The Emerging Role of CD8+ Tissue Resident Memory T (TRM) Cells in Antitumor Immunity: A Unique Functional Contribution of the CD103 Integrin

Cancer immunotherapy is aimed at stimulating tumor-specific cytotoxic T lymphocytes and their subsequent trafficking so that they may reach, and persist in, the tumor microenvironment, recognizing and eliminating malignant target cells. Thus, characterization of the phenotype and effector functions of CD8+ T lymphocytes infiltrating human solid tumors is essential for better understanding and manipulating the local antitumor immune response, and for defining their contribution to the success of current cancer immunotherapy approaches. Accumulating evidence indicates that a substantial subpopulation of CD3+CD8+ tumor-infiltrating lymphocytes are tissue resident memory T (TRM) cells, and is emerging as an activated tumor-specific T-cell subset. These TRM cells accumulate in various human cancer tissues, including non-small-cell lung carcinoma (NSCLC), ovarian and breast cancers, and are defined by expression of CD103 [αE(CD103)β7] and/or CD49a [α1(CD49a)β1] integrins, along with C-type lectin CD69, which most likely contribute to their residency characteristic. CD103 binds to the epithelial cell marker E-cadherin, thereby promoting retention of TRM cells in epithelial tumor islets and maturation of cytotoxic immune synapse with specific cancer cells, resulting in T-cell receptor (TCR)-dependent target cell killing. Moreover, CD103 integrin triggers bidirectional signaling events that cooperate with TCR signals to enable T-cell migration and optimal cytokine production. Remarkably, TRM cells infiltrating human NSCLC tumors also express inhibitory receptors such as programmed cell death-1, the neutralization of which, with blocking antibodies, enhances CD103-dependent TCR-mediated cytotoxicity toward autologous cancer cells. Thus, accumulation of TRM cells at the tumor site explains the more favorable clinical outcome, and might be associated with the success of immune checkpoint blockade in a fraction of cancer patients.

Cancer immunotherapy is aimed at stimulating tumor-specific cytotoxic T lymphocytes and their subsequent trafficking so that they may reach, and persist in, the tumor microenvironment, recognizing and eliminating malignant target cells. Thus, characterization of the phenotype and effector functions of CD8 + T lymphocytes infiltrating human solid tumors is essential for better understanding and manipulating the local antitumor immune response, and for defining their contribution to the success of current cancer immunotherapy approaches. Accumulating evidence indicates that a substantial subpopulation of CD3 + CD8 + tumor-infiltrating lymphocytes are tissue resident memory T (TRM) cells, and is emerging as an activated tumor-specific T-cell subset. These TRM cells accumulate in various human cancer tissues, including non-small-cell lung carcinoma (NSCLC), ovarian and breast cancers, and are defined by expression of CD103 [αE(CD103)β7] and/or CD49a [α1(CD49a)β1] integrins, along with C-type lectin CD69, which most likely contribute to their residency characteristic. CD103 binds to the epithelial cell marker E-cadherin, thereby promoting retention of TRM cells in epithelial tumor islets and maturation of cytotoxic immune synapse with specific cancer cells, resulting in T-cell receptor (TCR)-dependent target cell killing. Moreover, CD103 integrin triggers bidirectional signaling events that cooperate with TCR signals to enable T-cell migration and optimal cytokine production. Remarkably, TRM cells infiltrating human NSCLC tumors also express inhibitory receptors such as programmed cell death-1, the neutralization of which, with blocking antibodies, enhances CD103-dependent TCR-mediated cytotoxicity toward autologous cancer cells. Thus, accumulation of TRM cells at the tumor site explains the more favorable clinical outcome, and might be associated with the success of immune checkpoint blockade in a fraction of cancer patients.
