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The continuous recirculation of naïve T cells and their subsequent migration to tissue following activation is crucial for maintaining protective immunity against invading pathogens. The preferential targeting of effector and memory T cells to tissue is instructed during priming and mediated by cell surface expressed adhesion receptors such as integrins. Integrins are involved in nearly all aspects of T cell life, including naive T cell circulation, activation, and finally effector T cell trafficking and localization. Recent research has revealed that microenvironmental factors present during T cell priming result in the specific regulation of adhesion/integrin and chemokine receptor expression. Once antigen-experienced T cells enter tissue, further changes in integrin expression may occur that is critical for T cell localization, retention, effector function, and survival. This review will discuss the function of integrin expression on T cells and the multiple roles integrins play on naïve T cells and in directing effector T cell trafficking to non-lymphoid sites in order to maintain protective adaptive immunity at body barriers.
In contrast to the relatively static adherent cells composing the major organ systems in the body, many cells of the immune system are in a dynamic state that involves movement and directed recruitment throughout the body. This diverse set of highly specialized cells patrols the host, defending it against foreign invasion. T cells are critical not only in combating initial encounters with pathogens, but also in establishing immunological memory. In the naïve state, T cells preferentially recirculate through the secondary lymphoid organs (SLOs) of the body.1 It is there that naïve T cells encounter cognate peptides embedded in the grooves of self-major histocompatility proteins presented by dendritic cells (DC) and, under the appropriate conditions, differentiate into effector T cells. Both the environment and strength of this initial encounter determine the subsequent functionality of the effector population. In contrast to naïve T cells, activated effector T cells can move into peripheral non-lymphoid sites, where they enact their protection function. This non-lymphoid entry is made possible by regulated expression of adhesion molecules (integrins and selectins) and chemokine receptors, as well as site-specific endovascular expression of their ligands. Once in tissue, integrin adhesion receptors play additional, less well defined, but likely equally important roles in retention, localization, effector function, and survival. There is also a second level of regulation of integrin expression that is based on the tissue microevironment. These roles have been more challenging to explore experimentally but are of great relevance to our understanding of T cell function and maintenance. This review will focus on the regulation of integrin expression and the role of integrins in targeting and retaining T cells in non-lymphoid sites.
Integrins are widely expressed cell surface adhesion molecules that mediate cell-extracellular matrix (ECM) and cell-cell interactions.2 Integrins are composed of heterodimeric membrane spanning α and β chains.3 In the unstimulated state, T cells have little apparent adhesion to integrin ligands. However, upon activation through the T cell receptor (TCR) or chemokine receptors, a cascade of signaling events leads to enhanced integrin functionality.4;5 This response is referred to as “inside-out” signaling. It involves the translocation of proteins to integrin cytoplasmic domains and the assembly of multiprotein complexes. The formation of these complexes results in activation and clustering of integrins, thus enhancing both affinity and avidity of integrins for their ligands. These cytoplasmic complexes link integrins to the actin cytoskeleton, as well as intracellular signaling pathways. In this way, the well recognized adhesive function of integrins is linked to the less well understood ability of integrins to transduce signaling into the cell (“outside-in signaling”).
Naïve T cells express a fairly homogeneous array of cell surface molecules that promotes recirculation through the SLOs of the body, including the spleen, peripheral lymph nodes (pLN), mesenteric lymph node (mLN) and peyer’s patches (PP) of the small intestine. Naïve T cells express low levels of the αLβ2 (LFA-1), α4β1 (VLA-4) and α4β7 (LPAM) integrins, which bind ICAM-1, VCAM-1, and MAdCAM-1, respectively (Fig. 1).6 The interaction of αLβ2 with ICAM-1 is important for T cell entry into pLN and T cell interactions with antigen-presenting cells (APCs). The α4β1 ligand VCAM-1 is expressed at low levels throughout the vasculature, but becomes upregulated on a wide array of tissue during inflammation.7 α4β7 is also reported to bind with low affinity to VCAM-1 in vitro and under some circumstances may play a role in pLN entry.8–10 MAdCAM-1, the major ligand for α4β7 is preferentially expressed at steady state in the mLN and PP, where it promotes entry of naïve T cells into these sites through a high affinity interaction.11–13
It has been 45 years since Gowans and Knight first demonstrated that small lymphocytes from the blood enter into SLOs and are returned to the circulation via efferent lymphatic vessels.14 They proposed that the homing of lymphocytes to lymphoid tissue was via the “special affinity” of small lymphocytes for the endothelium of the post-capillary venules. Over the ensuing years the major molecular players mediating this “special affinity” were identified as lymphocyte expressed integrins, selectins, and most recently chemokine receptors coordinated with endothelial expression of their ligands.1;6;13;15 Thus, the array of specific homing molecules expressed on T cells promotes re-circulation through the tissue whose vascular endothelium expresses the appropriate receptors.
Naïve T cells preferentially recirculate through LNs in an integrin dependent manner. In LNs, specialized high endothelial venules (HEVs) provide the ‘landing strip’ for circulating naive T cells. The HEVs promote both selective adhesion to and subsequent transmigration (entry) into LNs. This interaction has been extensively reviewed previously and is highly integrin dependent.13;16 The next section will discuss what is currently known about the role for integrins in this process.
