Interaction of the integrin αE(CD103)β7 expressed on tumor-infiltrating lymphocytes (TIL) with E-cadherin on epithelial tumor cells is required to trigger polarized exocytosis of cytotoxic granules in TIL that elicit tumor cell lysis. In this study, we investigated the functional and signaling properties of CD103 and its individual contribution to T-cell–mediated cancer-cell killing. Our results indicated that the binding of CD103 on tumor-specific CTL to immobilized recombinant E-cadherin-Fc is sufficient to induce the polarization of cytolytic granules, whereas the degranulation of cytolytic granules also requires the coengagement of the T-cell receptor. Moreover, minimal CD103 triggering promotes the phosphorylation of the ERK1/2 kinases and phospholipase Cγ1 (PLCγ1). Inhibiting PLCγ blocks granule relocalization, decreasing T-cell receptor–mediated cytotoxicity. Thus, our results emphasize a unique costimulatory role of CD103 in tumor-specific CTL activation by providing signals that promote T-cell effector functions needed to specifically target and lyse cancer cells. Cancer Res; 71(2); 328–38. ©2011 AACR.

CTLs play a crucial role in immunity against cancers through their ability to kill neoplastic cells following the interaction of their T-cell receptor (TCR) with a specific peptide-major histocompatibility complex class I (pMHC-I) complex on tumor cells. This process is mainly mediated by a synaptic exocytosis of cytotoxic granules, also called secretory lysosomes, containing perforin and granzymes. Cytotoxicity proceeds through a multistep mechanism including an integrin-mediated adhesion of effector T cells to antigen-presenting cells (APC), polarization of the microtubule organizing center (MTOC) toward the target, and movement of lytic granules along the microtubules toward the MTOC (1). Lysis then occurs through the polarized release of the content of cytotoxic granules, a process also referred to as degranulation (2). Upon T cell–target cell contact, the remodeling of the actin cytoskeleton, reorganization of the cell surface, and repositioning of cytoplasmic proteins result in the formation of a so-called immune synapse (IS). TCR and associated signaling molecules are clustered at the center of the interface, the central supramolecular activation complex (cSMAC; ref. 3), whereas the adhesion/costimulatory molecules are localized at the peripheral (p)SMAC (4). Among adhesion/costimulatory molecules, lymphocyte function–associated antigen-1 (LFA-1; CD11a/CD18 or αL2 integrin) on CTL plays a critical role in TCR-mediated lysis by interacting with the intercellular adhesion molecule (ICAM)-1 on APC and directing the release of cytolytic granules to the surface of target cells near the cSMAC (1, 5).

Much less is known about the role of αE(CD103)β7 integrin in CTL-mediated cytotoxicity. We previously reported that CD103 plays an essential role in TCR-dependent tumor-cell killing through interaction with its ligand, the epithelial cell marker E-cadherin on target cells (6). This integrin has also been associated with the cytotoxicity of CD8+ T cells in several pathologies, including graft-versus-host disease (GVHD; 7), allogeneic transplant rejection (8–10), and autoimmune diseases (11, 12). CD103 is frequently expressed on intraepithelial lymphocytes (IEL; 13) as well as on a subpopulation of CD3+/CD8+ TIL (6, 14–16), CD4+/CD25+ Treg cells (17), and dendritic cells (18). It is locally induced by transforming growth factor (TGF)-β1 subsequent to T-cell activation (6, 19, 20). The actual function of αEβ7 integrin on T lymphocytes is not well understood. A role in T-cell homing into epithelia has been suggested, but most evidence favors a role for CD103 in the retention of T-lymphocyte populations in epithelial tissues by engaging E-cadherin (20, 21). However, the individual contribution of CD103 to intratumoral T-cell retention and CTL-mediated cancer cell destruction remains to be elucidated. In this report, we investigated the functional and signaling properties of CD103 and its direct participation in TCR-dependent tumor-cell killing. Our results indicate that the adhesion of αEβ7 integrin on CTL to E-cadherin–coupled beads is sufficient to induce the polarization of cytolytic granules and that degranulation is triggered following TCR engagement. This study defines a costimulatory signal for tumor-infiltrating T cells that is required along with TCR engagement to trigger the cytolytic granule lysis of cancer cell targets.

