The multifunctional nature of CD103 (αEβ7 integrin) signaling in tissue-resident lymphocytes

Abstract

Intestinal tissue-resident lymphocytes are critical for maintenance of the mucosal barrier and to prevent enteric infections. The activation of these lymphocytes must be tightly regulated to prevent aberrant inflammation and epithelial damage observed in autoimmune diseases, yet also ensure that antimicrobial host defense remains uncompromised. Tissue-resident lymphocytes express CD103, or αE integrin, which dimerizes with the β7 subunit to bind to E-cadherin expressed on epithelial cells. Although the role of CD103 in homing and retention of lymphocytes to and within peripheral tissues has been well characterized, the molecular signals activated following CD103 engagement remain understudied. Here, we highlight recent studies that elucidate the functional contribution of CD103 in various lymphocyte subpopulations, either as an independent signaling molecule or in the context of TCR co-stimulation. Finally, we will discuss the gaps in our understanding of CD103 biology and the therapeutic potential of targeting CD103 on tissue-resident lymphocytes.

INTRODUCTION

Tissue-resident lymphocytes are essential to provide immunosurveillance and contribute to the maintenance of the intestinal mucosal barrier. In the gut, a single layer of epithelial cell lines the intestinal tract to provide a physical barrier between microbes, dietary antigens, and the underlying mucosa. Within the epithelium resides a subset of lymphocytes referred to as intraepithelial lymphocytes (IELs), which contribute to the first line of defense against invasive microorganisms. Induced IELs are recruited from the periphery after antigen exposure; these IELs express T cell receptor (TCR) αβ and either CD8αβ or CD4 (1). CD8αβ TCRαβ IELs are largely tissue-resident memory (Trm) lymphocytes. In contrast, natural IELs including those that express CD8αα TCRαβ and TCRγδ are considered to be major histocompatibility complex (MHC)-independent and exhibit innate-like functions. Finally, a small fraction of CD4 CD8αα IELs are regulatory T cells (Tregs) that upregulate CD8αα expression following downregulation of ThPOK (2). Regardless of the subset, activation of these tissue-resident IELs must be tightly regulated to prevent aberrant inflammation and/or damage to the epithelium, as observed in inflammatory bowel disease (IBD) and celiac disease (3–5). However, IELs also function to limit microbial infection and lyse infected enterocytes as a key component of mucosal host defense (6).

αE (ITGAE) integrin, or CD103, dimerizes exclusively with β7 (ITGB7) and is expressed on the majority of tissue-resident lymphocytes. To date, the only known ligand for CD103 is E-cadherin (E-cad) (7, 8), an adherens junction protein expressed by epithelial cells. CD103 is largely implicated for its role in the homing and retention of lymphocytes in peripheral tissues (9–13) with less focus on the biological contribution of CD103 ligation beyond enhancing antigen-mediated killing by CD8 T cells. In this review, we will highlight recent findings regarding the molecular mechanisms by which engagement of CD103 leads to independent or co-stimulatory cellular responses, the known signaling pathways activated downstream of CD103/E-cad binding, and how these signals impact lymphocyte function in the context of gastrointestinal disease. Finally, we will define remaining gaps in knowledge regarding CD103 biology that may inform the development of therapeutics targeting CD103/E-cad interactions to improve clinical outcomes in the context of gastrointestinal infection, inflammation, and cancer.

Regulation of CD103 Expression

As mentioned in the INTRODUCTION, CD103 is widely used as a marker to identify tissue-resident immune cells in various organs such as the skin, lungs, and gut (14). CD103 expression is induced on peripheral lymphocytes as a result of local transforming growth factor (TGF)-β production within the tissue microenvironment (15, 16). The role of TGF-β in promoting CD103 expression has been demonstrated in multiple studies: 1) overexpression of TGF-β by tumor cells results in higher CD103 expression in tumor-infiltrating lymphocytes (TILs), 2) T cells with impaired TGF-β type II receptor signaling fail to upregulate CD103 upon migration into the tissue (9, 13, 15, 17), 3) TGF-β is sufficient to induce CD103 in cultured human leukocytes in vitro, and 4) T cell activation in conjunction with TGF-β results in CD103 expression by cytotoxic T lymphocytes (CTL) (9, 15, 18–20).

