Homing of CD8+ T lymphocytes to the tumor microenvironment is an important step for mounting a robust antitumor immune response. TGFβ is responsible for CD103 (αEβ7) integrin induction in activated intraepithelial CD8+ T lymphocytes. However, the interplay between TGFβ and CD103 and their contribution to T-cell infiltration and antitumor activity remain unknown. Here, we used viable human lung tumor slices and autologous tumor antigen-specific T-lymphocyte clones to provide evidence that CD103 is directly involved in T-lymphocyte recruitment within epithelial tumor islets and intratumoral early T-cell signaling. Moreover, TGFβ enhanced CD103-dependent T-cell adhesion and signaling, whereas it inhibited leukocyte function-associated antigen (LFA)-1 (αLβ2) integrin expression and LFA-1-mediated T-lymphocyte functions. Mechanistic investigations revealed that TGFβ bound to its receptors (TGFBR), which promoted the recruitment and phosphorylation of integrin-linked kinase (ILK) by TGFBR1. We further show that ILK interacted with the CD103 intracellular domain, resulting in protein kinase B (PKB)/AKT activation, thereby initiating integrin inside-out signaling. Collectively, our findings suggest that the abundance of TGFβ in the tumor microenvironment may in fact engage with integrin signaling pathways to promote T-lymphocyte antitumor functions, with potential implications for T-cell-based immunotherapies for cancer. Cancer Res; 76(7); 1757–69. ©2016 AACR.

Adequate positioning of tumor antigen-specific T cells into tumor tissues plays a central role in an effective antitumor immune response. Hence, the number of CD8+ T cells found in contact with tumor cells is usually associated with good outcome in cancer patients (1). Recruitment of T lymphocytes within the tumor involves multiple sequential steps regulated by molecular interactions with several stromal cell types and coordinated by chemotactic agents, cell surface adhesion molecules, and extracellular matrix (ECM) proteins. Integrins are one of the major families of adhesion molecules that mediate cell–ECM and cell–cell interactions (2). Adhesion of T cells to ECM proteins or cellular integrin ligands is fundamental for lymphocyte development, migration, extravasation, and activation (3, 4). Among the integrin family members, LFA-1 (αL2, CD11a/CD18) is essential for lymphocyte homing in lymphoid and nonlymphoid tissues, transmigration, and antigen-specific T-cell responses (3). On naive T lymphocytes, LFA-1 has weak affinity for its ligands, the intercellular adhesion molecules (ICAM). However, T-cell stimulation through T-cell receptor (TCR) or chemokine receptors triggers an “inside-out” signal that results in LFA-1 activation by inducing integrin-extended conformation and clustering, thereby increasing its affinity for its ligands (5). Firm adhesion of LFA-1 to ICAM-1 (CD54) initiates an “outside-in” signal that has costimulatory functions in TCR signaling, thus contributing to T-cell stimulation, spreading, and cytotoxicity (6, 7).

The αE(CD103)β7 integrin, hereafter referred to as CD103, plays a crucial role in TCR-mediated cytokine secretion and cytotoxic activity toward epithelial tumors by interacting with its ligand, epithelial cell marker E-cadherin, on target cells (8, 9). This integrin is essential in controlling CD8+ tumor-infiltrating lymphocyte (TIL) activities, not only by promoting effector T-cell adhesion to tumor cells, but also by triggering intracellular signaling events that costimulate TCR signals (10). A role for CD103 in T-cell homing into epithelia has been suggested (11, 12); however, accumulating evidence indicates a role for CD103 in retention of T-lymphocyte subpopulations in epithelial tissues (13). Consistently, CD103 mediates arrest of T lymphocytes by interacting with E-cadherin on epithelial tumors (9) and recruiting CCR5 at the immune synapse (IS) with cancer cells (14). Moreover, an enhanced CD103+ TIL subset correlates with improved patient survival in ovarian (15) and lung (16) carcinomas and increased intraepithelial lymphocyte infiltration, suggesting that CD103 promotes recruitment of TIL within epithelial tumor islets. However, the true contribution of CD103 to promoting T-cell infiltration into tumor regions, and the influence of TGFβ, abundant within the tumor microenvironment and responsible for ITGAE gene expression in TCR-engaged CD8+ T lymphocytes (8, 17, 18), on integrin functions remain poorly understood. Using viable human tumor slices (19) and autologous tumor antigen-specific CTL clones, we show here that CD103 promotes T-cell recruitment within epithelial tumor regions and enhances intratumoral T-cell early signaling. We also show that TGFβ upregulates CD103-dependent T-cell effector functions by triggering TGFBR1 (activin receptor-like kinase-5, ALK5)-mediated phosphorylation of ILK and its subsequent binding to CD103, thus initiating inside-out signaling leading to activation of the integrin and strengthening of CD103-E-cadherin adhesion.

Tumor cell lines, T-cell clones, and freshly isolated TIL

IGR-Heu and IGR-Heu-ICAM-1 tumor cell lines were derived in one of our laboratories in 1996 as previously described (8). Heu171 (CD103+/LFA-1+) and H32-22 (CD103/LFA-1+) T-cell clones were generated from autologous TIL and peripheral blood lymphocytes (PBL), respectively. Cells were regularly tested and authenticated by immunofluorescence analysis and cytotoxic assay including in this work. Fresh CD8+ TIL were isolated using a FACS ARIA cell sorter.

Recombinant molecules, antibodies, and chemical inhibitors

Recombinant (r) E-cadherin-Fc (rE-cadh-Fc), rICAM-1-Fc and rFibronectin-Fc molecules were provided by R&D Systems. rCXCL12 and rTGFβ was purchased from PeproTech and Abcys, respectively. Anti-CD103 and anti-β2 mAb were purchased from Beckman Coulter, and PE-conjugated anti-CD103 from eBiosciences. Anti-CD3 (UCHT1) was provided by Ozyme and anti-CD8 by BioLegend. Anti-ILK and anti-phosphorylated (phospho)-ILK (Ser-246) mAb were purchased from Millipore. Anti-TGFBRI and secondary mAb were provided by Santa Cruz Biotechnology. Anti-AKT/PKB and anti-phospho-AKT were purchased from Cell-Signaling. Anti-KLRG1 was a gift from H Pircher. TGRBR1 kinase inhibitor SB-431542 (SB) and PI3K inhibitor Wortmannin were supplied by Sigma-Aldrich. Phospho-AKT inhibitor MK-2206 was purchased from Millipore and ILK inhibitor QLT-0267 (QLT) from Quadra Logic Technology.

Confocal microscopy analyses

For T-cell adhesion, poly-l-lysine slices were coated with rE-cadh-Fc, rICAM-1-Fc, or rFibronectin-Fc (5 μg/mL) overnight at 4°C. T cells, nonstimulated or stimulated with rCXCL12 (100 nmol/L) or rTGFβ (2 ng/mL), were incubated on precoated slices as described (8, 9). Cells shape index was calculated as the ratio of the longer to the shorter axis measured via Image J software. For protein polarization, lymphocytes, untreated or pretreated with SB (1 μmol/L), QLT (20 μmol/L), or with rTGFβ were preincubated with IGR-Heu cells, or rE-cadh-Fc-coupled protein G-Dynabeads, at a 2:1 effector to target cell (E:T) ratio and then plated on poly-(l-lysine)-coated coverslips. Cells were stained with mouse anti-CD103, rabbit anti-phospho-ILK, or goat anti-TGFBRI mAb, followed by anti-mouse-Alexa Fluor-546, anti-rabbit-Alexa Fluor-647 or anti-goat-Alexa Fluor-488 Ab, respectively. Coverslips were analyzed using a fluorescence microscope with ×20 (adhesion) or ×63 (spreading and protein relocalization) lenses as described (9).

Tumor slices and T-cell recruitment experiments

Xenograft tumor samples were embedded in low-gelling-temperature agarose as reported (19). Lymphocytes were stained with SNARF and added onto the cut surface of each slice. In some experiments, T cells were preincubated with neutralizing anti-CD103 (anti-αE, 10 μg/mL), anti-β2 (10 μg/mL), or anti-CD3 (1 μg/mL), or treated with rTGFβ. Slices were then stained with anti-E-cadherin mAb followed by a secondary Ab (Alexa Fluor-488). Images were acquired with a ×4 or ×10 (S fluor; Nikon) objective and MetaVue imaging software (Universal Imaging).