Tumor cell line and T-cell clones

The IGR-Heu lung cancer cell line was established as described (22). The Heu171 and H32-8 T-cell clones were isolated from autologous TIL (22) and peripheral blood lymphocytes (PBL) respectively (23).

Ex vivo TIL were freshly isolated from human lung tumor specimens after mechanical dissociation and a small-size–based selection of mononuclear cells using a FACSVantage cell sorter.

Antibodies, recombinant molecules, and chemical inhibitors

Monoclonal antibodies (mAbs) directed against CD8 and CD103 were purchased from Immunotech or eBiosciences. Anti-CD107a and anti-CD3 (UCHT1) mAbs were provided by Becton–Dickinson. Anti-phospho-extracellular signal–regulated kinases 1 and 2 (ERK1/2; pThr202/204) mAb, anti-ERK1/2, anti-phospho-PLCγ1 (pSer1248), and anti-PLCγ1 Ab were purchased from Cell Signaling Technology. Anti-actin and secondary Ab were purchased from Santa Cruz Biotechnology. Anti-granzyme B mAb and rhodamin–phalloidin were purchased from Invitrogen.

The human recombinant (r) E-cadherin-Fc and ICAM-1-Fc were provided by R&D Systems. The phosphatidylinositol 3 kinase (PI3K) inhibitor Wortmannin, the ERK1/2 inhibitor U0126, the protein kinase C (PKC) inhibitor piceatannol, and the PLCγ inhibitor U-73122 and its inactive analogue U-73343 were purchased from CalBiochem.

Flow cytometric analysis and cytotoxicity assay

Phenotypic analyses of T cells were performed by direct immunofluorescence using a FACS Calibur flow cytometer. Data were processed using CellQuest software (BD Biosciences). For the granule exocytosis assay, T cells were stimulated with a combination of surface-bound UCHT1 mAb and rE-cadherin-Fc or rICAM-1-Fc in flat-bottom 96-well plates. CD107a externalization was then assessed.

The cytotoxic activity of the T-cell clones was measured by a conventional 4-hour 51Cr-release essay (6). The inhibition of lysis was assessed by preincubating effector cells with indicated inhibitors for 1 hour at room temperature.

Western blot analysis

T cells (5.106) were incubated at 37°C with rE-cadherin-Fc, rICAM-1-Fc, or autologous tumor cells. Total cellular extracts were obtained by cell lysis in ice-cold lysis buffer (HEPES 10 mmol/L pH 7.4, NaCl 150 mmol/L, 1% CHAPS, 1% glycerol) supplemented with a cocktail of anti-proteases (Roche) and orthovanadate (2 mmol/L) for 30 minutes at 4°C. Equivalent amounts of protein extracts (30 μg) were denatured in Laemmli buffer, separated by SDS–PAGE on 4%–20% precise protein gel (Thermo Scientific), and transferred onto a nitrocellulose membrane (Pierce/Perbio). After the saturation of the nonspecific binding sites by incubating the blot for 1 hour in TBS containing 20 mmol/L Tris-HCL, 5% nonfat dry milk, and 0.1% Tween 20, the membrane was probed with a specified primary Ab followed by appropriate secondary HRP-conjugated Ab and then revealed by chemoluminescence with SuperSignal WestPico substrate (Pierce/Perbio).

Confocal microscopy

E-cadherin–coupled or uncoupled Protein G-Dynabeads (Invitrogen) and T-cell clones were plated on poly-(L-lysine)-coated coverslips (Sigma-Aldrich) at a 1:5 E:T ratio. Cells were then fixed and permeabilized as described (6). They were then stained with anti-granzyme B mAb and rhodamin–phalloidin, followed by Alexa-Fluor-488–coupled secondary mAb; nuclei were stained with TO-PRO-3 iodide (Molecular Probes). Coverslips were mounted with Fluoromount-G (SouthernBiotech) and analyzed by fluorescence microscopy as described (6).