TGF-β ligation to its receptor results in the phosphorylation of Smad2/3, after which phosphorylated Smad3 binds to the promoter region of CD103 in CD8 T cells (21). Since Smad7 interferes with the recruitment and phosphorylation of Smad2/3, transgenic mice overexpressing Smad7 exhibit defective TGF-β signaling and reduced expression of CD103 on IELs (22). In addition to TGF-β, the transcription factor Runx3 was shown to regulate CD103 expression on CD8 single-positive thymocytes and dendritic epidermal T cells (23, 24). Runx3 also plays an important role in the differentiation of CD103-expressing Trm populations (25) by allowing chromatin accessibility within Itgae and other TGF-β regulated genes (26). In addition, T-bet and Tcf1 can directly bind Itgae regulatory regions and inhibit TGF-β-mediated CD103 expression (27, 28). Together, these studies demonstrate that TGF-β-Smad3 signaling and the expression of Runx3 are critical for the induction of CD103 expression on lymphocytes (Fig. 1).

Figure 1.

TGF-β signaling and Runx3 regulate CD103 expression. Activation of TGF-βR1 promotes the recruitment and phosphorylation of Smad2/3. Phosphorylated Smad3 and Runx3 bind to the CD103 promoter to induce transcription. Smad7 negatively regulates Smad2/3 signaling and can inhibit CD103 expression. TGF-β, transforming growth factor β. Image created with BioRender and published with permission.

Bidirectional CD103 Signaling

Activation of integrins occurs through two distinct signaling mechanisms: 1) “inside-out” in which intracellular signals downstream of GPCR or TCR signaling promote talin binding to the cytoplasmic tail of the β subunit of integrin, which results in an conformational change to expose the ligand binding region of the extracellular domain, or 2) “outside-in” signaling that is initiated following a high-affinity interaction between the integrin extracellular domain and its binding partner or cations (29). Current evidence suggests that CD103 activation can occur through both mechanisms. Activation of the TCR, CC motif chemokine receptor 9 (CCR9), and TGF-βR1 induces “inside-out” signaling and increases the avidity of the binding between CD103 and E-cad (30–32). Although the molecular mechanisms associated with TCR- or CCR9-mediated CD103 activation remain to be elucidated, signaling through TGF-βR1 promotes PI3K-mediated phosphorylation of integrin-linked kinase (ILK), which subsequently binds to the intracellular tail of CD103 to promote “inside-out” activation (32) (Fig. 2A). Phosphorylation of Akt downstream of TGF-βR1/ILK signaling also contributes to CD103 binding to E-cad; however, the molecular mechanisms by which Akt promotes the confirmational change of CD103 remains unclear. In this study, the authors posit that TGF-β signaling through ILK and Akt strengthened the interaction between CD103/E-cad, which in turn induces “outside-in” signaling to promote CTL adhesion, migration, and cytolytic capacity.

Figure 2.

Bidirectional signaling leading to CD103 activation. A: “inside-out” signaling of CD103 and its known molecular mechanisms, leading to the confirmational change of CD103. B: “outside-in” signaling of CD103 and its known molecular mechanisms, leading to cellular functions. Image created with BioRender and published with permission.

“Outside-in” signaling occurs following high-affinity binding of an integrin to its ligand and mediates cellular responses such as spreading, proliferation, and effector functions through phosphorylation of molecules and/or binding to extracellular matrix. CD103 binding to immobilized recombinant E-cad induced “outside-in” signaling to promote the phosphorylation of the proline-rich tyrosine kinase 2 (Pyk2) and the focal adhesion-associated adaptor protein, paxillin in a Src kinase-dependent manner (33). As a result, phosphorylated paxillin binds to the intracellular tail of clustered CD103, thus enhancing the migratory and effector functions of CD8 T cells. Although TCR coengagement results in CTL degranulation for targeted cell lysis, which may be suggestive of “inside-out” signaling, a later study demonstrated that E-cad engagement alone was sufficient to promote extracellular signaling regulated kinase (ERK) and phospholipase C γ 1 (PLCγ) phosphorylation, resulting in the polarization of cytolytic granules (34). These studies demonstrate that CD103 can be activated via bidirectional signaling, and thus may induce differential downstream signaling pathways in lymphocytes depending on whether CD103 is activated in conjunction with, or independently of, TCR activation (Fig. 2B).