Keywords: CD8 tissue resident memory T (TRM) cells, CD103 integrin, cytotoxic T lymphocytes, onco-immunology, cancer immunotherapy Abbreviations: CTL, cytotoxic T lymphocytes; CTLA, cytotoxic T-lymphocyte-associated antigen; PD-1, programmed cell death-1; IFN, interferon; LFA-1, lymphocyte-function-associated antigen-1; mAb, monoclonal antibodies; NSCLC, nonsmall-cell lung carcinoma; MHC-I, major histocompatibility complex class I; TCR, T-cell receptor; TIL, tumor-infiltrating lymphocytes. TRM Cells in Antitumor Immunity Frontiers in Immunology | www.frontiersin.org August 2018 | Volume 9 | Article 1904 iNTRODUCTiON CD8 + T lymphocytes play an essential role in defense against cancers through recognition by T-cell receptors (TCR) of specific antigenic peptides presented on the surface of malignant cells by major histocompatibility complex class I (MHC-I) molecules, and elimination of the tumor target, mainly by releasing the content of cytolytic granules containing perforin and granzymes.
To destroy their target, cytotoxic T lymphocytes (CTL) must first migrate to the tumor site, infiltrate the tumor tissue, and interact with the cancer cell, to finally trigger effector functions leading to transformed cell eradication. Integrins and their ligands (1) play a crucial role in promoting antitumor T-cell activities by regulating T-cell migration and retention within the tumor, adhesion to antigen-presenting cells and co-stimulation resulting in CTL activation and functions (2). Cytokines and chemokines are also involved in coordinating circulation, homing, retention, and activation of T lymphocytes. Although some of them are known to contribute to tumor cell proliferation and dissemination by inhibiting tumor-specific T-cell responses, others promote infiltration and activation of T lymphocytes in a hostile tumor ecosystem, resulting in tumor cell destruction (3). In this regard, TGF-β, abundant in the tumor microenvironment, was reported to be an immunosuppressive factor used by malignant cells to escape from the immune response (4). This cytokine inhibits expression of lymphocyte-function-associated antigen-1 (LFA-1, also known as αLβ2 or CD11a) integrin and LFA-1-mediated T-cell functions (5). Paradoxically, this cytokine induces CD103 (also known as αEβ7 or HML-1) integrin expression on activated intraepithelial CD8 + T lymphocytes, and enhanced CD103-dependent T-cell adhesion and signaling (6,7). LFA-1 and CD103 are the predominant integrins expressed by intraepithelial T lymphocytes (IEL) and CD8 + tumorinfiltrating lymphocytes (TIL). While the contribution of LFA-1 and its ligand ICAM-1 (CD54) to TCR-mediated CTL activities is well documented (8), much less is known about the role of CD103 and its ligand, the epithelial cell marker E-cadherin, to T-cell-mediated cytolytic activity. CD103 has been associated with cytotoxicity of CD8 + T cells in several human pathologies, including graft-versus-host disease (GVHD) (9), allogeneic transplant rejection (10)(11)(12), autoimmune diseases (13,14), and cancer (6,15). This integrin, together with the activation marker CD69 and the integrin CD49a [also known as α1β1 or very late antigen-1 (VLA-1)], defines a recently identified subtype of CD8 + T lymphocytes called "tissue-resident memory T (TRM) cells, " possibly endowed with potent cytotoxic activities. Moreover, there is an emerging consensus that TRM cells frequently accumulate in multiple human tumors, especially of epithelial origin, and play an essential role in tumor-specific T-cell responses and, likely, in control of malignant diseases. TRM cells are also surrogate markers of the efficacy of cancer vaccines (16,17), and a low number of this T-cell subset among TIL may correlate with failure of immune checkpoint blockade therapy in most cancer patients. In this review, we focus on CD8 + TRM cells accumulating in human solid tumors, mainly non-smallcell lung carcinoma (NSCLC), and current insight implicating CD103 integrin in regulating TRM functions and CTL-mediated antitumor immune responses, with potential prognosis and immunotherapeutic applications.