The adhesion pathway promoting T cell movement out of the circulation into LNs has been divided into a series of at least four major steps: rolling, activation, firm adhesion, and transmigration. Integrins have been implicated in some aspect of each of these four steps. The interaction of naïve T cells with HEVs occurs under conditions of vascular shear flow and requires expression of selectins, chemokine receptors and integrins on the T cell.17 For naïve T cells, engagement of CD62L (L-selectin) with peripheral node addressin (PNAd) on HEVs results in weak tethering that promotes rolling of the T cell on the endothelial surface. This weak tethering greatly enhances the probability of encountering chemokine ligands present on the vascular lumen. The CCR7 chemokine receptor, which is expressed by all naïve T cells, interacts with CCL19 or CCL21 displayed by the HEV. Ligand binding to CCR7 results in G-protein coupled, pertussis toxin-sensitive signaling that mediates extremely rapid activation of integrins and firm adhesion to the vascular endothelium.18 CD62L-dependent signaling on T cells may also play a role in activating integrins.19;20 Firm integrin-mediated attachment of naïve T cells is critical to subsequent transmigration through the endothelium into the LN. In pLNs under homeostatic conditions, αLβ2 is particularly critical for firm adhesion to ICAM-1 expressed on the resting endothelium. In the PP of the small intestine, this interaction is also dependent on the activation of α4β7 and firm adhesion to MAdCAM-1, which is preferentially expressed in this tissue. 21 In addition to serving as an α4β7 ligand, MAdCAM-1 also contains a mucin-like domain for binding CD62L.22 Therefore, if appropriately glycosylated, MAdCAM-1 can also participate in the initial rolling step via interaction with CD62L.
The interaction of MAdCAM-1 with α4β7 may also play a role in the initial tethering response that initiates naïve T cell adhesion to HEVs under shear flow.23 α4β7 is also reported to be capable of mediating T cell tethering and rolling on VCAM-1.24 However, the specific contribution of α4 integrins to initiating T cell interactions with HEVs in vivo remains unclear. Integrins also exhibit “cross-talk” whereby one integrin is thought to regulate the function of other co-expressed integrin heterodimers. Ligation of α4β1 has been reported to further enhance αLβ2-mediated adhesion to ICAM-1, while another report has documented negative regulation of α4β1 following αLβ2 ligation.25;26 This works highlights the complexity of integrin function during interactions with cells expressing multiple integrin ligands.
After firm adhesion to the vascular endothelium, the process of de-adhesion or release of integrin-integrin ligand interaction is also vital. Without this de-adhesion event, cells are unable to migrate on and subsequently transmigrate through endothelium into LNs. A membrane-proximal GFFKR sequence in the cytoplasmic tail of integrin α subunits has been proposed to form a putative salt bridge that restrains integrins in an inactive conformation. Mice expressing αL integrin with a mutation in the GFFKR sequence express constitutively active αLβ2 integrin. Lymphocytes isolated from these mice exhibit enhanced adhesion to ICAM-1, but diminished transendothelial migration.27 Similar findings have been reported in mice with a salt bridge disrupting mutation in the α4 integrin subunit, resulting in active α4 integrins.28 Another mouse with a β7 integrin ectodomain mutation expresses constitutively active α4β7.29 T cells from both of these mice have enhanced firm adhesion to the vasculature of the gut, but enter inefficiently into intestinal sites.
The final and less well understood step in the homing cascade is transmigration. Though traditionally thought of as the passage of the lymphocyte between endothelial cells (paracelluar), it is becoming more apparent that lymphocytes can also migrate directly through endothelial cells (transcellular).30–32 Both β1 and β2 integrins are thought to be involved in these processes via interaction with endothelial junction integrin ligands such as JAM-B and JAM-A, respectively.17 Of interest, the endothelial cell also depends on ligation of integrin ligands to form a docking structure that further strengthens the interaction of α4β1 and αLβ2 with VCAM-1 and ICAM-1.33 Signaling mediated by ICAM-1 and VCAM-1 may then act to stimulate the vascular endothelial cell, further facilitating leukocyte transmigration.34
Another possible role for integrins in promoting transmigration was recently reported. Autotaxin (ATX), a phospholipid producing enzyme, was identified as a HEV-produced soluble integrin ligand that binds chemokine activated α4 integrins on human T cells.35 ATX’s phopholipid product, lysophophatidic acid (LPA), promotes T cell migration in vitro and injection of an enzymatically inactive ATX decreases short term homing to LNs.
Genetic models in the mouse and characterization of rare human immunodeficiencies have provided striking evidence that integrins and appropriate control of integrin function are essential for proper T cell recirculation. Naïve T cells isolated from knockout mice lacking either the αL or β2 integrin subunits have dramatically reduced numbers of naïve T cells in the pLN and migrate inefficiently to these sites.10;36 An array of mutations in the human β2 integrin gene that lead to reduced or absent β2 integrin expression is the hallmark of leukocyte adhesion deficiency-I (LAD-I).37;38 LAD-I patients suffer from recurrent bacterial infections secondary to a decreased ability of leukocytes to enter into inflamed tissue. More recently, several patients have been described with clinical symptoms similar to LAD-I patients. Patients with this variant of LAD-I, now classified as LAD type III (LAD-III), also exhibit a bleeding disorder. Integrin expression is normal on leukocytes and platelets from these patients, but integrin activation is impaired.39 Mutations in two distinct genes have been identified, the Rap1 guanine nucleotide exchange factor CalDAG-GEFI, and kindlin-3, an integrin-binding protein that cooperates with talin to promote integrin activation.40–42 This group of diseases provides a striking example of how integrins and the signaling pathways regulating their function are of vital importance for appropriate leukocyte localization and immune function.