For video imaging, CTL or freshly isolated TIL were stained with LysoTracker Red (Invitrogen) and added to Protein G-Dynabeads coated or not with human rE-cadherin-Fc or rICAM-1-Fc at a 1:5 E:T ratio in glass-bottom culture dishes (MatTek). In inhibition experiments, CTLs were pretreated with the pharmacological agent for 1 hour at room temperature prior to coincubation with beads. Lysosome polarization was defined by the accumulation of LysoTracker Red in the contact area between effector cells and Protein-Fc-coupled beads.

For in situ TIL staining, approximately 1 mm thick human lung tumor slices from 3 different lung cancer specimens were fixed in 100% methanol for 10 minutes at −20°C and then stained with a combination of phycoerythrin-conjugated anti-CD103, allophycocyanin-conjugated anti-CD8 mAb, and SYTOX Green nucleic acid stain (Invitrogen). Tumor slices were then analyzed as described above.

Involvement of PI3K/ERK and PLC/PKC pathways in CD103-dependent TCR-mediated cytotoxicity

The cytotoxic activity of the tumor-specific T-cell clones Heu171 and H32-8 is dependent on the interaction of αE(CD103)β7 integrin on effector cells with E-cadherin on the autologous lung cancer cell line IGR-Heu (6). Figure 1B indicates that the cytotoxicity of Heu171 (CD103high) and H32-8 (CD103low) CTLs, derived respectively from patient TIL and PBL, toward the E-cadherin+/ICAM-1–specific target was positively correlated with the surface expression level of CD103 (Fig. 1A). To investigate whether the mitogen-activated protein kinase (MAPK)–signaling pathway is involved in TCR-mediated tumor cell lysis, we analyzed the involvement of ERK1/2 using a specific inhibitor of MAPK/ERK kinases (MEK)1/2. As shown in Figure 1B, preincubation of both clones with the MEK1/2 inhibitor U0126 at 100 μmol/L, inhibits their cytolytic function. A weak inhibition was also observed with the specific inhibitor of the PI3K Wortmannin at 1 μm (Fig. 1B).

Figure 1.

The role of PI3K/ERK and PLC/PKC signaling pathways in CD103-dependent T-cell clone–mediated lysis. A, surface expression of αE(CD103)β7 integrin on 2 tumor-specific T-cell clones derived from a lung cancer patient TIL (Heu171) and PBL (H32-8). Immunofluorescence analysis was done with anti-CD103 (black fill) mAb or an isotypic control (without fill). Mean fluorescence intensity values are in parentheses. B, the cytotoxic activity of the Heu171 and H32-8 clones toward the specific IGR–Heu tumor cell line was determined by a conventional 4-hour 51Cr-release assay at indicated E:T ratios. The T-cell clones were preincubated either in a medium or with indicated concentrations of PI3K inhibitor Wortmannin, ERK1/2 inhibitor U0126, PKC inhibitor piceatannol, PLCγ inhibitors U-73122 and U-73343 inactive form, or a vehicle control. Data shown are representative of 3 independent experiments.

Figure 1.

The role of PI3K/ERK and PLC/PKC signaling pathways in CD103-dependent T-cell clone–mediated lysis. A, surface expression of αE(CD103)β7 integrin on 2 tumor-specific T-cell clones derived from a lung cancer patient TIL (Heu171) and PBL (H32-8). Immunofluorescence analysis was done with anti-CD103 (black fill) mAb or an isotypic control (without fill). Mean fluorescence intensity values are in parentheses. B, the cytotoxic activity of the Heu171 and H32-8 clones toward the specific IGR–Heu tumor cell line was determined by a conventional 4-hour 51Cr-release assay at indicated E:T ratios. The T-cell clones were preincubated either in a medium or with indicated concentrations of PI3K inhibitor Wortmannin, ERK1/2 inhibitor U0126, PKC inhibitor piceatannol, PLCγ inhibitors U-73122 and U-73343 inactive form, or a vehicle control. Data shown are representative of 3 independent experiments.