Single-cell calcium measurement

The calcium response was measured in the same tumor slice with T cells loaded with 1 μmol/L Fura-2AM (Molecular Probes). One of the two clones was also labeled with 5-chloromethylfluorescein diacetate (CMFDA; 5 nmol/L). Fura-2AM-loaded lymphocytes and infiltrated into tumor slices were alternatively excited every 10 s at 350 and 380 nm on an inverted Eclipse TE300 microscope equipped with a ×20 objective and a Metafluor imaging system. Emissions at 510 nm were used for the analysis of Ca2+ responses. Values were represented as a ratio: fluorescence intensity at 350 nm/fluorescence intensity at 380 nm.

Western blot and immunoprecipitation experiments

Lymphocytes, untreated or pretreated with rTGFβ, were unstimulated or stimulated with plastic-coated rE-cadh-Fc. Equivalent amounts of protein extracts were separated by SDS-PAGE and transferred to a nitrocellulose membrane as described (10). Blots were then probed with rabbit anti-phospho(Ser-246)-ILK, anti-phospho(Ser-473)-AKT/PKB, anti-AKT/PKB or anti-β-actin-peroxidase followed by secondary HRP-conjugated Ab.

For immunoprecipitation, T-cell extracts were incubated for 2 h at 4°C with cross-linked anti-TGFBRI or anti-CD103 beads, or protein G-Sepharose beads prebound with control goat or mouse IgG. Beads were then washed and eluted with reducing agent at 95°C.

Flow cytometric analysis and cytotoxicity assay

For granule exocytosis, T cells, untreated or pretreated with inhibitors or cultured with rTGFβ, were stimulated with a combination of rE-cadh-Fc or rICAM-1-Fc (2.5 μg/mL) and UCHT1 mAb (0.4 μg/mL) in the presence of anti-CD107 mAb and monensin A (10). A high concentration of anti-CD3 (10 μg/mL) was used as a control. Cytotoxicity was evaluated with a conventional 51Cr-release assay (8).

Statistical analysis

Statistical analyses were performed using the two-tailed Student t-test. Two groups were considered as significantly different if P < 0.05.

Regulation of CD103-dependent T-cell adhesion and motility by TGFβ

Thus far, little is known about the involvement of CD103 in T-cell migration and regulation of integrin activities by TGFβ. Hence, experiments were performed to compare the effect of TGFβ on T-cell adhesion and the migratory behavior of the CD103+/LFA-1+ TIL clone Heu171 (8) on immobilized human rE-cadh-Fc or rICAM-1-Fc. The rFibronectin-Fc was included as a negative control. rCXCL12, rCCL5, and rCCL20 chemokines, the respective receptors (CXCR4, CCR5, and CCR6), of which are expressed on the T-cell clone surface, were used as positive controls. Indeed, chemokines can trigger integrin-dependent adhesion of lymphocytes by initiating an inside-out signaling leading to integrin activation (20, 21). Results indicated that T lymphocytes stimulated with rCXCL12 (Fig. 1A), rCCL5, or rCCL20 (Supplementary Fig. S1A), adhered more efficiently to both rE-cadh-Fc and rICAM-1-Fc than untreated cells. Of note, incubation of T cells for a short period of time (30 min) with the three chemokines had no effect on LFA-1 and CD103 integrin expression levels (data not shown). Remarkably, treatment of T lymphocytes with rTGFβ induced a sharp increase in T-cell adhesion to rE-cadh-Fc starting from 24 h of culture whereas it inhibited lymphocyte adhesion to rICAM-1-Fc (Fig. 1A). Treatment with rTGFβ of CD8+ TIL freshly isolated from three independent tumors also induced an increase in T-cell adhesion to E-cadherin-Fc whereas it inhibited adhesion to rICAM-1-Fc (Fig. 1B). Opposing effects of TGFβ on the adhesion strength of CD103+/LFA-1+ TIL clone and CD103/LFA-1+ PBL counterpart (clone H32-22) to autologous E-cadherin+/ICAM-1 (IGR-Heu) and E-cadherin+/ICAM-1+ (IGR-Heu-ICAM-1) tumor cells, respectively, were also observed under flow conditions (Supplementary Fig. S1B). These results correlated with upregulation of CD103 expression and, in contrast, downregulation of LFA-1 following T-cell incubation with the cytokine (Supplementary Fig. S2A). Notably, T-cell clones, untreated or pretreated with rTGFβ, failed to express detectable levels of the E-cadherin inhibitory receptor KLRG1 (data not shown).

Figure 1.

Adhesion and filopodia formation are increased following CD103+/LFA-1+ T-cell stimulation with rCXCL12 or rTGFβ. A, adhesion to rE-cadh-Fc or rICAM-1-Fc of the TIL clone, untreated or pretreated with rCXCL12 for 30 min, or with rTGFβ for indicated time points, was analyzed by confocal microscopy. rFibronectin-Fc was used as a negative control. Bottom, mean numbers of adherent lymphocytes per field. Adhesion was evaluated by counting adherent lymphocytes stained with TO-PRO-3 iodide. Statistical analyses are from three independent experiments (see Supplementary Table S1). Bars, 100 μm; *, P < 0.01; and **, P < 0.001. B, T-cell adhesion was increased following treatment of freshly isolated TIL with rTGFβ. Adhesion of CD8+ TIL (including up to 60% of CD103+ T cells), untreated or pretreated with rTGFβ for 96 h, to rE-cadh-Fc or rICAM-1-Fc. Right, mean numbers of adherent cells per field. Results are from one experiment out of three performed with fresh TIL from three different patients. Bars, 500 μm. C, migratory behavior of CD103+/LFA-1+ lymphocytes, untreated or pretreated with rCXCL12 or rTGFβ, seeded on recombinant molecules. Polymerized F-actin was visualized with rhodamine phalloidin. Arrows, filopodia and/or cell protrusions. Bottom, T-cell shape index. Statistical analyses are from three independent experiments. Bars, 5 μm; *, P < 0.01; **, P < 0.001; and ***, P < 0.001.

Figure 1.

Adhesion and filopodia formation are increased following CD103+/LFA-1+ T-cell stimulation with rCXCL12 or rTGFβ. A, adhesion to rE-cadh-Fc or rICAM-1-Fc of the TIL clone, untreated or pretreated with rCXCL12 for 30 min, or with rTGFβ for indicated time points, was analyzed by confocal microscopy. rFibronectin-Fc was used as a negative control. Bottom, mean numbers of adherent lymphocytes per field. Adhesion was evaluated by counting adherent lymphocytes stained with TO-PRO-3 iodide. Statistical analyses are from three independent experiments (see Supplementary Table S1). Bars, 100 μm; *, P < 0.01; and **, P < 0.001. B, T-cell adhesion was increased following treatment of freshly isolated TIL with rTGFβ. Adhesion of CD8+ TIL (including up to 60% of CD103+ T cells), untreated or pretreated with rTGFβ for 96 h, to rE-cadh-Fc or rICAM-1-Fc. Right, mean numbers of adherent cells per field. Results are from one experiment out of three performed with fresh TIL from three different patients. Bars, 500 μm. C, migratory behavior of CD103+/LFA-1+ lymphocytes, untreated or pretreated with rCXCL12 or rTGFβ, seeded on recombinant molecules. Polymerized F-actin was visualized with rhodamine phalloidin. Arrows, filopodia and/or cell protrusions. Bottom, T-cell shape index. Statistical analyses are from three independent experiments. Bars, 5 μm; *, P < 0.01; **, P < 0.001; and ***, P < 0.001.