CD103: Implication of Recruitment and Retention

Based on observations that deletion of CD103 reduces the number of tissue-resident lymphocytes in vivo (9–12), the interaction between CD103 and E-cad is implicated in the selective recruitment of lymphocytes to epithelial barriers and anchoring of lymphocytes to the epithelium. However, the requirement for CD103 in tissue homing was challenged when Austrup and colleagues (35) showed that in vitro cultured CD103⁺ T cells failed to migrate to the gut and other peripheral organs. This suggested that the observed reduction in the number of tissue-resident cells is likely due to impaired retention, and this is supported by infection studies that allow tracking of antigen-specific T cell dynamics. CD103 is not expressed on circulating effector lymphocyte populations or required for T cell trafficking into the tissue, but CD103-deficient Trm populations in the skin, brain, and intestinal epithelium are gradually lost after infection is resolved, indicating a role for CD103 in retention/maintenance of tissue-resident lymphocytes (9, 13, 36). However, this is not absolute, as the intestinal lamina propria contains CD103⁺ Trm cells that do not require CD103 for their retention within the tissue (9, 37). In addition, many tissues like the liver, salivary gland, and lamina propria contain bona fide Trm cells that are CD103⁻ (38, 39). Together, these studies demonstrate that the expression of CD103 is not essential for the trafficking of lymphocytes to the gut and other tissues, and is likely not a definitive marker of all tissue residents as CD103⁻ Trms are found in various tissues. This highlights the need to determine the functional significance suggesting that CD103, as these data suggest it could be an essential molecule for other cellular functions and not necessarily crucial for immune cells to exist in the tissue environment.

CD103 Functions Both in a Co-Stimulatory Manner and Independently to Promote Lymphocyte Function

The molecular mechanisms by which CD103/E-cad engagement promotes the cross talk between epithelial cells and lymphocytes remain largely understudied. This is particularly relevant in the context of antigen-specific responses as well as those that are MHC-independent. Regarding antigen-dependent activation, the involvement of CD103 in enhancing CD8 T cell cytolytic capacity is well documented (15, 34, 40). Ligation of CD103 by E-cad leads to the polarization of lytic granules and lowers the activation threshold for the local polarized release of granzymes, resulting in more efficient killing of E-cad-bearing target cells (Table 1) (34, 52). However, CD103 is also involved in enhancing the function of other lymphocyte populations. For example, CD103⁺ Tregs exhibit more potent suppressive capacity compared with those not expressing CD103, and are critical in limiting allergic contact hypersensitivity (43), colitis (44), and antigen-induced arthritis in animal models (45). Furthermore, coculture of CD103⁺ CD4⁺ T cells isolated from the gastric mucosa with H. pylori-primed dendritic cells promotes lymphocyte proliferation and proinflammatory cytokine production that is abrogated by antibody-mediated blockade of CD103 (46). The engagement of CD103⁺ CD8⁺ thymocytes with E-cad-expressed primary thymic epithelial cells leads to enhanced proliferation; however, E-cad binding to CD103 alone is insufficient to induce proliferation indicating the necessity for a second signal (47). Taken together, it is clear that CD103 acts as a co-stimulatory molecule in several lymphocyte subsets to enhance proliferation and effector function.

Table 1.

Functional contribution of CD103⁺ tissue-resident lymphocytes. Engagement of CD103 with E-cad on CD103⁺ lymphocytes, with or without TCR co-stimulation

Lymphocyte	Cellular Function	Tissue	Requirement for TCR Co-Stimulation	References
CD8	Enhanced cytotoxicity	Tumors and grafts	Yes	(15, 41, 42)
Regulatory T cell (Treg)	Enhanced suppression	Skin, colon, and tumor	Unknown	(18, 43–45)
CD4	Enhanced proliferation and Th1/17 cytokine production	Gastric mucosa	Yes	(46)
Thymocytes	Enhanced proliferation	Thymus	Unknown	(47)
γδ T cells	Facilitating apoptotic enterocyte shedding via degranulation-independent granzyme release	Villous epithelium	No	(48)
	Regulating duration of IEL/epithelial contact	Villous epithelium and small intestinal tumors	No	(49–51)

E-cad, E-cadherin; TCR, T cell receptor.