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Next, we analyzed the involvement of the PLC/PKC pathway in specific T-cell clone-mediated killing. Results show that pretreatment of CTL clones with the PKC inhibitor piceatannol at 100 μmol/L and the PLCγ inhibitor U-73122 at both 0.2 μm and 2 μm dramatically inhibited tumor cell lysis (Fig. 1B). In contrast, a marginal effect was observed with the inactive analogue U-73343, which was used as a negative control. It should be noted that none of the pharmacological reagents had any effect on T-cell viability, as assessed by propidium iodide staining (data not shown). These results suggest that both the PI3K/ERK and PLC/PKC signaling pathways are involved in CD103-dependent TCR-mediated cytotoxicity.

Adhesion of CD103 on CTL clones to immobilized E-cadherin-Fc results in phosphorylation of ERK1/2 and PLCγ1

Accumulating evidence indicates that integrins play a key role in T-cell–mediated cytotoxicity by promoting effector cell adhesion to target cells but also by triggering intracellular signaling events through interaction with their respective ligands. Thus far, little is known about the signaling properties of the αEβ7 integrin and its direct contribution to CTL functions. We therefore investigated whether the engagement of CD103 on Heu171 (CD103high) and H32-8 (CD103low) clones with plastic-coated human rE-cadherin-Fc is able to transduce intracellular signals by monitoring the phosphorylation of the ERK1/2 and PLCγ1 proteins. Western blot analyses indicated that the ligation of CD103 on both CTL clones to immobilized E-cadherin-Fc was sufficient to trigger the phosphorylation of PLCγ1 (at residue serine 1248) and ERK1/2 (Fig. 2A). It should be noted that both CTL clones fail to express the inhibitory ligand KLRG1 (6); however, the engagement of an unknown molecule to E-cadherin-Fc cannot be excluded. In this regard, the CD103high Heu171 TIL clone displayed a stronger phosphorylation of both PLCγ1 and ERK1/2 than the CD103low H32-8 PBL clone, supporting the direct engagement of the αEβ7 integrin. Importantly, the clustering of the LFA-1 (αLβ2 integrin) costimulatory molecule (24–28) on T-cells to immobilized rICAM-1-Fc also triggered the phosphorylation of ERK1/2 and PLCγ1 (Supplementary Fig. S1A).

Figure 2.

Engagement of immobilized E-cadherin-Fc with its ligand on CTL clones results in the activation of ERK1/2 and PLCγ signaling pathways. A, Western blot analysis of ERK1/2 and PLCγ1 protein phosphorylation following the stimulation of CD103high Heu171 and CD103low H32-8 clones with plastic-coated rE-cadherin-Fc or autologous tumor cells. Top panels: total protein extracts were prepared from effector cells incubated with IGR-Heu (at 5:1 E:T ratio) or plastic-coated E-cadherin-Fc (5 μg/mL) for the indicated time-points. Actin was used as a loading control. Bottom panels: the normalization of phosphorylated proteins relative to total proteins. Data shown represent 1 of 3 independent experiments. B, the potentiation of ERK1/2 phosphorylation following the coengagement of CD103 and TCR. Top panels: the H32-8 clone was incubated in the presence of anti-CD3 mAb (1 μg/mL), immobilized E-cadherin-Fc (5 μg/mL), or a combination of both reagents for indicated times. An IgG1 isotypic control (1 μg/mL) was included. Cell extracts were analyzed by Western blot using the specified Ab. Actin was used as a loading control. Bottom panels: the normalization of phosphorylated proteins relative to total proteins. Data shown correspond to 1 representative experiment of 3. E-cadh, E-cadherin.

Figure 2.

Engagement of immobilized E-cadherin-Fc with its ligand on CTL clones results in the activation of ERK1/2 and PLCγ signaling pathways. A, Western blot analysis of ERK1/2 and PLCγ1 protein phosphorylation following the stimulation of CD103high Heu171 and CD103low H32-8 clones with plastic-coated rE-cadherin-Fc or autologous tumor cells. Top panels: total protein extracts were prepared from effector cells incubated with IGR-Heu (at 5:1 E:T ratio) or plastic-coated E-cadherin-Fc (5 μg/mL) for the indicated time-points. Actin was used as a loading control. Bottom panels: the normalization of phosphorylated proteins relative to total proteins. Data shown represent 1 of 3 independent experiments. B, the potentiation of ERK1/2 phosphorylation following the coengagement of CD103 and TCR. Top panels: the H32-8 clone was incubated in the presence of anti-CD3 mAb (1 μg/mL), immobilized E-cadherin-Fc (5 μg/mL), or a combination of both reagents for indicated times. An IgG1 isotypic control (1 μg/mL) was included. Cell extracts were analyzed by Western blot using the specified Ab. Actin was used as a loading control. Bottom panels: the normalization of phosphorylated proteins relative to total proteins. Data shown correspond to 1 representative experiment of 3. E-cadh, E-cadherin.