Close modal

Chemokines generate both pro- and anti-adhesive intracellular signaling events, whose equilibrium is relevant to cell movement (21). Therefore, we analyzed morphological changes in CD103+/LFA-1+ lymphocytes cultured on recombinant molecule-coated surfaces in the absence or presence of rCXCL12, rCCL5, or rCCL20. Compared to untreated T cells, rCXCL12-treated cells overlaid on rE-cadh-Fc or rICAM-1-Fc displayed more active motility behavior characterized by greater polarized cell morphology (Fig. 1C). Indeed, an increase in the percent of cells with larger protrusion sizes was obtained when T lymphocytes were stimulated with rCXCL12 and plated on either of the molecules. Similar results were observed with rCCL5 and rCCL20 (Supplementary Fig. S2B). In contrast, starting from 24 h of treatment, TGFβ induced a strong increase in lymphocyte locomotion behavior when T cells were exposed to rE-cadh-Fc, but not to rICAM-1-Fc (Fig. 1C). In addition, live-microscopy imaging revealed that motility was enhanced when T cells were pretreated with rTGFβ before culturing them on rE-cadh-Fc, whereas it was compromised when the same pretreated cells were added onto rICAM-1-Fc (Supplementary Fig. S2C and Movies). These results suggest that TGFβ promotes T-cell adhesion and cytoskeleton rearrangement by strengthening the interaction of CD103 with E-cadherin.

TGFβ enhances CD103-mediated T-cell recruitment in epithelial tumor islets

Next, experiments were undertaken to assess the influence of CD103 on T-cell recruitment in tumor regions via our previously described approach using tumor slices (19) obtained from E-cadherin+ lung tumor cell line, untransfected or transfected with ICAM-1, engrafted into immunodeficient NOD-SCID mice. Fluorescent-labeled autologous CD103+/LFA-1+ and CD103/LFA-1+ CTL were overlaid onto fresh tumor slices generated from either E-cadherin+/ICAM-1 or E-cadherin+/ICAM-1+ tumors. Slices were stained with anti-E-cadherin mAb to distinguish epithelial from stromal regions (Supplementary Fig. S3A), and then lymphocytes that infiltrated epithelial tumor regions and the stroma were counted. Results indicate that the CD103+/LFA-1+ TIL clone was much more efficiently recruited within E-cadherin+/ICAM-1 tumor islets than the CD103/LFA-1+ PBL clone, which was concentrated within stromal regions (Fig. 2A). Moreover, treatment of the CD103+/LFA-1+ clone with TGFβ enhanced T-cell recruitment in tumor regions starting from 24 h of preincubation. In contrast, treatment of the CD103/LFA-1+ PBL clone with TGFβ alone was unable to induce CD103 expression (8) and thus it had no effect on T-cell recruitment within tumor islets (data not shown). Recruitment of the TIL clone in tumor regions was dependent on CD103 and TCR engagement, because neutralizing anti-CD103 and anti-CD3 mAb dramatically blocked lymphocyte migration by inhibiting T-cell adhesion to E-cadherin+ target cells and peptide–MHC–I complex recognition by TCR, respectively. In contrast, neutralizing anti-β2 mAb, used as a negative control, had no effect on T-cell distribution (Fig. 2A).

Figure 2.

Recruitment of CD103+/LFA-1+ and CD103/LFA-1+ lymphocytes within epithelial tumor regions of human lung tumor slices. A, fluorescent-labeled CD103+/LFA-1+ T cells, untreated or pretreated with neutralizing anti-CD103, anti-β2, or anti-CD3 mAb, or with rTGFβ, were plated on E-cadherin+/ICAM-1 tumor (IGR-Heu) slices. Stromal (S) and tumor (T) areas, identified by brightness contrast and confirmed by E-cadherin labeling (see Supplementary Fig. S3A). Lymphocytes were stained with SNARF. Right, percentages of cells inside tumor areas are from three independent experiments. B, CD103+/LFA-1+ or CD103/LFA-1+ lymphocytes, untreated or pretreated with neutralizing anti-CD103, anti-CD3, or anti-β2 mAb, or with rTGFβ, were overlaid onto E-cadherin+/ICAM-1+ (IGR-Heu-ICAM-1) tumor slices. Right, percentages of T cells inside tumor areas are from three independent experiments out of 6. Bars, 100 μm; *, P < 0.01; **, P < 0.001; and ***, P < 0.0001.

Figure 2.

Recruitment of CD103+/LFA-1+ and CD103/LFA-1+ lymphocytes within epithelial tumor regions of human lung tumor slices. A, fluorescent-labeled CD103+/LFA-1+ T cells, untreated or pretreated with neutralizing anti-CD103, anti-β2, or anti-CD3 mAb, or with rTGFβ, were plated on E-cadherin+/ICAM-1 tumor (IGR-Heu) slices. Stromal (S) and tumor (T) areas, identified by brightness contrast and confirmed by E-cadherin labeling (see Supplementary Fig. S3A). Lymphocytes were stained with SNARF. Right, percentages of cells inside tumor areas are from three independent experiments. B, CD103+/LFA-1+ or CD103/LFA-1+ lymphocytes, untreated or pretreated with neutralizing anti-CD103, anti-CD3, or anti-β2 mAb, or with rTGFβ, were overlaid onto E-cadherin+/ICAM-1+ (IGR-Heu-ICAM-1) tumor slices. Right, percentages of T cells inside tumor areas are from three independent experiments out of 6. Bars, 100 μm; *, P < 0.01; **, P < 0.001; and ***, P < 0.0001.

Close modal

We then analyzed the location of both T-cell clones plated on E-cadherin+/ICAM-1+ tumor slices and the effect of TGFβ on this process. Transfection of tumor cells with ICAM-1 only slightly increased recruitment of the CD103+/LFA-1+ clone in tumor islets, whereas it strongly enhanced CD103/LFA-1+ clone migration (Fig. 2B). Moreover, anti-β2 and anti-CD3 mAb strongly inhibited PBL clone recruitment, whereas anti-CD103, anti-β2, or a combination of both mAb blocked TIL clone migration toward tumor regions. Notably, pertussis toxin, a bacterial toxin that blocks signaling from G protein–coupled receptors including chemokine receptors had no effect on T-cell distribution (data not shown). In contrast, treatment of the CD103/LFA-1+ clone with TGFβ resulted in profound inhibition of T-cell recruitment within E-cadherin+/ICAM-1+ tumor islets, in particular after 96 h of preincubation (Fig. 2B). These results strongly suggest that CD103 is directly involved in T-lymphocyte recruitment in tumor islets and that TGFβ upregulates its functions, whereas it has opposing effects on LFA-1.

TGFβ potentiates intratumoral early signaling of CD103+ lymphocytes

Imaging of the intracellular calcium increase is a powerful means of monitoring the dynamics of T-lymphocyte activation. We therefore evaluated the role of CD103 and the influence of TGFβ on the Ca2+ response triggered in CTL CD103+/LFA-1+ and CD103/LFA-1+. T-cell clones, untreated or pretreated with rTGFβ, were labeled with Fura-2 (red) to monitor calcium response. The CD103/LFA-1+ clone was also labeled with CMFDA fluorescent dye (green; thus appearing in yellow) in order to identify each T-cell population on the same slice. The two clones were then added simultaneously to tumor slices, untransfected or transfected with ICAM-1. Results indicated that CTL CD103+/LFA-1+, added to the E-cadherin+/ICAM-1 slice and recruited within tumor islets, displayed a much stronger calcium response than CTL CD103/LFA-1+ (Fig. 3A). Moreover, neutralizing anti-CD103 induced a decrease in the calcium level of the TIL clone, whereas anti-β2 mAb, used as a negative control, had no effect. Remarkably, treatment of the TIL clone with rTGFβ induced a sharp increase in the T-cell calcium response. An increase in CD103+/LFA-1+ Ca2+ response was also observed when the T-cell clone was pretreated with TGFβ and plated on a monolayer of E-cadherin+/ICAM-1 IGR-Heu tumor cells, which was inhibited when T lymphocytes were preincubated with neutralizing anti-CD103 mAb (Supplementary Fig. S3B). In contrast, treatment of the CD103/LFA-1+ PBL clone with TGFβ alone failed to induce CD103 expression and thus a marginal calcium response was observed when T cells were added to E-cadherin+/ICAM-1 tumor cells (data not shown).

Figure 3.