SLOs serve as the major meeting place for lymphocytes and their potential cognate antigen. With the advent of in vivo imaging techniques such as two-photon microscopy, much knowledge has been gained regarding the factors involved in T cell movement and APC interaction within the LN environment.43–45 Imaging of explanted LNs and intravital inguinal LN preparations has revealed that T cells migrate rapidly in the LN with an average velocity of 11 μm/min and peaking around 30 μm/min.46 In comparison, fibroblasts migrate at less than 1 μm/min.47 Thus, naïve T cells are among the most motile cells in the body. During movement, T cells take on a polarized, oblong shape while moving in a stop-and-go manner. The random-walk behavior exhibited by T cells is due primarily to CCR7-dependent migration through the fibroblastic reticular cells (FRC), the LN stromal cells that are a critical component of a structural network in the T cell-rich areas of LNs.48 These FRCs provide “guided randomness” that promotes high probability encounters with FRC-associated DCs, which can present lymph borne antigen.49 FRCs also form a network in the spleen that guides the entry of T cells into the T cell zone of the splenic white pulp.50
The LN is structurally composed of a 3-dimensional scaffolding mesh with a collagen I core surrounded by microfibrils of ECM molecules.49 Interestingly, the contiguous network of FRCs nearly completely ensheaths these ECM structural components, effectively concealing them from direct contact with T cells.51 Thus, though several potential ECM integrin ligands are highly abundant in the LN, they are generally not thought to be accessible to T cells. Following exit from the blood, T cells are known to crawl along the FRCs and quickly localize to the paracortex or T cell zone of the LN.48 This motility and precise localization are dependent on T cell CCR7 binding to CCL19 or CCL21.52;53 These are both secreted by FRCs and bound to surface glycosaminogylcans, being in highest concentration in the paracortex.48
Although FRCs express both VCAM-1 and ICAM-1,54;55 integrins are not essential for naïve T cell motility in LNs. Woolf et al. demonstrated that in the absence of shear flow (as in the LN environment), immobilzed CCL21 promoted pertussis toxin-sensitive random T cell migration at a velocity similar to that observed in vivo using two-photon imaging.55 Interestingly, polarization of αLβ2 to the leading edge pseudopodia and α4 integrins to the trailing edge uropod was observed during migration. In vivo imaging revealed that loss of αLβ2 expression on T cells or ICAM-1 on FRC only reduced migration by 15%. There was no additional inhibitory effect on motility with the addition of an α4 integrin-specific blocking antibody. Thus, interactions of T cell integrins with ligands expressed on FRCs are thought to play a minor role for basal T cell motility in the LN.55 Intriguingly, under conditions of shear flow (as might be present when T cells are interacting with HEVs), immobilized CCL21 promoted firm integrin-mediated adhesion. Firm adhesion of integrins thus seems to be dependent on an activation event (chemokine signal) coupled to environmental force (blood flow).56
Repeated encounters of CD4 T cells with MHC class II positive cells in the LN environment has also been shown to be vital for maintaining T cell motility.57 In the absence of low affinity self-ligand/MHC class II-TCR interaction, T cells demonstrate a 50% reduction in motility and loss of basal activity of the Rap1 GTPase. Although Rap1 is a known activator of integrins,58 the role of integrins in the motility promoted by tonic low level signaling induced by TCR:self-ligand interactions is currently unclear.
During the early phases of a primary immune response, T cells are retained or sequestered in the activated LN.59 The interaction of the phospholipid spingosine-1-phosphate (S1P) with sphingosine-1-phosphate receptor 1 (S1P1) is critical for T cell sequestration.60 Although integrins mediate T cell contact with APCs (see below), combined inhibition of both αLβ2 integrin and α4β1 integrin does not alter T cell retention in LNs following subcutaneous antigen deposition.61
In order for naïve T cells to achieve competent activation, they must interact with DCs presenting cognate antigen recognized by the TCR. T cells interact transiently with DCs during the first 8 hours of the T cell immune response, but then convert to a behavior characterized by prolonged contact with DCs that can last up to several hours.62;63 These stable contacts are dependent on ICAM-1 expression in host environment and have functional consequences for the durability of the T cell immune response. In ICAM-1-deficient mice, TCR transgenic OT-I CD8 T cells only interact transiently (less than 10 min) with mature DCs presenting cognate antigen.64 While the loss of prolonged T-DC interactions did not affect T cell proliferation and clonal expansion, it did inhibit the survival of activated CD8 T cells and the generation of effective memory. Thus, prolonged T-DC contacts mediated by ICAM-1 (and presumably αLβ2 on the T cell) are critical for naïve T cells to receive the appropriate educational signals to differentiate into effector and memory T cells.
When a T cell interacts with a DC displaying cognate antigen, a series of spatial and temporal molecular reorganization events occurs between the T cell and DC. These organizational events form a microscopically distinct structure at the contact site termed the immunological synapse (IS). The IS is a “bullseye” type structure where the TCR and other TCR signaling kinases and adapters localize to the center of the bullseye (termed central supramolecular activation clusters or cSMAC), while integrins and the integrin-associated protein talin localize to an outer ring-like structure that surrounds the cSMAC (termed pSMAC for peripheral supramolecular activation clusters).65–67 In vitro, the formation of the IS occurs over a time frame of 20–30 minutes, well after biochemical signaling events initiated by TCR stimulation can be detected.68 Subsequent studies demonstrated that T cell activation is initiated and sustained in microclusters composed of the TCR and active signaling kinases that form at the periphery of what eventually becomes the mature IS. These microclusters travel from the periphery of the T-APC contact site to the cSMAC.68–70 Although the cSMAC has been proposed to be a site where TCR signaling is terminated,69;70 recent studies have shown that the strength of antigen stimulation determines whether signaling is sustained or terminated in the cSMAC.71 This suggests that the cSMAC fine-tunes the immune response, sustaining signals from weak antigenic stimuli while terminating signals from strong antigenic stimuli.
Although integrins can clearly transduce signals that augment TCR signaling and promote specific T cell differentiation events,72–74 the precise function of integrins in the IS above and beyond the facilitation of adhesion between the T cell and DC remains unclear. αLβ2 integrin ligation leads to the reorganization of actin into a ring-shaped structure called an “actin cloud”. Tyrosine phosphorylated proteins accumulated with αLβ2 integrin in this novel actin structure, and formation of the actin cloud enhances TCR signaling.75 In addition to αLβ2, the α4β1 integrin has also been reported to localize to the T-APC contact site during T cell activation, even under conditions where the APC does not express VCAM-1.76 Recent studies have shown that T cell adhesion to the α4β1 integrin ligand VCAM-1 retards the actin-dependent movement of signaling microclusters containing the adapter protein SLP-76 to the cSMAC.77 This work is consistent with the fact that TCR signaling is initiated at the periphery of the contact site, where integrins are localized, and suggests that integrins may promote efficient T cell activation by dampening the termination of TCR signaling from microclusters that move to the cSMAC.