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To further corroborate the above observations, we stimulated the H32-8 clone with either plastic-coated UCHT1 mAb, immobilized E-cadherin-Fc, or a combination of both reagents. As expected, a suboptimal concentration of anti-CD3 mAb induced a weak phosphorylation of ERK1/2 15 minutes after TCR triggering (Fig. 2B). Interestingly, CD103 ligation to E-cadherin-Fc also triggered the phosphorylation of ERK, which was strongly increased by concomitant TCR stimulation (Fig. 2B). These results further emphasize a role of CD103 in T-cell adhesion to epithelial tissues and suggest a costimulatory function of this integrin in the TCR signaling of activated CTLs, similar to LFA-1 (1, 5, 26, 29).

Binding of CD103 to E-cadherin-Fc is sufficient to trigger cytotoxic granule polarization through a PLCγ-dependent pathway

Our previous data indicated that the αEβ7 integrin is recruited at the IS formed between TIL and epithelial tumor cells and that its interaction with E-cadherin is required for cytolytic granule polarization and subsequent exocytosis resulting in effective target cell lysis (6). To determine the direct contribution of CD103 to TCR-mediated tumor-cell killing, we first tested whether the binding of CD103 on Heu171 CTL to rE-cadherin-coupled beads was efficient to induce cytotoxic granule polarization at the contact zone. Confocal microscopy analyses using a granzyme B-specific mAb showed that CD103high T-cells bound to E-cadherin–coupled but not to –uncoupled beads and that drastic relocalization of cytolytic granules occurred at the interface between CTL and rE-cadherin beads (Fig. 3A). Polarization of cytotoxic granules was also observed in the CD103low H32-8 T-cell clone incubated with rE-cadherin-coated beads (data not shown). Moreover, video-imaging experiments performed with a vital lysosomal marker, LysoTracker probe, showed a prompt attachment of the CD103high Heu171 clone to rE-cadherin–conjugated beads and a subsequent movement of stained lysosomes at the contact area (Fig. 3B, see also movies in supplemental material). This was found in 97% ± 3.3% of analyzed conjugates formed between Heu171 and E-cadherin–coupled beads (n = 63; Fig. 3C). In contrast, a CD103 CTL clone, H32–22 (6), used as a negative control was unable to adhere to E-cadherin–coupled beads, and thus, no lysosome polarization was observed (data not shown). Notably, the ligation of LFA-1 on Heu171 CTL to rICAM-1–coupled beads also induced cytotoxic granule relocalization at the contact zone, as shown by confocal microscopy with a LysoTracker probe (see Supplementary Fig. S1B).

Figure 3.

Minimal interaction of the αEβ7 integrin on the TIL clone with rE-cadherin–coated beads is sufficient to induce lytic granule polarization. A, conjugates formed between the Heu171 TIL clone and E-cadherin–coated beads were analyzed by confocal microscopy after 15 minutes of incubation. Granule polarization, as defined by the accumulation of granzyme B in the contact area between effector T cells and E-cadherin–coated beads, was followed up with anti-granzyme B mAb (green fluorescence). Nuclei were stained with TO-PRO-3 iodide (blue fluorescence) and polymerized actin with rhodamin–phalloidin (red fluorescence). Scale bars, 10 μm. B, time-lapse gallery (intervals of approximately 150 seconds) of lysosome polarization in the presence or absence of U-73122 (0.2 μmol/L), U-73343 (0.2 μmol/L), or a vehicle control. The Heu171 TIL clone was labeled with a LysoTracker Red probe and conjugates formed with E-cadherin-Fc–coated or -uncoated beads were followed up by confocal microscopy. Scale bars, 10 μm. Arrows point to lysosome polarization at the contact area between CTL and rE-cadherin–coated beads. C, percentages of CTL displaying lysosome relocalization during conjugate formation between the Heu171 and rE-cadherin–coated beads, after preincubation of T-cell clones with U-73122, U-73343, or a vehicle control. Data show mean ± SD of 4 different fields and are representative of 3 independent experiments. Numbers (n) of analyzed conjugates are indicated. E-cadh, E-cadherin

Figure 3.