Calcium response of T cells within epithelial islets of lung tumor slices. A, CD103+/LFA-1+ and CD103/LFA-1+ CTL were loaded with Fura-2 probe (top, red). CD103/LFA-1+ T cells were also labeled with CMFDA (green) to distinguish them from CD103+/LFA-1+ cells. Thus, CD103+/LFA-1+ lymphocytes appeared in red, whereas CD103/LFA-1+ lymphocytes appeared in yellow (a merge of red and green dyes) in color merge panels. Loaded T cells were added to a tumor slice 1 h before the recording. Calcium response, represented in pseudo-colors (from green, average calcium level to red, strong calcium level), of CD103+/LFA-1+, untreated or pretreated with neutralizing anti-CD103 or anti-β2, or with rTGFβ for 96 h, and CD103/LFA-1+ lymphocytes within E-cadherin+/ICAM-1 (IGR-Heu) tumor areas. White dotted circles correspond to CD103+/LFA-1+ responding cells. Right, quantification of intracellular Ca2+. B, calcium response of CD103+/LFA-1+ and CD103/LFA-1+ lymphocytes, untreated or pretreated with neutralizing anti-CD103 or anti-β2 mAb, or with rTGFβ, in tumor islets of E-cadherin+/ICAM-1+ (IGR-Heu-ICAM-1) slices. White dotted and full circles correspond to CD103+/LFA-1+ and CD103/LFA-1+ responding lymphocytes, respectively. Right, quantification of intracellular calcium response. Statistical analyses are from three independent experiments. Bars, 100 μm; **, P < 0.001; and ***, P < 0.0001.

Figure 3.

Calcium response of T cells within epithelial islets of lung tumor slices. A, CD103+/LFA-1+ and CD103/LFA-1+ CTL were loaded with Fura-2 probe (top, red). CD103/LFA-1+ T cells were also labeled with CMFDA (green) to distinguish them from CD103+/LFA-1+ cells. Thus, CD103+/LFA-1+ lymphocytes appeared in red, whereas CD103/LFA-1+ lymphocytes appeared in yellow (a merge of red and green dyes) in color merge panels. Loaded T cells were added to a tumor slice 1 h before the recording. Calcium response, represented in pseudo-colors (from green, average calcium level to red, strong calcium level), of CD103+/LFA-1+, untreated or pretreated with neutralizing anti-CD103 or anti-β2, or with rTGFβ for 96 h, and CD103/LFA-1+ lymphocytes within E-cadherin+/ICAM-1 (IGR-Heu) tumor areas. White dotted circles correspond to CD103+/LFA-1+ responding cells. Right, quantification of intracellular Ca2+. B, calcium response of CD103+/LFA-1+ and CD103/LFA-1+ lymphocytes, untreated or pretreated with neutralizing anti-CD103 or anti-β2 mAb, or with rTGFβ, in tumor islets of E-cadherin+/ICAM-1+ (IGR-Heu-ICAM-1) slices. White dotted and full circles correspond to CD103+/LFA-1+ and CD103/LFA-1+ responding lymphocytes, respectively. Right, quantification of intracellular calcium response. Statistical analyses are from three independent experiments. Bars, 100 μm; **, P < 0.001; and ***, P < 0.0001.

Close modal

We then monitored Ca2+ signaling of both clones plated on E-cadherin+/ICAM-1+ tumor slices. As expected, a strong calcium response was obtained with the CD103+/LFA-1+ clone, which was inhibited by both anti-CD103 and anti-β2 blocking mAb. Transfection of tumor cells with ICAM-1 induced a strong Ca2+ increase in the CD103/LFA-1+ clone, which was blocked by neutralizing anti-β2. Importantly, treatment of the PBL clone with TGFβ strongly inhibited early T-cell signaling (Fig. 3B). These results suggest a crucial role of CD103 in regulating TCR-mediated activities within a TGFβ-rich tumor ecosystem and that TGFβ is involved in the CD103 signaling pathway.

TGFβ regulates CD103 activation via ILK phosphorylation

The signaling pathway of CD103 and its contribution to T-cell migration and functions within epithelial tissues are unknown. ILK, a serine/threonine kinase, has been reported to mediate integrin signaling and to play a role in leukocyte recruitment (22) and T-cell chemotaxis (23). Moreover, a role for TGFβ in regulating ILK expression and activation has been reported (24). We therefore analyzed the involvement of ILK in CD103 signaling and the effect of TGFβ on integrin activation in CD103+/LFA-1+ CTL. Initial experiments indicated that TGFβ upregulated ILK gene transcription starting from 6 h of T-cell clone treatment (Supplementary Fig. S3C). Moreover, stimulation of the clone with immobilized rE-cadh-Fc and/or rTGFβ triggered phosphorylation of ILK at Ser 246 (Fig. 4A), which was strongly inhibited by the ILK inhibitor QLT, the TGFBR1 inhibitor SB, the PI3K inhibitor Wortmannin (Fig. 4B), and anti-CD103 mAb (Supplementary Fig. S3D). Notably, inhibition of ILK phosphorylation resulted in a marked decrease in CTL adhesion on immobilized rE-cadh-Fc (Supplementary Fig. S3E). Stimulation of the TIL clone with rE-cadh-Fc also induced phosphorylation of the PKB/AKT at Ser-473, which was strongly inhibited by the AKT inhibitor MK-2206 and QLT, SB, and Wortmannin (Fig. 4C). Of note, incubation of lymphocytes for a short period of time (1 h) with all inhibitors had no effect on integrin expression level and T-cell viability, as checked by CD103 and annexin V labeling, respectively (data not shown).

Figure 4.

Engagement of CD103 on CD103+/LFA-1+ lymphocytes with rE-cadh-Fc–induced phosphorylation of ILK and AKT. A, engagement of CD103 with rE-cadh-Fc or treatment of the CD103+/LFA-1+ clone with TGFβ-induced phosphorylation of ILK. Lymphocytes, untreated or pretreated with rTGFβ for indicated time points, were stimulated with immobilized rE-cadh-Fc and then protein extracts were analyzed by Western blot using anti-phospho-ILK or anti-ILK mAb. F-actin was used as a loading control. Right, normalization of phospho-ILK relative to total ILK. B, Wortmannin (Wortm), SB, and QLT inhibit ILK phosphorylation induced by CD103 engagement with rE-cadh-Fc or rTGFβ stimulation. CD103+/LFA-1+ lymphocytes were pretreated with PI3K, TGFBR1, or ILK inhibitors and then stimulated with immobilized rE-cadh-Fc or with rTGFβ. T-cell protein extracts were analyzed by Western blot. Bottom, normalization of phospho-ILK relative to ILK. C, Wortmannin, SB, QLT, and MK-2206 inhibit AKT phosphorylation induced by CD103 engagement with rE-cadh-Fc. CD103+/LFA-1+ lymphocytes were pretreated for 1 h with either of the inhibitors and then stimulated with immobilized rE-cadh-Fc. Protein extracts were analyzed by Western blot using indicated mAb. Right, normalization of phospho-AKT relative to AKT. Statistical analyses are from three independent experiments.

Figure 4.

Engagement of CD103 on CD103+/LFA-1+ lymphocytes with rE-cadh-Fc–induced phosphorylation of ILK and AKT. A, engagement of CD103 with rE-cadh-Fc or treatment of the CD103+/LFA-1+ clone with TGFβ-induced phosphorylation of ILK. Lymphocytes, untreated or pretreated with rTGFβ for indicated time points, were stimulated with immobilized rE-cadh-Fc and then protein extracts were analyzed by Western blot using anti-phospho-ILK or anti-ILK mAb. F-actin was used as a loading control. Right, normalization of phospho-ILK relative to total ILK. B, Wortmannin (Wortm), SB, and QLT inhibit ILK phosphorylation induced by CD103 engagement with rE-cadh-Fc or rTGFβ stimulation. CD103+/LFA-1+ lymphocytes were pretreated with PI3K, TGFBR1, or ILK inhibitors and then stimulated with immobilized rE-cadh-Fc or with rTGFβ. T-cell protein extracts were analyzed by Western blot. Bottom, normalization of phospho-ILK relative to ILK. C, Wortmannin, SB, QLT, and MK-2206 inhibit AKT phosphorylation induced by CD103 engagement with rE-cadh-Fc. CD103+/LFA-1+ lymphocytes were pretreated for 1 h with either of the inhibitors and then stimulated with immobilized rE-cadh-Fc. Protein extracts were analyzed by Western blot using indicated mAb. Right, normalization of phospho-AKT relative to AKT. Statistical analyses are from three independent experiments.