PHeNOTYPiC AND MOLeCULAR FeATUReS OF T RM CeLLS iN TUMORS
It is now generally agreed that a population of TRM cells accumulates in tumors of epithelial origin, such as ovarian, pancreatic, colorectal, and lung tumors (15,(18)(19)(20), as well as those of nonepithelial origin, including malignant glioma and melanoma (21,22). These TRM cells express a broad range of integrins and chemokine receptors, probably involved in their migration to the tumor site, and may interfere with their egress from the tumor tissue. Transcriptional studies pointed to expression of CXCR3 and CXCR6 by TRM cells infiltrating human lungs (23). Intratumoral TRM cells express high levels of CCR5 and CCR6 chemokine receptors that may confer T-cell homing to the inflammatory tumor microenvironment (15). Moreover, CCR5 is recruited at the immune synapse formed between T cells and tumor target cells upon interaction of CD103 with E-cadherin, promoting retention of TRM cells at the tumor site by inhibiting their sensitivity to a CCL5 chemotactic gradient (7). By contrast, TRM cells do not express CX3CR1, a chemokine receptor that mediates transmigration through the endothelium, supporting the hypothesis that this T-cell population has reached its final destination and does not need to exit from the lung tissue (23). Lung tumor TRM lack expression of lymph node homing receptors CCR7 and CD62L, as well as the receptor for sphingosine 1-phosphate, S1PR1 (15), which mediates the egress of T cells from lymphoid organs (24). Indeed, downregulation of SIPR1 appears to be a prerequisite for retention of CD8 + CD103 + TRM cells in peripheral tissues (25,26).
With regard to adhesion/costimulatory molecules, the expression profile of intratumoral TRM cells seems to be compatible with their capacity to reside in tumor tissue and their inability to recirculate in the bloodstream. In melanoma, CD8 + TRM cells were found to co-express CD69, CD103, and VLA-1 (CD49a or α1β1 integrin), with the latter reported to cause long-term retention of activated T cells in peripheral tissues (27). Human lung tumor CD8 + TRM cells are characterized by downregulation of CD28 and upregulation of CD69 and CD103 and CD49a integrins, which are most likely induced by TGF-β in the tumor microenvironment (15,28). TGF-β plays a pivotal role in formation and maintenance of TRM, at least in part via induction of CD103. Indeed, TGF-β is directly involved in CD103 expression in tumor-specific T cells upon engagement of TCR with specific tumor peptide-MHC-I complexes (7), through binding of Smad2/3 and NFAT-1 transcription factors to promoter and enhancer elements of the ITGAE gene, which encodes the CD103 (αE) subunit (29). This cytokine is also involved in dampening expression of the LFA-1 integrin on TIL, thus participating in T-cell residency within the tumor (15,30). In LCMV chronic infection, but not acute infection, TGF-β signaling inhibits migration of CD8 + effector T lymphocytes from the spleen to the gut by dampening expression of integrin α4β7 during the formation phase of TRM cells (31). Consequently, CD8 + Tgfbr2 −/− T cells migrate normally to the intestine, but their retention in the gut epithelium is impaired. In contrast, TGF-β signaling does not impact α4β7 integrin expression and T-cell migration to the gut after acute bacterial infection (32). Moreover, E-cadherin, which is downregulated by TGF-β in cancer cells during epithelial-to-mesenchymal transition [for a review see Ref. (33)], appeared to promote accumulation of a subset of CD8 + memory T cells in murine submandibular glands by a mechanism independent of CD103 (34). This cytokine has been identified as a potential therapeutic target in cancer because of its role in supporting tumor progression and in inducing immunosuppression. In this regard, it has been shown that targeting the TGF-β pathway inhibits tumor growth by promoting antitumor immunity associated with increased CD8 + T-cell numbers (35). However, the consequence of such cancer immunotherapy approaches on TRM cells, the maintenance of which is dependent of TGF-β, has not been addressed.
T-cell inhibitory receptors are important for maintaining self-tolerance and regulating the immune response in peripheral tissues (36). Among these immune checkpoints, cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and Tim-3 appeared to be associated with tumor antigen-specific CD8 + T-cell dysfunction in melanoma patients (37). CD103 + TRM cells have been shown to express a wide range of inhibitory receptors, such as CTLA-4, Tim-3, and programmed cell death-1 (PD-1), associated with their capacity to maintain peripheral tolerance (25,38). Data from our group and other groups revealed that intratumoral CD8 + CD103 + TRM cells frequently express PD-1, Tim-3, and Lag-3, which are likely involved in their exhausted state and their dysfunctioning at the tumor site (15,28,39,40). Notably, TGF-β is also involved in PD-1 induction on CD8 + T cells, contributing to T-cell anergy and a sustained tolerance (41). Neutralization of TGF-β results in downregulation of PD-1 expression in T cells causing graft rejection. Mechanistically, PD-1 is regulated by the NFATc1 transcription factor (42), and is enhanced by a TGF-β/ SMAD3-dependent signaling pathway (43). Expression of PD-1 on TIL is described as a biomarker of CD8 + tumor-reactive T cells in cancer patients (44). Thus, the PD-1 + status of tumor TRM cells suggests that they are enriched with antigen-specific CD8 + T cells that may be used as targets in cancer immunotherapy.