Minimal interaction of the αEβ7 integrin on the TIL clone with rE-cadherin–coated beads is sufficient to induce lytic granule polarization. A, conjugates formed between the Heu171 TIL clone and E-cadherin–coated beads were analyzed by confocal microscopy after 15 minutes of incubation. Granule polarization, as defined by the accumulation of granzyme B in the contact area between effector T cells and E-cadherin–coated beads, was followed up with anti-granzyme B mAb (green fluorescence). Nuclei were stained with TO-PRO-3 iodide (blue fluorescence) and polymerized actin with rhodamin–phalloidin (red fluorescence). Scale bars, 10 μm. B, time-lapse gallery (intervals of approximately 150 seconds) of lysosome polarization in the presence or absence of U-73122 (0.2 μmol/L), U-73343 (0.2 μmol/L), or a vehicle control. The Heu171 TIL clone was labeled with a LysoTracker Red probe and conjugates formed with E-cadherin-Fc–coated or -uncoated beads were followed up by confocal microscopy. Scale bars, 10 μm. Arrows point to lysosome polarization at the contact area between CTL and rE-cadherin–coated beads. C, percentages of CTL displaying lysosome relocalization during conjugate formation between the Heu171 and rE-cadherin–coated beads, after preincubation of T-cell clones with U-73122, U-73343, or a vehicle control. Data show mean ± SD of 4 different fields and are representative of 3 independent experiments. Numbers (n) of analyzed conjugates are indicated. E-cadh, E-cadherin

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Next, we analyzed T-cell infiltration in human non–small cell lung carcinoma (NSCLC) specimens and the behavior of freshly purified CD8+/CD103+/granzyme B+ TIL coincubated with E-cadherin–coated beads. Remarkably, size-selected freshly isolated TIL mostly displayed a CD8+/CD103+/granzyme B+ phenotype (Fig. 4A) and exhibited lytic granule polarization toward the contact zone with E-cadherin–conjugated beads (Fig. 4B, see also movies in supplemental material). This was found in 87% of analyzed conjugates formed between freshly purified LysoTracker+ TIL and rE-cadherin–coupled beads (n = 16). However, these interactions appeared to be more transient than those observed with the TIL clone Heu171. To assess the lytic potential of CD8+/CD103+ TIL, we analyzed their localization within fresh tumor slices and their capacity to secrete granzyme B when in contact with tumor cells. Confocal microscopy analysis revealed conjugates formed between CTLs and autologous tumor cells, with synaptic relocalization of CD103 and the release of granzyme B into the target (Fig. 4C). These data further emphasize a key role for CD103 in the formation of a functional presynapse and strongly suggest that CD103+/CD8+ T cells are effective in vivo.

Figure 4.

Ligation of CD103 on freshly isolated CD8+ TIL with rE-cadherin–coated beads induces lytic granule polarization A, phenotypic analysis of freshly isolated TIL. Immunofluorescence analysis was done with anti-CD3, anti-CD103, anti-CD8, and anti-granzyme B mAb. SSC, side scatter; FSC, forward scatter. B, freshly isolated TIL were labeled with a LysoTracker Red probe and conjugates formed with E-cadherin–coated beads were followed up by confocal microscopy. Time-lapse gallery (intervals of approximately 150 seconds) of lysosome polarization in the presence of uncoated beads (top) or E-cadherin-Fc–coated beads (bottom). Scale bars, 10 μm. Bead aggregates most likely result from the homotypic bonds between adjacent E-cadherin–coated beads. Arrows indicate the granule relocalization at the contact zone between TIL and rE-cadherin–coated beads. The data shown correspond to 1 experiment of 3. C, analysis of CD103+ TIL behavior within a fresh human lung tumor specimen. Tumor slices were stained with a combination of anti-granzyme B (green fluorescence), anti-CD103 (red fluorescence) mAb, and SYTOX Green (blue fluorescence, nuclei). Scale bar, 10 μm. Data shown correspond to 1 representative experiment of 3.