Close modal

Next, we evaluated the consequences of CD103 engagement with its ligand on ILK localization in CD103+/LFA-1+ TIL plated on a rE-cadh-Fc-coated slide. Results indicate that the CD103–E-cadherin interaction resulted not only in T-cell spreading, but also in recruitment of phospho-ILK, together with CD103, at the leading edge of migrating CTL presenting a high actin cytoskeleton rearrangement (Fig. 5A). Moreover, ILK and TGFBR1 inhibitors induced a marked decrease in phospho-ILK recruitment at the leading edge. Together, these results suggested that TGFBR1 serine kinase catalyzed, even in the absence of its ligand, phosphorylation of ILK through a PI3K-dependent pathway and that phospho-ILK and phospho-AKT were involved in CD103 signaling.

Figure 5.

Engagement of CD103 on the T-cell surface with E-cadherin resulted in polarization of phospho-ILK and its subsequent binding to CD103. A, engagement of CD103 with immobilized rE-cadh-Fc resulted in polarization of phospho-ILK at the leading edge. CD103+/LFA-1+ T cells, untreated or pretreated with QLT or SB, were plated on plastic-coated rE-cadh-Fc, and then lymphocytes were analyzed for intracellular protein distribution by confocal microscopy using anti-phospho-ILK, anti-CD103, or anti-F-actin. Right, percentages of T cells displaying polarized phospho-ILK at the leading edge (n = 40). B, the CD103-E-cadherin interaction induced recruitment of phospho-ILK at the IS. Conjugates between CD103+/LFA-1+ CTL, untreated or treated with QLT or SB, and IGR-Heu tumor cells were analyzed for CD103, phospho-ILK, and TGFBRI polarization. Right, percentages of cells displaying polarized proteins (n = 35). Statistical analyses are from one experiment out of three. Bars, 5 μm. **, P < 0.001. C, ILK binds to CD103. Protein extracts from CD103+/LFA-1+ lymphocytes, stimulated with immobilized r-E-cadh-Fc for 30 min or rTGFβ for 96 h, were immunoprecipitated (IP) using anti-CD103 mAb and then immunoblotted (WB) with anti-ILK. An IgG negative control was included. D, TGFBR1 binds to ILK. CD103+/LFA-1+ cells were treated with rTGFβ and then protein extracts were immunoprecipitated with anti-TGFBR1, followed by immunoblotting with anti-ILK. Results are from one experiment out of three. E, TGFBR1 does not bind to CD103. Protein extracts from CD103+/LFA-1+ lymphocytes were immunoprecipitated with anti-TGFBR1 mAb and then immunoblotted with anti-CD103. The CD103/LFA-1+ clone and IgG were used as negative controls. Right, CD103 detected in the supernatant of CD103+/LFA-1+ CTL.

Figure 5.

Engagement of CD103 on the T-cell surface with E-cadherin resulted in polarization of phospho-ILK and its subsequent binding to CD103. A, engagement of CD103 with immobilized rE-cadh-Fc resulted in polarization of phospho-ILK at the leading edge. CD103+/LFA-1+ T cells, untreated or pretreated with QLT or SB, were plated on plastic-coated rE-cadh-Fc, and then lymphocytes were analyzed for intracellular protein distribution by confocal microscopy using anti-phospho-ILK, anti-CD103, or anti-F-actin. Right, percentages of T cells displaying polarized phospho-ILK at the leading edge (n = 40). B, the CD103-E-cadherin interaction induced recruitment of phospho-ILK at the IS. Conjugates between CD103+/LFA-1+ CTL, untreated or treated with QLT or SB, and IGR-Heu tumor cells were analyzed for CD103, phospho-ILK, and TGFBRI polarization. Right, percentages of cells displaying polarized proteins (n = 35). Statistical analyses are from one experiment out of three. Bars, 5 μm. **, P < 0.001. C, ILK binds to CD103. Protein extracts from CD103+/LFA-1+ lymphocytes, stimulated with immobilized r-E-cadh-Fc for 30 min or rTGFβ for 96 h, were immunoprecipitated (IP) using anti-CD103 mAb and then immunoblotted (WB) with anti-ILK. An IgG negative control was included. D, TGFBR1 binds to ILK. CD103+/LFA-1+ cells were treated with rTGFβ and then protein extracts were immunoprecipitated with anti-TGFBR1, followed by immunoblotting with anti-ILK. Results are from one experiment out of three. E, TGFBR1 does not bind to CD103. Protein extracts from CD103+/LFA-1+ lymphocytes were immunoprecipitated with anti-TGFBR1 mAb and then immunoblotted with anti-CD103. The CD103/LFA-1+ clone and IgG were used as negative controls. Right, CD103 detected in the supernatant of CD103+/LFA-1+ CTL.

Close modal

ILK recruitment and phosphorylation by TGFBR1 triggers binding to CD103

Because the interaction of ILK with β-integrins is important for intracellular signaling, we first monitored the localization of phospho-ILK and TGFBR1 in conjugates formed between CD103+/LFA-1+ T cells and E-cadherin+/ICAM-1 tumor cells. Results revealed polarization of phospho-ILK, together with CD103 and TGFBR1, at the IS, which was inhibited by QLT and SB (Fig. 5B), as well as siRNA targeting E-cadherin (Supplementary Fig. S4A). Consistently, shRNA targeting ILK also inhibited CD103 polarization (Supplementary Figs. S4B and S4C) and AKT phosphorylation (data not shown). In contrast, treatment of the TIL clone with rTGFβ induced an increase in synaptic relocalization of CD103, phospho-ILK, and TGFBR1 (Supplementary Fig. S4D).

To explore a potential link between TGFBR1, phospho-ILK, and CD103, we searched for physical interactions between these proteins. Therefore, we performed immunoprecipitation experiments using anti-CD103 or anti-TGFBR1 mAb and immunobloting with anti-ILK. Results revealed that ILK bound to CD103 (Fig. 5C) and TGFBR1 (Fig. 5D), and that TGFβ, and to a lesser extent rE-cadh-Fc, enhanced ILK linking to CD103. Notably, no direct interaction between CD103 and TGFBR1 was observed, because CD103 was detected only in the supernatant of immunoprecipitation with anti-TGFBR1 (Fig. 5E). These results further support the hypothesis that the interaction of TGFβ with its receptors induces recruitment and phosphorylation of ILK by TGFBR1 and its subsequent binding to the CD103 intracellular domain, resulting in AKT phosphorylation and thereby initiating integrin inside-out signaling.

TGFβ regulates CD103-dependent CTL activities via PI3K–ILK–AKT pathway

Experiments were then conducted to evaluate the contribution of PI3K–ILK–AKT pathway in CD103-mediated T-cell functions. Using the tumor slice system, results indicated that Wortmannin, SB, QLT, and MK-2206 inhibited T-cell recruitment in epithelial tumor islets (Fig. 6A). We then analyzed the effect of all the inhibitors and the influence of TGFβ on CD103-dependent T-cell clone degranulation. As shown in Fig. 6B, CD103+/LFA-1+ T cells cultured with rTGFβ and stimulated with a combination of anti-CD3 plus rE-cadh-Fc expressed higher surface levels of CD107a than T cells exposed to a combination of anti-CD3 and rICAM-1-Fc. Moreover, Wortmannin, SB, QLT, and MK-2206 induced a decrease in T-cell degranulation triggered by a combination of anti-CD3 plus rE-cadh-Fc, whereas they had no effect on CD107a expression induced by a high dose of anti-CD3. Consistently, SB and QLT strongly inhibited cytotoxic granule polarization at the contact area between CD103+/LFA-1+ CTL and rE-cadh-Fc-coupled beads (Supplementary Fig. S5A). In addition, although TGFβ inhibited granzyme B polarization at the contact area between the TIL clone and rICAM-Fc-coupled beads, it had no effect on serine protease relocalization at the contact zone with rE-cadh-Fc-coated beads.

Figure 6.