Although integrins such as αLβ2 do not appear to be essential for T cell motility in LNs, αLβ2 is differentially expressed on the surface of a migrating T cell.78 At the leading edge, αLβ2 is in an intermediate affinity state, while αLβ2 in the mid-cell region is associated with talin and capable of mediating firm adhesion to ICAM-1. It has recently been proposed that migrating T cells can receive TCR signals through the formation of transient contacts with APCs designated as “kinapses”.79 The actin-regulatory protein WASp mediates conversion from kinapses to stable synapses, while the PKCθ isoform mediates conversion from stable synapses to kinapses.80 The relationship between kinapses and the differential localization of αLβ2 integrin of differing affinities on the surface of a migrating T cell is currently unclear. However, PKCθ has recently been shown to regulate TCR-mediated activation of LFA-1 via regulation of the Rap1 GTPase.81
The discovery that T cells harvested from the intestine of sheep preferentially return to the intestine laid the foundation for a model of site-directed trafficking of T cells.82 Subsequently, naïve and effector/memory T cells were demonstrated to possess different trafficking patterns.83 Following activation, T cells upregulate molecules important for homing to non-lymphoid sites, and decrease the LN homing molecules CD62L and CCR7. It was thus postulated that differential integrin expression on effector/memory T cells imparted altered homing properties from their naïve precursors. The fact that human memory T cells displayed higher and varied integrin expression than naïve T cells led to the proposal that during T cell priming the local environment may provide a molecular “imprint” endowing cells with a selective pattern of homing molecules.84 Additional analysis revealed that the α4β1 and α4β7 integrins maintained reciprocal expression on circulating human CD4 memory T cells.11;85 T cells with high levels of α4β7 but low levels of the β1 integrin subunit migrated preferentially into mucosal tissues such as the PP and mLN, whereas T cells with high α4β1 levels but low β7 integrin subunit expression preferentially migrated through peripheral non-mucosal tissues such as the skin.86;87 The factors within local lymphoid environments that control the differential expression of integrins on activated T cells are beginning to be elucidated.
The α4β7 integrin ligand MAdCAM-1 is expressed by the HEVs of the mLN, PP, and the venules of the small and large intestine12;88 and promotes the entry of α4β7-expressing cells into intestinal sites.89 Mice lacking β7 integrin have decreased numbers of PP and intestinal T cells, and reduced migration to the gut.90;91 Inflammation results in enhanced expression of MAdCAM-1 on venules in the intestine, resulting in increased entry of α4β7-positive cells into the gut. Patients with inflammatory bowel disease have both enhanced expression of MAdCAM-1 and an increased number of infiltrating T cells.88 Furthermore, blocking antibodies against α4β7 attenuate some mouse models of intestinal inflammation and are clinically effective in the treatment of human ulcerative colitis.92;93 In addition to α4β7, a role for α4β1 and αLβ2 in mediating T cell entry into the intestine during inflammation has also been reported.94–96
The restricted expression of MAdCAM-1 in the gut suggests that it is critical to understand the factors that control the expression of α4β7 during T cell activation responses. Adoptive transfer approaches using OT-II TCR transgenic T cells demonstrated that priming in differential SLOs resulted in differential upregulation of adhesion molecules.97 In pLNs, T cells downregulated α4β7 and expressed high levels of a P-selectin ligand following antigen challenge. In contrast, T cells activated in mLNs expressed high levels of α4β7 and became responsive to CCL25, a chemokine expressed in the small intestine that promotes firm adhesion of α4β7 to MAdCAM-1.98 These results suggest that the mLN environment is conducive to the generation of α4β7+ effector T cells.
Similar results were observed in in vitro experiments utilizing DCs isolated from different LNs. When compared to DCs isolated from pLNs, the addition of DCs isolated from mLNs to anti-CD3 stimulated T cell cultures resulted in a higher proportion of T cells expressing high levels of α4β7 integrin.99 Similar results were reported using DCs isolated from PPs.100 In these studies, the expression of other integrins such as β1 and αL was not altered. T cells activated in vitro by DCs isolated from PPs also had enhanced homing to intestinal lymphoid sites when compared to T cells isolated by pLN-derived DCs. T cells activated by PP DCs showed particularly enhanced trafficking to the non-lymphoid small intestine. In contrast, T cells activated by pLN DCs exhibited preferential trafficking to pLNs.
The soluble mediator inducing gut-homing molecules was subsequently identified as the vitamin A metabolite, all-trans retinoic acid (ATRA). Iwata et al. demonstrated that the addition of ATRA enhanced the expression of α4β7 and the CCR9 chemokine receptor on CD4 T cells following activation with anti-CD3 and anti-CD28 antibodies.101 DCs isolated from mLN or PP express several isoforms of the ATRA-producing enzymes, retinal dehydrogenase (RALDH). Inhibition of these enzymes reduced the ability of intestinal DCs to induce α4β7 expression in vitro. This further supports a role for these DCs in the production of ATRA and induction of α4β7 integrin and other gut-homing molecules on T cells (Fig. 2A).
ATRA likely regulates T cell integrin expression at the transcriptional level, as a retinoic acid receptor (RAR) antagonist inhibited the ability of PP DCs to induce α4β7 expression.101 RARs primarily form heterodimers with retinoid X receptors (RXR).102 ATRA binds RAR with high affinity and this complex translocates to the nucleus, where it binds to RA response elements (RARE). Although the β7 integrin gene is reported to have a RARE half site,101 α4 integrin mRNA was the only integrin message consistently elevated following activation by PP DCs.103 In contrast, transcripts for β7, β1, or αE integrin were not altered significantly. This suggests that ATRA-mediated changes in α4β7 expression are likely to be controlled at the level of α4 integrin. The possibility of further post-transcriptional, translational, or protein level regulation is still an area of active investigation.