Functional studies showed that CD8 + CD103 + TIL are able to secrete inflammatory cytokines, including interferon (IFN)γ and TNFα (28,46). Moreover, interaction of CD103 with E-cadherin on tumor target cells optimizes cytokine release, since siRNA targeting E-cadherin partially inhibited IFNγ production (61). Cytotoxicity experiments indicated that freshly isolated CD103 + TIL were able to kill autologous tumor cells following neutralization of the PD-1-PD-L1 interaction with anti-PD-1 or anti-PD-L1 blocking antibodies (15). This cytotoxic activity is most likely mediated by CD103 + T cells, since anti-CD103 neutralizing monoclonal antibodies (mAb) compromise this function. Consistently, cytotoxicity of CD103 + T-cell clones toward autologous E-cadherin + tumor cells is inhibited anti-CD103 blocking mAb (6). Another noteworthy aspect of our contribution to the field is the demonstration that CD103 is an important molecule required for polarization of cytotoxic granules at the immune synapse formed between CTL clones and autologous tumor cells, and that siRNA targeting E-cadherin inhibited TCRmediated target cell killing (6). Moreover, CD103 contributes to recruitment of CD103 + TRM cells within epithelial tumor islets, and intratumoral early T-cell signaling (30).
A role for the VLA-1 integrin in the differentiation and functions of TRM cells was reported in a mouse tumor model (27). VLA-1 + T cells, co-expressing or not CD103, secreted high levels of IFNγ upon re-stimulation, and this cytokine production was impaired by anti-VLA-1 or anti-CD103 mAb. Moreover, blockade of VLA-1 or CD103 severely compromised control of tumor growth in vivo. Similar studies revealed that CD8 + CD103 + TRM cells accumulate and protect mice against melanoma in a CD103-dependent manner, and these TRM cells play a pivotal role in perpetuating antitumor immunity (22). Conversely, it has been reported that anti-latency-associated peptide (LAP) antibodies targeting the LAP/TGF-β complex induce a decrease in CD8 + CD103 + T cells in mouse spleen and lymph nodes, and that this peculiar T-cell subset displays a tolerogenic feature (62). Murine CD8 + CD103 + regulatory T cells have also been described in autoimmune diseases where they are induced by TGF-β and display suppressive activities independently of granzyme B (63). Moreover, CD8 + CD103 + T cells are crucial for prevention of chronic GVHD lupus in mice by suppressing T helper and B cell responses through a non-cytotoxic mechanism involving TGFβ and IL-10 signals (64). However, further studies are needed to permit the distinction between human CD8 + CD103 + CTL and CD8 + CD103 + T regulatory cells, even though granzyme B expression appears as a good marker, and determine the exact contribution of both subsets in autoimmune [for a review see Ref. (65)] and cancer diseases.
On naive T lymphocytes, integrins have weak affinity for their ligands. However, stimulation of T lymphocytes through TCR or chemokine receptors initiates an "inside-out" signal that induces integrin activation by triggering integrin-extended conformation and clustering, thereby enhancing their affinity for their ligands. Firm adhesion of integrins to their ligands triggers an "outside-in" signal that has costimulatory functions in TCR signaling, thereby contributing to T-cell activation, migration, and cytotoxicity (71)(72)(73). Until recently, the signaling pathways of CD103 integrin and the molecules involved in its bidirectional activation were not clearly elucidated. Like the other integrins, CD103 activation is regulated by TCR engagement. In this context, it has been shown that cross-linking of TCR on IEL or cell treatment with phorbol myristate acetate increased the avidity of CD103 for E-cadherin and provided a mechanism for lymphocyte adherence and activation (74). Furthermore, the CCR9 ligand, CCL25, induced CD103-mediated adhesion of CD8 + IEL to E-cadherin, suggesting a role for this chemokine receptor/ chemokine pair in promoting functions of CD103 via inside-out signaling (75). Similarly, the CCL7 chemokine has been shown to favor adhesion and retention of CD103-expressing T cells during renal allograft rejection, by promoting the adhesive properties of CD103 (76).