Figure 4.

Ligation of CD103 on freshly isolated CD8+ TIL with rE-cadherin–coated beads induces lytic granule polarization A, phenotypic analysis of freshly isolated TIL. Immunofluorescence analysis was done with anti-CD3, anti-CD103, anti-CD8, and anti-granzyme B mAb. SSC, side scatter; FSC, forward scatter. B, freshly isolated TIL were labeled with a LysoTracker Red probe and conjugates formed with E-cadherin–coated beads were followed up by confocal microscopy. Time-lapse gallery (intervals of approximately 150 seconds) of lysosome polarization in the presence of uncoated beads (top) or E-cadherin-Fc–coated beads (bottom). Scale bars, 10 μm. Bead aggregates most likely result from the homotypic bonds between adjacent E-cadherin–coated beads. Arrows indicate the granule relocalization at the contact zone between TIL and rE-cadherin–coated beads. The data shown correspond to 1 experiment of 3. C, analysis of CD103+ TIL behavior within a fresh human lung tumor specimen. Tumor slices were stained with a combination of anti-granzyme B (green fluorescence), anti-CD103 (red fluorescence) mAb, and SYTOX Green (blue fluorescence, nuclei). Scale bar, 10 μm. Data shown correspond to 1 representative experiment of 3.

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We then assessed the contribution of PLCγ activity in cytotoxic granule accumulation in the contact zone between effector T cells and E-cadherin–conjugated beads. Results obtained with Heu171 CTL indicate that the PLCγ inhibitor U-73122 dramatically inhibited the polarization of acidic lysosomes (Fig. 3B, right panels). In contrast, the inactive analogue of the PLCγ inhibitor U-73343, PI3K inhibitor Wortmannin, and MEK1/2 inhibitor U0126 had no effect (Fig. 3B, right panels, and data not shown). Lytic granule relocalization was observed in 93% ± 5.4% of conjugates formed between U-73343–treated Heu171 and E-cadherin–coupled beads (n = 50) and 92% ± 6.8% for vehicle control–treated cells (n = 52) but only in 47% ± 4.4% of conjugates formed by U-73122-treated Heu171 (n = 68; Fig. 3C). These results strongly suggest that the CD103/E-cadherin interaction directly contributes to TCR-mediated cytotoxicity, not only by favoring the adhesion of effector T cells to epithelial target cells but also by triggering granule polarization at the IS formed between the CTL and tumor cells.

Coengagement of TCR and CD103 promotes cytotoxic granule exocytosis

Next, we analyzed whether integrin-ligand binding is sufficient to promote lytic granule exocytosis by the TIL clone when cultured in plastic dishes coated with E-cadherin-Fc alone or in combination with suboptimal concentrations of UCHT1 mAb, measured by the evaluation of lysosomal-associated membrane glycoprotein-1 (LAMP-1, CD107a) externalization (30–32). Immunofluorescence analyses did not reveal any CD107a induction at the surface of Heu171 CTL stimulated with either E-cadherin-Fc alone (2.5 μg/mL) or with low concentrations of UCHT1 alone (0.4 and 2 μg/mL) even after 1 hour of incubation (Fig. 5A). Interestingly, a combination of immobilized E-cadherin-Fc and suboptimal concentrations of anti-CD3 mAb–triggered CD107a externalization, which was abrogated by the pretreatment of the effector T cells with the PLCγ inhibitor U-73122, but not the inactive U-73343 analogue. MEK1/2 inhibitor U0126 and PI3K inhibitor Wortmannin had much weaker blocking effects than the U-73122 inhibitor (Fig. 5B). Remarkably, a combination of immobilized rICAM-1-Fc and suboptimal concentrations of UCHT1 also triggered CD107a externalization on Heu171 CTL (see Supplementary Fig. S2). These data indicate that the coengagement of CD103 or LFA-1 with TCR following the recognition of the specific pMHC-I complex on epithelial tumor cells is required to trigger cytotoxic granule polarization and subsequent exocytosis leading to target cell killing.