Phospho-ILK and phospho-AKT are involved in CTL activities. A, recruitment of fluorescent-labeled CD103+/LFA-1+ lymphocytes, untreated or pretreated with Wortmannin, SB, QLT, or MK-2206 inhibitors, within epithelial tumor regions of lung tumor (IGR-Heu) slices. Stromal (S) and tumor (T) areas were identified by brightness contrast. Right, percentages of T cells inside tumor areas were determined from three independent experiments. Bars, 100 μm. B, CD103+/LFA-1+ lymphocytes, untreated or pretreated with rTGFβ for 96 h or with indicated inhibitors for 1 h, were incubated with a combination of rE-cadh-Fc plus a suboptimal concentration of anti-CD3 mAb. A combination of a suboptimal concentration of anti-CD3 plus rICAM-1-Fc or a high concentration of anti-CD3 was used as controls. Data correspond to percentages of CD107a+ lymphocytes from two independent experiments out of three. C, cytotoxic activity of CD103+/LFA-1+ CTL, untreated or pretreated with rTGFβ and then with indicated inhibitors, toward autologous E-cadherin+/ICAM-1 (IGR-Heu) tumor cells. D, cytotoxicity of CD103+/LFA-1+ CTL, untreated or pretreated with rTGFβ and then either left in medium or preincubated with neutralizing anti-CD103 mAb, toward autologous E-cadherin+/ICAM-1 tumor cells. E, cytotoxic activity of CD103/LFA-1+ CTL, untreated or pretreated with rTGFβ, toward autologous E-cadherin+/ICAM-1+ (IGR-Heu-ICAM-1) tumor cells. *, P < 0.01 and **, P < 0.001.

Figure 6.

Phospho-ILK and phospho-AKT are involved in CTL activities. A, recruitment of fluorescent-labeled CD103+/LFA-1+ lymphocytes, untreated or pretreated with Wortmannin, SB, QLT, or MK-2206 inhibitors, within epithelial tumor regions of lung tumor (IGR-Heu) slices. Stromal (S) and tumor (T) areas were identified by brightness contrast. Right, percentages of T cells inside tumor areas were determined from three independent experiments. Bars, 100 μm. B, CD103+/LFA-1+ lymphocytes, untreated or pretreated with rTGFβ for 96 h or with indicated inhibitors for 1 h, were incubated with a combination of rE-cadh-Fc plus a suboptimal concentration of anti-CD3 mAb. A combination of a suboptimal concentration of anti-CD3 plus rICAM-1-Fc or a high concentration of anti-CD3 was used as controls. Data correspond to percentages of CD107a+ lymphocytes from two independent experiments out of three. C, cytotoxic activity of CD103+/LFA-1+ CTL, untreated or pretreated with rTGFβ and then with indicated inhibitors, toward autologous E-cadherin+/ICAM-1 (IGR-Heu) tumor cells. D, cytotoxicity of CD103+/LFA-1+ CTL, untreated or pretreated with rTGFβ and then either left in medium or preincubated with neutralizing anti-CD103 mAb, toward autologous E-cadherin+/ICAM-1 tumor cells. E, cytotoxic activity of CD103/LFA-1+ CTL, untreated or pretreated with rTGFβ, toward autologous E-cadherin+/ICAM-1+ (IGR-Heu-ICAM-1) tumor cells. *, P < 0.01 and **, P < 0.001.

Close modal

We then evaluated the effect of TGFβ, PI3K, TGFBR1, phospho-ILK, and phospho-AKT inhibitors on T-cell clone-mediated cytotoxicity toward target cells. Incubation of the CD103+/LFA-1+ clone with rTGFβ enhanced E-cadherin+/ICAM-1 IGR-Heu tumor cell killing (Fig. 6C), which was inhibited in the presence of neutralizing anti-CD103 (Fig. 6D), whereas Wortmannin, SB, QLT, and MK-2206 inhibited target cell lysis (Fig. 6C). Notably, all inhibitors had no effect on T-cell clone-mediated CD3-redirected cytotoxicity of the P815 target (Supplementary Fig. S5B). In contrast, culture of the CD103/LFA-1+ CTL clone in the presence of TGFβ dramatically inhibited E-cadherin+/ICAM-1+ IGR-Heu-ICAM-1 target cell lysis (Fig. 6E). Notably, TGFβ had no effect on granzyme B expression levels in both T-cell clones (data not shown). These results further emphasize the role of PI3K, TGFBR1, phospho-ILK, and phospho-AKT/PKB in CD103 signaling and the positive effect of TGFβ in antitumor CTL-mediated activities.

In this report, we identified CD103 and its ligand E-cadherin as important adhesion molecules stimulating recruitment and effector functions of activated CD8+/CD103+ TIL inside human lung tumors. In agreement with our previous studies, we also show that CD8+/CD103 T cells accumulate preferentially within the tumor stroma and display inefficient antitumor activities (19). Along the same line, it has been reported that mouse CD103−/− T cells failed to infiltrate allograft islets, suggesting that CD103 promotes intragraft migration of CD8+ effectors into epithelial islets (25). Our data are consistent with a key role for CD103 in promoting entry of tumor antigen-specific CD8+ T cells into epithelial tumor islets and in triggering antitumor T-cell functions. Recognition of tumor antigen by TCR also plays an important role, because anti-CD3 mAb inhibited T-cell recruitment into tumor regions. It is possible that CD103 contributes to intratumoral scanning of target cells for cognate antigen recognition.

CD103 and LFA-1 are the predominant integrins expressed by TIL and both are likely involved in T-cell adhesion and migration within and toward epithelial tumors, respectively. Indeed, we show here that LFA-1, through an interaction with ICAM-1, also induced T-cell infiltration within tumor nests. However, tumors often express low levels of ICAM (26) and produce high amounts of TGFβ (27), thus altering LFA-1 expression and functions (ref. 28 and this study). Indeed, this cytokine shifted the integrin expression profile of tumor-specific TIL from LFA-1high/CD103low to LFA-1low/CD103high. Thus, CD103 most likely corresponds to an important integrin for adjusting T-cell adhesion and migratory potential in the tumor ecosystem, where TGFβ is abundant. Along this line, it has been reported that murine CD103 determines cell shape and motility, and confers the ability to move in an actin-polymerization-dependent manner (29). These results suggest that CD103-mediated cell movement probably involves signaling interactions with components of the cytoskeleton. Likewise, we show here that human CD103 confers the capacity to CD8+ T cells to form protrusions/filopodia in an E-cadherin–dependent fashion. Moreover, TGFβ strongly optimizes T-cell adhesion and motility by enhancing CD103 expression levels and initiating intracellular signals, resulting in integrin activation.

TGFβ is known to inhibit cell growth in benign cells but promotes progression in cancer cells (30). Moreover, TGFβ signaling in peripheral T lymphocytes is essential for tolerance and homeostasis by restraining TCR activation-dependent Th1, cytotoxic, and NKT cell differentiation program (31, 32). This cytokine, produced by stromal and tumor cells, is also described as an immunosuppressive factor used by malignant cells to escape from the immune response and to compromise CTL activities (33). Paradoxically, TGFβ is involved in promoting antitumor CD8+ T-cell effector functions within epithelial tumors by inducing CD103 upon TCR engagement and by adjusting its expression levels. TGFβ also plays an essential role in the formation and long-term persistence of tissue-resident memory T cells (TRM), at least in part via induction of CD103 (34). Importantly, our data indicate that TGFβ also contributes to T-cell migration toward epithelial tumor islets and subsequent cancer cell destruction. This paradoxical effect of TGFβ on T-cell functions is most likely associated with the use in this study of CD103+/LFA-1+ lymphocytes whereas most of the previous studies were based on peripheral T cells, which generally express a CD103/LFA-1+ phenotype. Consistently, we show here that TGFβ is responsible for arrest of LFA-1-dependent T-cell migration within stromal regions by preventing integrin expression and functions. It has been reported that TGFβ inhibits phosphorylation of Erk and, thereby, LFA-1–mediated T-cell adhesion and chemotaxis (28, 35). In contrast, this cytokine contributes to CD103 signaling by upregulating ILK expression and inducing its phosphorylation. TGFβ initiates signaling by binding to TGFBR, leading to formation of a hetero-tetrameric complex composed of TGFBR1 and TGFBR2 dimers, in which TGFBR2 catalyzes phosphorylation of the cytoplasmic domain of TGFBR1 (36). In addition to the Smad pathway, TGFβ utilizes multiple non-Smad pathways, including PI3K/AKT pathway, to regulate a wide range of cell functions (37). We show here that TGFBR1 recruits and phosphorylates ILK, which then binds to the αE subunit of CD103, leading to AKT/PKB phosphorylation and thus initiating integrin inside-out signaling. It has been reported that ILK associates with TGFBR2 and that ILK gene inactivation results in a decrease in TGFBR2 signaling (38).