Several groups have had similar findings concerning the role of ATRA in inducing α4β7 integrin expression.104 DCs derived from the small intestine that express the αE (CD103) integrin subunit are believed to be a source of ATRA, as they enhance RAR signaling and induce both α4β7 and CCR9 expression on responding CD4 and CD8 T cells.105–109 The conditioning of αE integrin postive DCs to produce ATRA is thought to be mediated by intestinal epithelial cells prior to DC migration to the mLN or PP.110;111 mLN stromal cells are also reported to participate in the imprinting of gut-homing T cells through further production of ATRA, as they express high levels of RALDH1/RALDH3.112;113
The OX40-OX40L costimulatory pathway has also been implicated in regulating α4β7 integrin expression following T cell activation (Fig. 2A).89 An anti-OX40L antibody inhibited the ability of mLN-derived DCs to promote α4β7 integrin expression on T cells both in vitro and in vivo.105;114 This suggests that OX40-OX40L signaling synergizes with ATRA to promote the upregulation of α4β7 on activated T cells.
Antigen concentration has also been shown to vary the ability of cultured mLN DCs to induce α4β7 expression on CD8 T cells (Fig. 2A).109 In a reciprocal fashion, higher antigen doses resulted in less α4β7 expression. The mechanism underlying antigen dose-dependent inhibition of α4β7 expression is unknown, but it seems to be independent of RAR mediated signaling. This suggests the possibility that additional undefined soluble factors are capable of mediating the inhibition of α4β7 expression at high antigen dose.
In addition to causing the upregulation of gut-homing molecules, ATRA has been shown to suppress expression of molecules that promote T cell trafficking to the skin.101 More recently, the vitamin D metabolite, 1,25 dihydroxy-VitD3 (1,25VitD3) was shown to mediate a reciprocal expression pattern of homing molecules. When present during T cell activation, 1,25VitD3 induced the skin-homing chemokine receptor CCR10 on human T cells and suppressed expression of α4β7 and CCR9 in the presence of ATRA (Fig. 2B).115 These findings point to opposing roles for ATRA and 1,25VitD3 in the regulation of gut versus skin-homing T cells. Expression of other skin homing molecules, such as P- and E- selectin ligands and CCR4, is upregulated in a 1,25VitD3 independent manner and their upregulation may involve other unidentified factors.116 There also seem to be some species differences in these effects, as 1,25VitD3 failed to inhibit the induction of α4β7 integrin on mouse T cells by ATRA or mLN DCs.109
The molecular mechanism whereby 1,25VitD3 suppresses α4β7 expression may involve cytoplasmic receptor competition.115;117 Both the RAR and the vitamin D receptor bind to the RXR prior to translocation to the nucleus. Thus, when 1,25VitD3 is present along with ATRA, there may be competition for RXR that inhibits downstream activation of RAREs by ATRA-bound retinoic acid receptor.118
The bone marrow vasculature, as well as stromal cells, express the α4β1 integrin ligand VCAM-1.119 Intravital microscopy has revealed that α4β1 mediates firm adhesion to the bone marrow.120 Inhibition of αLβ2 integrin function has little effect. However, in the absence of VCAM-1, firm adhesion was not completely abolished so other adhesion molecules are likely involved. Conditional VCAM-1 knockout mice have also demonstrated the importance of VCAM-1 expression for entry of T cells into the bone marrow.119 Thus, T cell entry into the bone marrow is thought to be highly dependent on the α4β1-VCAM-1 interaction. There is some evidence that α4β7 integrin may also be involved in homing to the inflamed bone marrow following irradiation through interaction with induced MAdCAM-1.121;122 As bone marrow stromal cells express VCAM-1, there may be a role for α4β1 integrin in enhancing both the retention of T cells and the delivery of survival cytokines (IL-7, IL-15) that are reported to be present in the bone marrow.123;124 Recent studies have highlighted an important role for the bone marrow as a reservoir for memory CD8 T cells,120 but the specific role of α4β1 integrin in maintenance/retention of memory T cells in the bone marrow remains undefined.
Both VCAM-1 and ICAM-1 are expressed on the vasculature of the inflamed brain. In particular, VCAM-1 is known to promote T cell entry through its interaction with α4β1 expressed on activated T cells.125 In accordance with these findings, the ability of brain autoreactive T cells to cause disease has been correlated with their expression of α4 integrin.126 Anti-α4 integrin antibodies have also been successful in the treatment of human multiple sclerosis (MS) in humans127–129 and inhibiting or reversing a CD4 T cell-mediated brain disease, experimental autoimmune encephalomyelitis (EAE), in mice.130–132
The site of priming of encephalogenic T cells is thought to be deep cervical LNs (cLN). 133 As the brain has no anatomical lymphatic drainage, the mechanism by which antigen is delivered to the cLN is unknown. Migration of DCs, parenchymal APCs, or the direct drainage of antigen have all been hypothesized.134 Priming of CD8 T cells in the cLNs following intracranial tumor implantation results in high expression of α4β1 and low expression of α4β7 (Fig. 2C).135 These cells also migrated better to the brain than cells primed in the inguinal LN. The specific factors involved in the ‘imprinting’ of brain-tropic α4β1-high α4β7-low T cells are unknown. These results are consistent with APC programming at the site of antigen capture and subsequent migration to the draining LN, where naïve T cells are activated and educated with specific homing preference. It is not known if CD4 T cells primed in a similar manner also develop similar integrin expression and brain tropism, as would be predicted based on EAE studies.