In contrast, natural IELs expressing the γδ TCR display antigen-independent functionality and uniformly express CD103. We have shown that γδ IELs migrate dynamically along the basement membrane to provide continuous surveillance of the villous epithelium (49). Although CD103 engagement influences lymphocyte shape and motility by promoting actin remodeling (53), we found that CD103-deficient γδ IELs exhibit enhanced migratory speed based on reduced retention within the lateral intercellular space (LIS) between adjacent epithelial cells (49). This suggests that CD103/E-cadherin binding is important for regulating the duration of IEL/epithelial contact (Table 1). As a result of the enhanced γδ IEL surveillance behavior in CD103-deficient mice, the frequency of acute Salmonella typhimurium translocation across the epithelium is reduced (50). Furthermore, administration of anti-γδ TCR antibody has no effect on γδ IEL migratory behavior in response to enteric infection (54) and suggests that CD103 signaling in this capacity does not require TCR activation (Table 1). More recently, we demonstrated that CD103 is required for prolonged interactions between γδ IELs and shedding apoptotic enterocytes both at steady state and in response to TNF exposure (48). Whereas TCR activation is required for γδ IEL degranulation, CD103 engagement by E-cad alone is sufficient to induce the extracellular secretion of granzyme (Gzm) A and GzmB. We found that γδ IEL CD103-mediated Gzm release is required for these lymphocytes to facilitate cell shedding. These observations highlight that CD103 can function independently of TCR activation, which is in line with the more “innate-like” function of these sentinel lymphocytes. It remains to be determined whether CD103 ligation also acts as a co-stimulatory molecule to amplify adaptive γδ IEL responses similar to conventional CD8 T cells. However, many of the downstream molecular pathways that regulate these lymphocyte responses following CD103 engagement, whether TCR-dependent or independent, remain to be elucidated.

CD103 in Gastrointestinal Disease

Intestinal host defense involves the coordinated response of both natural and induced IELs, as the speed and potency of this response are critical to prevent disruption of the epithelial barrier and unchecked pathogen replication and dissemination. γδ IELs are activated early after infection, and alterations in their surveillance behavior are observed within minutes to hours following Salmonella or Toxoplasma encounter, respectively (50, 54). Increased migration into the LIS following the loss of CD103 expression likely allows γδ IELs to elicit a highly localized and conserved antimicrobial response to limit pathogen translocation (50, 55). Trm cells can control pathogen replication in the intestinal tissue (38, 56), but the mechanism and role of CD103 in this process are less well defined. However, Trm cells in other tissues can undergo bystander activation by innate cytokines to support pathogen control (57). Subsequent processing and presentation of pathogen-derived antigens lead to the activation of Trm IELs to potentially eliminate infected enterocytes and produce cytokines that stimulate activation and recruitment of additional immune cells (58, 59).

Obesity can promote epithelial barrier dysfunction and low levels of chronic inflammation (60, 61). Feeding of a high-fat diet to young mice results in the downregulation of CD103 on CD4 and γδ IELs leading to a reduction in the persistence of IELs within the mucosa; however, IEL number and CD103 expression are restored following diet-induced weight loss (62). Although this study focuses on the role of CD103 in the retention or maintenance of these tissue-resident populations, these findings suggest that obesity can indirectly regulate CD103 expression and may have additional implications for tissue-resident lymphocyte function.