ILK is a serine–threonine kinase that interacts with intracytoplasmic domains of β1 and β3 integrins and cytoskeletal-associated proteins (39). This kinase is associated with integrin activation and intracellular signaling (40), and has been reported to mediate actin filament rearrangement and tumor cell migration and invasion through PI3K/AKT/Rac1 signaling (41). Indeed, ILK directly binds to AKT/PKB and contributes to its phosphorylation, thereby regulating several signaling pathways including spreading and migration (42). Consistently, our results indicated that ILK is directly involved in CD103 intracellular signaling by promoting phosphorylation of AKT/PKB and that the CD103–E-cadherin interaction induced phosphorylation of ILK and its subsequent polarization at the leading edge of migrating T cells. Implication of ILK in both inside-out and outside-in integrin signaling pathways (43, 44) and in β2 integrin-mediated polarization of lytic granules toward the target have also been reported (45). Our data suggest that ILK functions as a key component of a protein complex formed upon CD103 clustering that may connect extracellular ligands with the T-cell intracellular signaling complex. We also show here that TGFβ corresponds to an important factor that not only activates αE (CD103) subunit expression, but also locally regulates integrin activation and signaling via the ILK and AKT/PKB pathway. This leads to strengthening of CD103–E-cadherin interaction, thereby initiating outside-in signaling that promotes CD103-mediated T-cell functions such as T-cell spreading, migration, and cytotoxicity.

Overall, our data expand our knowledge of CD8+ T-cell locomotion within epithelial tissues and provide more insight into mechanisms that promote CD103 signaling within a TGFβ–rich tumor microenvironment. Thus, CD103–E-cadherin adhesive interactions regulate multiple local T-cell activities, including adhesion, Ca2+ response, and recruitment within epithelial tumor regions. This integrin is also involved in T-lymphocyte proliferation, cytokine secretion, cytotoxic IS maturation, and killing of epithelial target cells (8, 9, 14, 46). These findings are consistent with a general role of CD103 in promoting CD8 T-cell immune responses within epithelial tumors and may contribute to optimizing current T-cell–based cancer immunotherapy strategies.

No potential conflicts of interest were disclosed.

Conception and design: M. Boutet, F. Mami-Chouaib

Development of methodology: N. Théret, E. Donnadieu, F. Mami-Chouaib

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Boutet, L. Gauthier, M. Leclerc, G. Gros, V. de Montpreville, E. Donnadieu, F. Mami-Chouaib

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Boutet, L. Gauthier, E. Donnadieu, F. Mami-Chouaib

Writing, review, and/or revision of the manuscript: M. Boutet, L. Gauthier, N. Théret, F. Mami-Chouaib

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): F. Mami-Chouaib

Study supervision: F. Mami-Chouaib

The authors thank S. Salomé-Desmoulez for her help with confocal microscopy.