TGF-β is responsible for inducing CD103 integrin in CD8 + T lymphocytes (6,77) by regulating expression of both ITGAE (29,78) and ITGB7 (79) genes encoding αE and β7 chains, respectively. In addition, in contrast to all other integrins, TGF-β regulates CD103 activation and signaling within epithelial tissues (Figure 1). Indeed, we previously demonstrated that the interaction of TGF-β with its receptors TGFBR on the surface of CD8 + CD103 + T cells induces recruitment and phosphorylation of integrin-linked kinase (ILK) by TGFBR1 (activin receptor-like kinase-5) (30). We further showed that phosphorylated-ILK interacted with the CD103 subunit intracellular domain, resulting in phosphorylation of protein kinase B (PKB)/AKT, thereby initiating integrin inside-out signaling leading to activation of CD103 and strengthening of CD103-E-cadherin adhesion.
The mechanism regulating the CD103 outside-in signaling pathway is not fully understood. Studies from our group have shown that CD103-E-cadherin tight adhesion initiates an outside-in signal by promoting phosphorylation of the focaladhesion-associated adaptor protein paxillin and proline-rich tyrosine kinase-2 (Pyk2), and subsequent binding of phosphorylated-paxillin to the CD103 subunit tail (80). In addition, the adhesive interaction of E-cadherin with CD103 on TIL triggers phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and phospholipase C γ1 proteins, providing intracellular signals that promote CTL effector functions (60). These studies emphasize a unique costimulatory role of the CD103 integrin in activation of tumor-specific CTL, by triggering polarization of cytotoxic granules at the immune synapse and subsequent TCR-mediated cytotoxicity (60), and in proliferation of CD103 + thymocyte cells (81). Engagement of CD103 with E-cadherin also determines cell shape and motility of CD103 + lymphocytes (82), and recruitment of CD8 + TRM cells within epithelial tumor islets, in an actin-polymerization-dependent fashion (30,80). Moreover, TGF-β enhances T-cell adhesion and movement toward tumor regions by increasing CD103 expression levels and promoting intracellular T-cell signals leading to integrin activation (30). CD103 also contributes to retention of TRM cell subpopulations by interacting with E-cadherin and mediating arrest of T lymphocytes on epithelial tissues (32,61). Thus, CD103 appears to be a unique integrin for adjusting T-cell adhesion and migratory potential in a TGF-β-rich tumor microenvironment, as well as retention of tumor-specific CD8 + TRM cells and local antitumor effector functions (Figure 1).

PROGNOSTiC vALUe OF T RM CeLLS iN HUMAN CANCeRS
CD8 + CD103 + TRM cells have emerged as predictive markers of patient survival in several malignant diseases, including ovarian, lung, endometrial, and breast cancers (15,20,28,83,84). Indeed, in a large cohort of high-grade serous ovarian cancers (20) and a cohort of early-stage NSCLC (15), an enhanced CD103 + TIL subset correlated with improved patient survival. CD103 + TIL were also associated with a favorable prognosis in urothelial cell carcinoma of the bladder, and could represent a favorable prognostic predictor of overall and recurrence-free survival (83). In that retrospective study, CD8 + T cells were identified as the principal cellular sources of CD103, and the density of intratumoral CD103 + cells was inversely associated with tumor size. More recent studies also defined the CD103 integrin as a biomarker of good prognosis in cohorts of breast (85) and lung cancer (17,28,84). Notably, TRM infiltration in lung cancer correlated with better clinical outcome in both univariate and multivariate analyses, independently of CD8 + T cells (17). In addition, high numbers of intratumoral CD103 + TIL were FiGURe 1 | TGF-β induces CD103 expression in tumor-specific T cells and participates in integrin bidirectional signaling. Left: TGF-β controls CD103 expression in tumor antigen (Ag)-specific T cells upon interaction of T-cell receptor (TCR) with specific tumor peptide-major histocompatibility complex class I complexes, via a Smad-dependent pathway. TGF-β binds to TGFBR at the surface of CD8 + T lymphocytes and leads to recruitment and phosphorylation of Smad2 and Smad3 and their subsequent nuclear translocation. Transcription factors NFAT-1, translocated into the nucleus upon TCR engagement, and Smad2/3 bind to promoter and enhancer elements of the ITGAE gene, which encodes the CD103 (αE) subunit, and activates