Pathogen infection is often accompanied by an inflammatory response. Inflammation promotes the influx of both naïve and memory T cells into reactive LN.136 Global hyperthermia (fever) is a programmed physiological response driven by endogenous or exogenous pathogen-associated pyrogens during an infection. The acute phase reactant IL-6, which is produced during inflammation, can mediate this fever response and has been more recently shown to increase the expression of ICAM-1 on reactive LN HEVs, thereby promoting the influx of naïve and memory T cells.137 CCL21 expression was also increased, further promoting enhanced firm adhesion mediated by LFA-1 to ICAM-1.
It has been proposed that the presence of inflammation may additionally promote T cell homing promiscuity to non-lymphoid sites.89;138 This idea is supported by the known alterations of vascular endothelial expression of integrin ligands that occurs during acute/chronic inflammation or disease. LPS or TNFα treatment results in a global increase in VCAM-1 and ICAM-1 expression on vascular endothelial cells.7. VCAM-1 upregulation was particularly pronounced in the small intestine. This is consistent with other work demonstrating that inhibition of VCAM-1 function in some models of colitis is more effective than inhibition of MAdCAM-1 function.139 MAdCAM-1 expression in the gut is also enhanced following TNFα treatment.140 In chronic disease states such as inflammatory bowel disease and diabetes, MAdCAM-1 is additionally upregulated on the inflamed venules of non-intestinal sites, such as the liver, joints, eyes, skin, and pancreas.141;142 It has therefore been proposed that the “aberrant” homing of mucosal T cells expressing high levels of α4β7 to the liver may underlie the hepatic complications observed in inflammatory bowel disease.141 Thus, during inflammation the site specific expression of endovascular integrin ligands may be altered, potentially allowing the entry of T cells expressing a less specific set of integrin molecules.
Effector and memory T cells that access non-lymphoid tissue encounter a microenvironment that is quite distinct from that found in SLOs. While SLOs are densely packed with cells, the intercellular spaces in non-lymphoid tissue contain collagen fibrils and other ECM proteins.143 Thus, T cells in non-lymphoid tissue are much more likely to encounter and interact with ECM molecules. This suggests a more prominent role for ECM-binding integrins in controlling T cell function in non-lymphoid tissue.
Very little is known about the movement of T cells in non-lymphoid sites. Collagen matrices have been utilized in vitro to demonstrate that T cells exhibit a high rate of motility and form short-lived interactions with DCs.144 This pattern of motility is strikingly similar to the behavior of naïve T cells in LNs, as observed with two-photon imaging46;62. This suggests that T cells in non-lymphoid tissue may also exhibit high rates of basal motility. In addition, the presence of collagen may promote migratory behavior, as exposure of lymphoid cells to collagen in vitro stimulates cell migration.145 In vitro studies have provided conflicting results on the role of integrins in T cell motility through collagen matrices.146;147 In one study, antibodies specific for β1, β2, β3 and αV integrins did not alter T cell movement in collagen gels.148
Genetic ablation of all integrins expressed on wild-type mouse DCs has recently been shown to have no effect on DC migration in a three-dimensional matrix in vitro and in the ear dermis in vivo.149 The dispensability of integrins for DC movement was explained by a “flowing and squeezing” model, where the protrusive flow of the actin cytoskeleton drives the cell forward and a myosin-II contractile module propels the trailing nucleus through confined ECM spaces. These findings support the view of integrins as immobilizers rather than locomotive molecules on leukocytes. This predicts a role for integrins in tissue retention instead of migration. The phenotype or function of T cells from these global integrin-deficient mice was not reported.
In a murine CD4 T cell line, septins have recently been revealed to provide cortical rigidity and the absence of septin 7 results in defective motility.150 Septins are hypothesized to promote a stable platform from which actomycin filaments can apply force, resulting in forward protrusion of the leading edge. The role of integrins in this process remains unclear, although motility speed on ICAM-1 coated coverslips under shear flow was found to be septin-dependent.
Tissue resident effector/memory T cell exhibit distinct functions, such as the ability to rapidly produce cytokine, that are not seen in their lymphoid resident counterparts. The expression of specific integrins is also known to be altered following entrance into non-lymphoid sites. These phenotypic changes following T cell entry are dependent on factors in the non-lymphoid tissue microenvironment.151 The first place these phenotypic changes can be imparted upon T cells is during the transmigration process though the endothelium. αLβ2 integrin-dependent interactions between CD4 T cells and endothelial cells in vitro result in activation of the transcription factor AP-1 without sustained NF-κB activation.152 Transmigrated T cells also upregulate αL and α4 integrin, are more migratory and are hyperresponsive to antigenic stimulation. Several other reports have also demonstrated enhanced T cell sensitivity to antigen following transmigration through endothelium.153;154
Once in non-lymphoid sites, the microenvironmental milieu may further alter the function and phenotype of the tissue resident T cell. Following LCMV infection, splenic and intraepithelial lymphocyte (IEL) memory CD8 T cells demonstrate different function and phenotype. For example, the IELs express dramatically higher granzyme B, a marker of cytolytic function, and higher αEβ7 integrin expression. However, in vivo restimulation of αEβ7 high IELs following adoptive transfer into secondary recipient mice resulted in tissue-specific development of function and phenotype. Restimulated T cells harvested from the spleen were αEβ7 integrin low, while restimulated T cells harvested from the intestine were αEβ7 integrin high.155 Reversible alterations in memory T cell expression of α4 integrin in the peritoneum and αL integrin in the lung have also been described.156;157 This suggests that T cell function and integrin expression is dependent upon tonic/continuous environmental factors present in the particular tissue.
