vivo administration of VLA-4–activating Ab was found to cause immune deviation toward the Th1 phenotype, with simultaneous dampening of Th2-driven autoimmune nephritis (106).

These findings suggest that T lymphocytes maintain a nonprimed functional phenotype prior to their migration into the tissue site of danger, injury, or inflammation. Failure of such pathways could lead to unwanted tissue damage and/or immune-mediated damage at inflammation sites. These observations also support the recently proposed “second-touch hypothesis” (107), suggesting that signals emanating from LFA-1/ICAM-1 fine-tune the classical immune response by Notch1, possibly facilitating the delivery of effector lymphocytes primed to respond at the site of inflammation. Of note, protein tyrosine phosphatase nonreceptor type 22, also induced by LFA-1 contact, inhibits LFA-1 signaling in effector T cells (108), indicating the existence of an LFA-1 autoregulation mechanism that may have important implications in patients in whom disease susceptibility is determined by inherited phosphatase mutations that perturb integrin functions.

This review highlights the dynamic and complex nature of LFA-1 contact with its cognate ligands in terms of its role in fine-tuning T cell effector functions. Notably, T lymphocytes infiltrating the sites of inflammation do not migrate all at once at a single time point; they infiltrate continuously during the entire course of the immune reaction. Thus, following LFA-1 contact, stimulated T cells find, depending on when they arrive, completely different microenvironments and, therefore, could undergo dissimilar differentiation or regulation.

Further work is needed to define how transcriptional changes induced by LFA-1/ICAM-1 cross-linking observed in vitro also has a role in vivo. It should be noted that a continuous cross-talk between LFA-1 and other receptors (e.g., TCR, VLA-4, and growth factor receptors) occurs at multiple levels, from T cell arrest on the vascular wall to the extravasation of T lymphocytes and the regulation of transcriptional programs (e.g., induction of TGF-β and Notch gene signatures) that alter effector functions and subsequent cellular behavior. Thus, to gain a more thorough understanding of LFA-1 contact and downstream signaling in T lymphocytes, it will be necessary to dissect the relative contributions of LFA-1 and other cell surface receptors, such as TCR and VLA-4, by revisiting what we know about LFA-1 signaling and by examining immediate and medium-term molecular events in the presence of functional modulators mimicking physiological and several pathological conditions.

Despite compelling evidence for the nonclassical roles of LFA-1, several important questions remain outstanding. To what extent does LFA-1 contact contribute to the endogenous pool of Th cells? In what situations does the LFA-1 signal modulate the conversion of naive T lymphocytes to effector cells and in which organs and/or tissues does this peripheral conversion to effector cells take place? Is there any need to generate additional Th1 cells in the lymph nodes and spleen of a healthy human or animal? Further, the exact proportion of the effector T cell population in specific tissues also remains to be clearly defined. In this context, with the data suggesting LFA-1 signaling as a means of inducing unresponsiveness to TGF-β, activation of Notch pathway, and LFA-1 autoregulation by protein tyrosine phosphatase nonreceptor type 22, we are starting to see a more fine-tuned local immune system that is dependent on the tissue-specific environment, such as chemokine-mediated attraction and the genesis of TGF-β–unresponsive lymphocytes in inflamed tissue sites. These signaling mechanisms must be reversible once the lymphocyte has performed specific immune functions; otherwise, it could lead to chronic inflammation. Thus, the reversal mechanisms become important, duration of gene induction by LFA-1 contact becomes important, what switches off the effector functions of T cells and other counteracting pathways become important, and the role of other immune cell populations (e.g., APCs, dendritic cells, and Tregs) in the local milieu also need to be understood. Although these questions require further exploration, understanding LFA-1 signaling cascades warrants continued investigations. New, more effective gene-silencing techniques, such as CRISPR/Cas9 (109) or GapmeR (110), may provide more through dissection of such genetic effects in adoptive transfer and knockout models. Such studies will be crucial for identifying novel T cell fate-associated markers and will yield new insights into how heterogeneous T lymphocyte fates are specified during the complex orchestration of an immune response and immune-mediated diseases, particularly those characterized by abnormalities in local effector populations. However, the challenges ahead include a more precise understanding of the scope of LFA-1 signaling, as well as the mechanisms used by T lymphocytes to mount an immune response in physiological and pathological settings. Given the multiple biological effects of the LFA-1 signal, such information will be beneficial for the effective and safe clinical translation of therapies targeted toward LFA-1–activated signaling cascades.

Taken together, this review paints a picture of the complexity of LFA-1 downstream signaling in T cells that parses extracellular cues into an appropriate intracellular response. In addition to triggering signals necessary for the migration of T lymphocytes, LFA-1 contact with ICAM-1 modulates gene-expression profiles that program T cells to differentiate into specific effector phenotypes that are dependent on several other environment factors, such as chemokines and cytokines. Future studies will continue to build on and refine the existing knowledge about the LFA-1 receptor and will reveal many surprises in the years to come.

The authors have no financial conflicts of interest.