This work was supported by grants from the INSERM, Association pour la Recherche sur le Cancer (ARC), Institut National du Cancer (INCa), and Ligue contre le Cancer. M. Boutet was a recipient of a fellowship from Cancéropôle IDF and Gustave Roussy (SIRIC-SOCRATE).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Galon
J
,
Costes
A
,
Sanchez-Cabo
F
,
Kirilovsky
A
,
Mlecnik
B
,
Lagorce-Pages
C
, et al
Type, density, and location of immune cells within human colorectal tumors predict clinical outcome
.
Science
2006
;
313
:
1960
4
.
2.
Pribila
JT
,
Quale
AC
,
Mueller
KL
,
Shimizu
Y
. 
Integrins and T cell-mediated immunity
.
Annu Rev Immunol
2004
;
22
:
157
80
.
3.
Epler
JA
,
Liu
R
,
Shimizu
Y
. 
From the ECM to the cytoskeleton and back: how integrins orchestrate T cell action
.
Dev Immunol
2000
;
7
:
155
70
.
4.
van Kooyk
Y
,
Figdor
CG
. 
Avidity regulation of integrins: the driving force in leukocyte adhesion
.
Curr Opin Cell Biol
2000
;
12
:
542
7
.
5.
Constantin
G
,
Majeed
M
,
Giagulli
C
,
Piccio
L
,
Kim
JY
,
Butcher
EC
, et al
Chemokines trigger immediate beta2 integrin affinity and mobility changes: differential regulation and roles in lymphocyte arrest under flow
.
Immunity
2000
;
13
:
759
69
.
6.
Dustin
ML
,
Springer
TA
. 
T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1
.
Nature
1989
;
341
:
619
24
.
7.
Lek
HS
,
Morrison
VL
,
Conneely
M
,
Campbell
PA
,
McGloin
D
,
Kliche
S
, et al
The spontaneously adhesive leukocyte function-associated antigen-1 (LFA-1) integrin in effector T cells mediates rapid actin- and calmodulin-dependent adhesion strengthening to ligand under shear flow
.
J Biol Chem
2013
;
288
:
14698
708
.
8.
Le Floc'h
A
,
Jalil
A
,
Vergnon
I
,
Le Maux Chansac
B
,
Lazar
V
,
Bismuth
G
, et al
Alpha E beta 7 integrin interaction with E-cadherin promotes antitumor CTL activity by triggering lytic granule polarization and exocytosis
.
J Exp Med
2007
;
204
:
559
70
.
9.
Franciszkiewicz
K
,
Le Floc'h
A
,
Boutet
M
,
Vergnon
I
,
Schmitt
A
,
Mami-Chouaib
F
. 
CD103 or LFA-1 engagement at the immune synapse between cytotoxic T cells and tumor cells promotes maturation and regulates T-cell effector functions
.
Cancer Res
2013
;
73
:
617
28
.
10.
Le Floc'h
A
,
Jalil
A
,
Franciszkiewicz
K
,
Validire
P
,
Vergnon
I
,
Mami-Chouaib
F
. 
Minimal engagement of CD103 on cytotoxic T lymphocytes with an E-cadherin-Fc molecule triggers lytic granule polarization via a phospholipase Cgamma-dependent pathway
.
Cancer Res
2011
;
71
:
328
38
.
11.
Wagner
N
,
Lohler
J
,
Kunkel
EJ
,
Ley
K
,
Leung
E
,
Krissansen
G
, et al
Critical role for beta7 integrins in formation of the gut-associated lymphoid tissue
.
Nature
1996
;
382
:
366
70
.
12.
Lefrancois
L
,
Parker
CM
,
Olson
S
,
Muller
W
,
Wagner
N
,
Schon
MP
, et al
The role of beta7 integrins in CD8 T cell trafficking during an antiviral immune response
.
J Exp Med
1999
;
189
:
1631
8
.
13.
Schon
MP
,
Arya
A
,
Murphy
EA
,
Adams
CM
,
Strauch
UG
,
Agace
WW
, et al
Mucosal T lymphocyte numbers are selectively reduced in integrin alpha E (CD103)-deficient mice
.
J Immunol
1999
;
162
:
6641
9
.
14.
Franciszkiewicz
K
,
Le Floc'h
A
,
Jalil
A
,
Vigant
F
,
Robert
T
,
Vergnon
I
, et al
Intratumoral induction of CD103 triggers tumor-specific CTL function and CCR5-dependent T-cell retention
.
Cancer Res
2009
;
69
:
6249
55
.
15.
Webb
JR
,
Milne
K
,
Watson
P
,
Deleeuw
RJ
,
Nelson
BH
. 
Tumor-infiltrating lymphocytes expressing the tissue resident memory marker CD103 are associated with increased survival in high-grade serous ovarian cancer
.
Clin Cancer Res
2014
;
20
:
434
44
.
16.
Djenidi
F
,
Adam
J
,
Goubar
A
,
Durgeau
A
,
Meurice
G
,
de Montpreville
V
, et al
CD8+CD103+ tumor-infiltrating lymphocytes are tumor-specific tissue-resident memory T cells and a prognostic factor for survival in lung cancer patients
.
J Immunol
2015
;
194
:
3475
86
.
17.
Parker
CM
,
Cepek
KL
,
Russell
GJ
,
Shaw
SK
,
Posnett
DN
,
Schwarting
R
, et al
A family of beta 7 integrins on human mucosal lymphocytes
.
Proc Natl Acad Sci U S A
1992
;
89
:
1924
8
.
18.
Mokrani
M
,
Klibi
J
,
Bluteau
D
,
Bismuth
G
,
Mami-Chouaib
F
. 
Smad and NFAT pathways cooperate to induce CD103 expression in human CD8 T lymphocytes
.
J Immunol
2014
;
192
:
2471
9
.
19.
Salmon
H
,
Franciszkiewicz
K
,
Damotte
D
,
Dieu-Nosjean
MC
,
Validire
P
,
Trautmann
A
, et al
Matrix architecture defines the preferential localization and migration of T cells into the stroma of human lung tumors
.
J Clin Invest
2012
;
122
:
899
910
.
20.
Kinashi
T.
Intracellular signalling controlling integrin activation in lymphocytes
.
Nat Rev Immunol
2005
;
5
:
546
59
.
21.
Laudanna
C
,
Kim
JY
,
Constantin
G
,
Butcher
E
. 
Rapid leukocyte integrin activation by chemokines
.
Immunol Rev
2002
;
186
:
37
46
.
22.
Friedrich
EB
,
Sinha
S
,
Li
L
,
Dedhar
S
,
Force
T
,
Rosenzweig
A
, et al
Role of integrin-linked kinase in leukocyte recruitment
.
J Biol Chem
2002
;
277
:
16371
5
.
23.
Liu
E
,
Sinha
S
,
Williams
C
,
Cyrille
M
,
Heller
E
,
Snapper
SB
, et al
Targeted deletion of integrin-linked kinase reveals a role in T-cell chemotaxis and survival
.
Mol Cell Biol
2005
;
25
:
11145
55
.
24.
Janji
B
,
Melchior
C
,
Gouon
V
,
Vallar
L
,
Kieffer
N
. 
Autocrine TGF-beta-regulated expression of adhesion receptors and integrin-linked kinase in HT-144 melanoma cells correlates with their metastatic phenotype
.
Int J Cancer
1999
;
83
:
255
62
.
25.
Feng
Y
,
Wang
D
,
Yuan
R
,
Parker
CM
,
Farber
DL
,
Hadley
GA
. 
CD103 expression is required for destruction of pancreatic islet allografts by CD8(+) T cells
.
J Exp Med
2002
;
196
:
877
86
.
26.
Passlick
B
,
Pantel
K
,
Kubuschok
B
,
Angstwurm
M
,
Neher
A
,
Thetter
O
, et al
Expression of MHC molecules and ICAM-1 on non-small cell lung carcinomas: association with early lymphatic spread of tumour cells
.
Eur J Cancer
1996
;
32A
:
141
5
.
27.
Asselin-Paturel
C
,
Echchakir
H
,
Carayol
G
,
Gay
F
,
Opolon
P
,
Grunenwald
D
, et al
Quantitative analysis of Th1, Th2 and TGF-beta1 cytokine expression in tumor, TIL and PBL of non-small cell lung cancer patients.
Int J Cancer
1998
;
77
:
7
12
.
28.
Sela
U
,
Mauermann
N
,
Hershkoviz
R
,
Zinger
H
,
Dayan
M
,
Cahalon
L
, et al
The inhibition of autoreactive T cell functions by a peptide based on the CDR1 of an anti-DNA autoantibody is via TGF-beta-mediated suppression of LFA-1 and CD44 expression and function
.
J Immunol
2005
;
175
:
7255
63
.
29.
Schlickum
S
,
Sennefelder
H
,
Friedrich
M
,
Harms
G
,
Lohse
MJ
,
Kilshaw
P
, et al
Integrin alpha E(CD103)beta 7 influences cellular shape and motility in a ligand-dependent fashion
.
Blood
2008
;
112
:
619
25
.
30.
Zhang
Q
,
Yu
N
,
Lee
C
. 
Mysteries of TGF-beta Paradox in Benign and Malignant Cells
.
Front Oncol
2014
;
4
:
94
.
31.
Li
MO
,
Sanjabi
S
,
Flavell
RA
. 
Transforming growth factor-beta controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and -independent mechanisms
.
Immunity
2006
;
25
:
455
71
.
32.
Marie
JC
,
Liggitt
D
,
Rudensky
AY
. 
Cellular mechanisms of fatal early-onset autoimmunity in mice with the T cell-specific targeting of transforming growth factor-beta receptor
.
Immunity
2006
;
25
:
441
54
.
33.
Thomas
DA
,
Massague
J
. 
TGF-beta directly targets cytotoxic T cell functions during tumor evasion of immune surveillance
.
Cancer Cell
2005
;
8
:
369
80
.
34.
Gebhardt
T
,
Mackay
LK
. 
Local immunity by tissue-resident CD8(+) memory T cells
.
Front Immunol
2012
;
3
:
340
.
35.
Sela
U
,
Hershkoviz
R
,
Cahalon
L
,
Lider
O
,
Mozes
E
. 
Down-regulation of stromal cell-derived factor-1alpha-induced T cell chemotaxis by a peptide based on the complementarity-determining region 1 of an anti-DNA autoantibody via up-regulation of TGF-beta secretion
.
J Immunol
2005
;
174
:
302
9
.
36.
Shi
Y
,
Massague
J
. 
Mechanisms of TGF-beta signaling from cell membrane to the nucleus
.
Cell
2003
;
113
:
685
700
.
37.
Zhang
YE.
Non-Smad pathways in TGF-beta signaling
.
Cell Res
2009
;
19
:
128
39
.
38.
Vi
L
,
Boo
S
,
Sayedyahossein
S
,
Singh
RK
,
McLean
S
,
Di Guglielmo
GM
, et al
Modulation of type II TGF-beta receptor degradation by integrin-linked kinase
.
J Invest Dermatol
2015
;
135
:
885
94
.
39.
Hannigan
GE
,
Leung-Hagesteijn
C
,
Fitz-Gibbon
L
,
Coppolino
MG
,
Radeva
G
,
Filmus
J
, et al
Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase
.
Nature
1996
;
379
:
91
6
.
40.
Honda
S
,
Shirotani-Ikejima
H
,
Tadokoro
S
,
Maeda
Y
,
Kinoshita
T
,
Tomiyama
Y
, et al
Integrin-linked kinase associated with integrin activation
.
Blood
2009
;
113
:
5304
13
.
41.
Qian
Y
,
Zhong
X
,
Flynn
DC
,
Zheng
JZ
,
Qiao
M
,
Wu
C
, et al
ILK mediates actin filament rearrangements and cell migration and invasion through PI3K/Akt/Rac1 signaling
.
Oncogene
2005
;
24
:
3154
65
.
42.
Legate
KR
,
Montanez
E
,
Kudlacek
O
,
Fassler
R
. 
ILK, PINCH and parvin: the tIPP of integrin signalling
.
Nat Rev Mol Cell Biol
2006
;
7
:
20
31
.
43.
Grashoff
C
,
Aszodi
A
,
Sakai
T
,
Hunziker
EB
,
Fassler
R
. 
Integrin-linked kinase regulates chondrocyte shape and proliferation
.
EMBO Rep
2003
;
4
:
432
8
.
44.
Stevens
JM
,
Jordan
PA
,
Sage
T
,
Gibbins
JM
. 
The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin alpha 2 beta 1
.
J Thromb Haemost
2004
;
2
:
1443
52
.
45.
Zhang
M
,
March
ME
,
Lane
WS
,
Long
EO
. 
A signaling network stimulated by beta2 integrin promotes the polarization of lytic granules in cytotoxic cells
.
Sci Signal
2014
;
7
:
ra96
.
46.
Kutlesa
S
,
Wessels
JT
,
Speiser
A
,
Steiert
I
,
Muller
CA
,
Klein
G
. 
E-cadherin-mediated interactions of thymic epithelial cells with CD103+ thymocytes lead to enhanced thymocyte cell proliferation
.
J Cell Sci
2002
;
115
(Pt 23):
4505
15
.