Integrins likely also transduce signals to T cells upon engagement with ligands in the microenvironment. In non-lymphoid tissue, T cell interactions with ECM proteins such as fibronectin, laminin and collagen may provide signals that facilitate T cell activation responses. This hypothesis is supported by many studies demonstrating that β1 integrin-mediated interactions of T cells with ECM proteins can enhance TCR signaling.158
The gasterointestinal tract, skin, and lungs are in constant interaction with the environment. Therefore, the maintenance of T cells in a non-lymphoid site is of great importance for sustaining protective immunity as body barriers. The epithelium of these tissues provides the first layer of defense against pathogen invasion. Situated within and underneath the epithelial layers of these organs reside immune cells poised to recognize and destroy foreign organisms. To maintain such surveillance functions, cells need to be retained and positioned properly within each of these sites. Integrins implicated in retaining T cells in non-lymphoid tissue include the αEβ7 integrin, which binds to E-cadherin, and the collagen-binding integrins α1β1 (VLA-1) and α2β1 (VLA-2).
αEβ7 integrin was originally identified to be expressed by the vast majority of IELs and nearly 50% of T cells in the lamina propria of the small intestine.159–161 Notably, αEβ7 integrin is also expressed on subsets of both murine and human CD4 regulatory T cells.162;163 Unlike the other member of the β7 integrin subfamily, α4β7, αEβ7 integrin is not thought to be involved in the homing of T cells to the intestine and it does not interact with MAdCAM-1.164;165 E-cadherin is the only known ligand for αEβ7 integrin and it is expressed on the lateral and basolateral surfaces of epithelial cells.166 Naïve murine CD8 T cells also express αEβ7 integrin but this expression is quickly lost after activation.155 Interestingly, high level expression of αEβ7 integrin is subsequently induced upon entry into the small intestine in a TGF-β dependent fashion.167;168 The entry of α4β7-high T cells into the TGF-β rich environment of the intestine is reported to result in a switch from α4β7 expression to αEβ7 expression (Fig. 3A). The exact mechanism for this switch is currently unknown, but Smad7 transgenic mice, which have impaired TGF-β signaling, are unable to induce αEβ7 expression.169 These mice have reduced IELs similar to the αE integrin-deficient mice.170 Signaling from the CCR9 chemokine receptor upon engagement with CCL25 may be critical to enhancing αEβ7 expression and promoting adhesion to E-cadherin as well.171;172
The mechanism by which IELs are maintained in the intestine is still unclear. Almost all IELs express αEβ7 and αE integrin knockout mice have a reduced number of IELs.170 However, the severity of this phenotype does have some mouse strain dependence. IELs also express the α1β1 and α4β1 integrins, which have been proposed to mediate IEL binding to type IV collagen173 and intestinal mesenchymal cells,174 respectively. Although there is a 50% reduction in IELs in α1 integrin knockout mice,175 no reduction in IEL numbers was reported in β1 integrin-deficient bone marrow chimeric mice.176 The reason for this discrepancy may be due to mouse strain differences and/or compensation by other integrins or adhesion molecules.
The brain microenvironment has also been demonstrated to induce αEβ7 expression on tumor-reactive CD8 T cells, potentially enhancing their retention in the brain (Fig. 3B).177 Expression of αEβ7 integrin on CD8 T cells may play a more general role in promoting CD8 T cell function. CD8 T cells expressing αEβ7 integrin can kill E-cadherin positive cells and this killing is inhibited by an anti-αE integrin antibody.178 In addition, αEβ7 integrin expressed on cytotoxic CD8 T cells is recruited to the IS during interaction with E-cadherin positive tumor cells and is required for cytotoxic granule polarization and release.179 TGF-β in the tumor microenvironment may play an important role in regulating α4β7 expression on tumor-resident CD8 T cells.
The α1β1 (VLA-1) and α2β1 (VLA-2) integrins were initially discovered as antigens that were induced after long-term in vitro activation of human T cells.180–182 Both α1β1 and α2β1 mediate cell adhesion to collagen. α1β1 mediates adhesion to type IV collagen, while α2β1 mediates adhesion to type I collagen. As naïve T cells do not bind to collagen, even after acute stimulation signals that promote integrin-dependent adhesion to ICAM-1, VCAM-1 and fibronectin,183 the regulated expression of α1β1 and α2β1 on effector/memory T cells may be critical to promoting effective T cell adhesion to collagen found in non-lymphoid tissue. Initial studies using integrin blocking antibodies and α1 integrin-deficient mice clearly highlighted a role for collagen-binding integrins in various inflammatory models, as well as in a mouse model of arthritis.184 Subsequent studies in a variety of model systems have suggested multiple functions in non-lymphoid tissue for α1β1 and α2β1 expressed on activated T cells.
In the lung, α1β1 integrin enables both retention and survival of influenza specific CD8 T cells.185 Although α1β1 integrin is not required for T cell entry into the lung, many more T cells in the lung are α1β1 integrin-positive compared to activated T cells in the spleen. This suggests that α1β1 integrin expression may be specifically induced on CD8 T cells after their entry into the lung. α1β1 integrin-positive cells had reduced levels of apoptosis compared to the α1β1 integrin-negative population in the lung. Other work has supported this role for α1β1 integrin in promoting the survival of lung-resident CD8 T cells, potentially via signaling mechanisms that synergize with TNF Receptor II.186 Antibody-mediated inhibition of α1β1 integrin resulted in both a loss of cells from the lungs and reduced ability to protect against a secondary challenge.185 Mice deficient in α1 integrin expression also exhibit enhanced susceptibility to secondary virus challenge. α1β1 integrin-positive CD8 T cells isolated from the lung produce effector cytokines, such as IFN-γ and TNFα. CD4 T cells in the lung express higher levels of α2β1 integrin than CD8 T cells, and this is associated with CD4 T cell localization to interstitial spaces in the lung that express type I collagen. In vitro analysis of these CD4 T cells suggest a role for α2β1 in enhancing IFN-γ production.187 Thus, these studies suggest that collagen-binding integrins promote T cell survival, retention, and effector function in the lung parachyma, thus enhancing protective immunity.