CD103 expression (29). Right: TGF-β participates in CD103 intracellular signaling via a non-Smad-dependent pathway. Interaction of TGF-β with TGFBR on CD8 + CD103 + TRM cells induces recruitment and phosphorylation of integrinlinked kinase (ILK). Phosphorylated (P)-ILK interacts with the CD103 subunit intracellular domain, resulting in phosphorylation of protein kinase B/AKT and initiating integrin inside-out signaling leading to activation of CD103 (30). CD103-E-cadherin tight adhesion initiates an outside-in signal by promoting phosphorylation of paxillin (Pax) and Pyk2, and subsequent binding of phosphorylated-paxillin to the αE subunit tail where a phosphorylatable Ser (S) in the ES 1163 IRKAQL motif plays an important role (80). Adhesive interaction of E-cadherin with CD103 also triggers activation of PI3K/extracellular signal-regulated kinase (ERK) and phospholipase C/PKC pathways (60), providing intracellular signals that promote CD8 + TRM effector functions, including actin cytoskeleton reorganization, T-cell spreading and migration, cytokine release and polarized exocytosis of cytotoxic granules leading to target cell destruction. significantly associated with prolonged disease-free survival and overall survival in patients with pulmonary squamous cell carcinoma, but not in those with pulmonary adenocarcinoma (84). The epithelial localization of CD103 + TIL has an even more significant predictive value compared to the stromal location, suggesting that intraepithelial CD8 + CD103 + cells encompass a higher proportion of tumor-specific TRM cells (15,85). This intratumoral positioning of CD103 + TIL was correlated with expression of E-cadherin on tumor cells in bladder cancer (83), but not in ovarian or breast cancer (20,85). Moreover, this predominant location in intratumoral regions, rather than in the stroma, was associated with the capacity of CD103 to promote recruitment of TIL in epithelial tumor islets (30). Thus, TRM cells appear to be key components in antitumor immunity, and their presence at the tumor site predicts a favorable prognosis in many cancers of different histological types. Paradoxically, their dominant expression of checkpoint receptors suggests that may be functionally exhausted. However, their location in close contact with tumor cells, their ability to proliferate in situ, to produce granzyme B and other cytotoxic molecules and pro-inflammatory cytokines, support the hypothesis that TRM cells are enriched in tumorspecific CD8 + T cells that could trigger specific cytotoxic activity toward target cells in physiological conditions and following neutralization of PD-1-PD-L1 interaction, as we demonstrated ex vivo (15) and possibly also during anti-PD-1/anti-PD-L1 cancer immunotherapy. Accordantly, recent studies revealed an expansion of CD8 + CD103 + TRM cells during anti-PD-1 treatment in melanoma (86).

CONCLUDiNG ReMARKS
Overall, CD8 + TRM cells that accumulate in human tumor lesions appear to be important effectors in antitumor CTL responses. Their retention within the tumor ecosystem may control tumor growth and explain more favorable prognoses in certain cancer patients. Moreover, CD103 emerges as a key molecule in CD8 + TRM activation, the expression of which is probably adjusted in the tumor microenvironment by TGF-β. This integrin not only promotes T-cell adhesion to target cells through interaction with its unique known ligand E-cadherin but also provides positive signals triggering diverse T-cell effector functions, such as spreading, migration, proliferation, and cytotoxicity (Figure 1). Nevertheless, additional studies and tools are required to further decipher CD103 structure and bidirectional signaling, and to determine whether this integrin also undergoes conformational changes within the tumor ecosystem in order to control the affinity to its ligand E-cadherin and to regulate its functional properties. In this regard, identification of new partners and associated molecules controlling integrin intracellular signals and regulating the dynamics of CD103 are essential in order to optimize the antitumor reactivity of CD8 + TRM cells. They would also help to determine the true contribution of CD8 + CD103 + TRM cells and the identified costimulatory molecules in the success of immune checkpoint blockade immunotherapies in a minor subpopulation of cancer patients, and to improve current T-cell-based cancer immunotherapeutic approaches such as adoptive T-cell therapies (Figure 2).