IBD, which includes both ulcerative colitis (UC) and Crohn’s disease (CD), is a multifactorial disease associated with relapsing and remitting intestinal inflammation. In the T cell transfer model of colitis, CD103 is not required for homing of lymphocytes to the intestinal lamina propria and the transfer of either WT or CD103-deficient effector T cells results in equally severe disease (63). In contrast, the identification of CD8⁺ CD103⁺ Tregs revealed that these cells exhibit the capacity to attenuate ileitis and suppress T-cell-mediated colitis (64, 65). Although γδ T cells are typically considered to be protective in the context of IBD (3), Do and colleagues identified a peripheral population of CD103⁺ ${\mathrm{\alpha }}_{\mathrm{4}}{\mathrm{\beta }}_{\mathrm{7}}^{\mathrm{hi}}\mathrm{\gamma \delta }$ T cells that are proinflammatory and can exacerbate colitis (66). Genetic or antibody-mediated inhibition of β7 integrin ameliorates colitis (67, 68); however, specific targeting of β7 integrin will affect both α₄β₇ and α_Eβ₇ (CD103) integrins. While vedolizumab, a humanized monoclonal antibody that selectively targets α₄β₇, is efficacious in the treatment of moderate to severe UC and CD (69), etrolizumab, a humanized monoclonal antibody against β⁷ integrin, appeared to be promising in phase 2 clinical trial (70); but failed to yield consistent results in a phase 3 trial (71). It was thought that inhibiting both integrins would reduce intestinal inflammation by inhibiting lymphocyte trafficking to the gut, yet it is possible that blocking CD103 may be a double-edged sword and ultimately limit the protective functions of tissue-resident IELs, Trms, and Tregs.

In solid tumors, CD103 expression on CD8 TILs results in a favorable prognosis and increased overall survival (41). CD103 expression is thought to retain CTLs within the tumor epithelium (19), and a reduction of colonic CD103⁺ γδ IELs or Trm IELs is observed in patients with familial adenomatous polyposis (FAP) (72). Moreover, CD103 expression was also reduced in lamina propria T cells in patients with FAP. Deletion of CD103 on lymphocytes was recently shown to impair tumor immunosurveillance by reducing the frequency of IEL/epithelial interactions leading to an increase in small intestinal tumors in an APC^min model (51); however, this study did not directly address whether loss of CD103 affected cytolytic effector function. Furthermore, enrichment of TGF-β in the tumor microenvironment induces CD103 expression on tumor-infiltrating Tregs (18). Thus, the enhanced suppressive nature of CD103⁺ Tregs (44) may lead to increased tumor burden and negative prognosis, as observed in patients with ovarian and breast cancer (73, 74). Together, these findings indicate a need to better understand the functional contribution of CD103 within distinct lymphocyte subsets in the context of infection, chronic intestinal inflammation, and cancer.

CONCLUDING REMARKS

In summary, CD103 contributes to several aspects of protective T cell-mediated immunity in the gut including antigen-dependent activation, surveillance behavior, and control of enteric infection. These studies clearly indicate that CD103 expression on various lymphocyte populations has additional functions beyond tissue homing and retention. Despite the multifunctional contribution of CD103 to tissue-resident lymphocyte biology, much remains unknown regarding how positive and/or negative regulation of signaling downstream of CD103 contributes to the modulation of lymphocyte effector function. For example, inside-out activation of integrins can promote the release of calcium from intracellular stores (75), which may contribute to changes in lymphocyte motility, cytolytic function, and effector cytokine production. Moreover, our knowledge regarding how CD103 signaling in gut-resident lymphocytes is affected in response to different diets, celiac disease, or aging, is extremely limited. Studies to date suggest that the functional role of CD103 may differ based on lymphocyte subset; therefore, investigating the molecular pathways activated by CD103 engagement, either in response to co-stimulation or independent of TCR signaling, will significantly advance our understanding of how these tissue-resident cells are regulated and activated under homeostatic and pathological conditions at barrier sites.

GRANTS

This work was supported by the Agency for Science, Technology and Research (A*STAR) postdoctoral fellowship (to W.X.); the National Institutes of Health (NIH) Grants R01 AI153096 and R21 AI148900 (to T.B.); and NIH R01 DK119349, R21 DK123488, and R21 AI171959 (to K.L.E.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

W.X. and K.L.E. prepared figures; W.X. drafted manuscript; W.X., T.B., and K.L.E. edited and revised manuscript; W.X., T.B., and K.L.E. approved final version of manuscript.