α1β1 has also been shown to retain pathogenic type 1 effector T cells in the epidermis in a xenoplant model of psoriasis.188 In this model, α1 integrin-specific blocking antibodies prevented the development of psoriasis by preventing the accumulation of these cells in the epidermis. These intraepidermal α1β1 integrin-positive T cells were also shown to co-express αEβ7 integrin. The lack of expression of α1β1 on dermal T cells suggests that α1β1 integrin may be upregulated during passage through the collagen IV rich demo-epidermal basement membrane. Entry into the E-cadherin rich epidermis might also promote upregulation of αEβ7, which would facilitate epidermal localization of CD8 T cells in the skin. In addition, the αEβ7 integrin inducing molecule, TGF-β, is found in high concentration in areas underlying psoriatic lesions.189
Overall, there is emerging evidence that the microenvironment in non-lymphoid tissue can both modulate integrin expression on T cells and provide adhesive ligands that promote T cell retention, survival and effector function. Integrins such as αEβ7, α1β1 and α2β1 have been proposed to play unique roles in promoting T cell retention in non-lymphoid tissue. However, it is known that the presence of antigen is important for the retention of effector/memory T cells in non-lymphoid sites.190 In addition, MHC class II-positive DCs have recently been reported to migrate to non-lymphoid tissue and interact with memory CD4 T cells.191 Thus, TCR-mediated signals that activate integrins may allow multiple integrins expressed on effector T cells to promote retention in tissue via adhesion to APCs or ECM proteins.
The factors that control the movement of T cells out of non-lympohoid tissue into afferent lymphatics remain incompletely characterized. Expression of the CCR7 chemokine receptor is critical for migration of both DCs192;193 and T cells194;195 out of non-lymphoid tissue and into draining LNs. Studies with integrin-null DCs indicate that integrins are not required for DCs trafficking out of non-lymphoid tissue.149 However, recent work has revealed a role for S1P1 in retaining T cells in peripheral tissue via modulation of integrin function. 196 Treatment of T cells with the S1P1 agonist FTY720 inhibited migration into the afferent lymphatic vessel by promoting firm adhesion of T cells to the basal (tissue) side of afferent lymph vessels mediated by both αLβ2 and α4β1 integrins. This suggests a regulatory interplay between the S1P1 system and integrins, and that changes in S1P levels may control T cell egress out of non-lymphoid tissue via effects on integrin function.
Based on the homing preference of specific subsets of T cells and other leukocytes, therapies targeting integrins have made their way into the modern medical armamentarium. Natalizumab is a humanized anti-α4 integrin antibody that is approved for treatment of both multiple sclerosis and Crohn’s disease. Although preliclinical studies and clinical trials showed efficacy, 197 a few patients taking natalizumab developed a rare and often fatal demyelinating disorder called progressive multifocal leukoencephalopathy (PML).198 The development of PML in these patients may be due to the high level of immunosuppression induced by bi-therapy with natalizumab and interferon. Currently, natalizumab is approved as a monotherapy and no further cases of PML have been reported.199 As natalizumab targets both α4β1 and α4β7, there is interest in developing therapies that more specifically target certain integrins. An α4β7 specific antibody (MLN-02) has been shown to be effective in inducing clinical remission in the treatment of ulcerative colitis and active Crohn’s disease.93;200 Although the mechanism of action of these antibodies is presumed to be primarily at the level of modulating integrin-dependent functions that control T cell migration and localization, it is important to consider other potential effects of these antibodies on T cell function.201 Recent studies in the mouse suggest that antibodies against α4 integrin or α4 integrin deficiency can alter T cell activation and differentiation.76;202–206 Thus, additional research is clearly needed to more fully define the mechanism by which these therapies ameliorate disease.
The β7 integrin has also been used as target for the delivery of siRNA to gut-homing phenotype cells.207 In these experiments, siRNA targeting cyclin D1 was delivered in vivo via stabilized nanoparticles that had been coated with anti-β7 integrin antibody. Cyclin D1 knockdown in cells expressing β7 integrin resulted in a reversal of the development of experimental colitis in mice. The HIV cell surface glycoprotein, gp120, was recently shown to directly bind α4β7 potentially facilitating HIV entrance into CD4 T cells.208 Supporting this hypothesis, several but not all integrin blocking antibodies reduced viral replication in CD4 T cell cultures. This suggests the possibility that previously developed and clinically available therapies designed at blocking α4β7 may decrease the ability of HIV to spread by infecting new CD4 T cells. However, one recent study has shown that natilizumab does not inhibit HIV replication in CD4 T cell cultures.209
The involvement of integrins in all phases of the T cell life cycle calls for their regulated expression depending upon the current need of the cell. On naïve T cells, integrins promote efficient T cell entry into peripheral LNs. Although integrins are not required for motility in LNs, they provide adhesive forces that promote T cell interactions with APCs that are critical for effective T cell priming. The microenvironment within the LN is critical to determining the specific changes in integrin expression that occur as naïve T cells clonally expand and differentiate into effector T cells. While factors such as ATRA have been identified that promote expression of specific integrins such as α4β7, our understanding of these environmental signals that regulate integrin expression on activated T cells is still in its infancy. These environmental signals play a central role in defining the ability of effector T cells to access various non-lymphoid tissue sites. The environment within non-lymphoid tissue differs dramatically from the LN environment. Although there is now strong evidence suggesting that the non-lymphoid tissue environment can induce expression of certain integrins on T cells after their entry from the blood, the factors that control this induction of integrin expression remain unclear. This will be an important area of future investigation, as it is clear that integrins play an important role in the retention, survival and function of T cells in non-lymphoid tissue.
This work was supported by NIH grants AI031126, AI038474, and AI064271 (Y.S.), NIH grant F30 DK082139 (C.C.D.), NIH grant T32 CA009138 (J.S.M.) and the Harry Kay Chair in Biomedical Research (Y.S.).