Figure 5.

Engagement of both CD103 and TCR is required for lytic granule exocytosis. A, CD107a externalization on the surface of Heu171 CTL cocultured with the indicated concentration of immobilized E-cadherin-Fc, suboptimal concentrations of anti-CD3 mAb or a combination of both reagents. Immunofluorescence analyses were done at indicated time-points. B, the involvement of PI3K/ERK and PLC/PKC pathways in cytotoxic granule exocytosis. The Heu171 clone was incubated with indicated suboptimal concentrations of surface-bound anti-CD3 mAb or a combination of immobilized anti-CD3 mAb and E-cadherin-Fc (2.5 μg/mL), in medium alone, with vehicle control, or in the presence of indicated inhibitors. CD107a induction was monitored by immunofluorescence analysis. Data shown are representative of 3 independent experiments. E-cadh, E-cadherin.

Figure 5.

Engagement of both CD103 and TCR is required for lytic granule exocytosis. A, CD107a externalization on the surface of Heu171 CTL cocultured with the indicated concentration of immobilized E-cadherin-Fc, suboptimal concentrations of anti-CD3 mAb or a combination of both reagents. Immunofluorescence analyses were done at indicated time-points. B, the involvement of PI3K/ERK and PLC/PKC pathways in cytotoxic granule exocytosis. The Heu171 clone was incubated with indicated suboptimal concentrations of surface-bound anti-CD3 mAb or a combination of immobilized anti-CD3 mAb and E-cadherin-Fc (2.5 μg/mL), in medium alone, with vehicle control, or in the presence of indicated inhibitors. CD107a induction was monitored by immunofluorescence analysis. Data shown are representative of 3 independent experiments. E-cadh, E-cadherin.

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Integrins are cell surface receptors formed by α and β subunits that play critical roles in T-cell functions, including adhesion to APC, costimulation, migration to lymphoid organs and inflammation sites, and extravasation (33). Integrin adhesiveness is regulated by a dynamic process termed “inside-out” signaling, initiated by TCR and chemokine receptor stimulation, resulting in the clustering of individual integrin units and conformational changes in the integrin itself (34). In addition, ligand binding can generate downstream signals termed “outside-in” signaling that regulates the effects of other receptors, including TCR and growth factor receptors (35, 36). Multiple integrins are expressed on CTLs and can play an essential role in TCR-mediated killing by interacting with their respective ligands on specific target cells. Thus far, little is known about the contribution of the αE(CD103)β7 integrin to activation signals for CD8+ T-lymphocyte cytotoxicity besides its role in cell adhesion. In the present report, we have explored in more detail the role of CD103 in TCR-mediated cytotoxicity using well-characterized human CTL clones, recognizing a mutated α-actinin-4 epitope on autologous E-cadherin+/ICAM-1/MHC-Ilow tumor cells in an HLA-A2 context. The capacity of these clones to kill the cognate tumor was correlated with the αEβ7 integrin surface expression level and was abrogated by anti-CD103 neutralizing mAb or small interference (si)RNA targeting E-cadherin (6). We show here that, in these particular effector–target cell interactions, CD103 facilitates Ag recognition on epithelial tumors by favoring T-lymphocyte adhesion to specific tumor cells. We also show that CD103 ligation to E-cadherin induces costimulation in activated CTLs by triggering “outside-in” signals that integrate and/or synergistically cooperate with TCR/CD3-mediated signals.

The adhesiveness of the αEβ7 integrin to its ligand appeared to be regulated by “inside-out” signals, as CD103-expressing IEL have been shown to bind more avidly to E-cadherin after treatment with PMA or anti-CD3 mAb (37). In addition, a role of CD103 in transmitting “outside-in” signaling has been suggested by experiments indicating that anti-CD103 mAb increases T-cell proliferation in response to the suboptimal concentrations of anti-CD3 mAb (38–40) and induces a redirected lysis of Fc-receptor–bearing target cells (41, 42). The T-cell clones used in the present study and, more importantly, freshly isolated CD8+ TIL display high CD103 affinity for ligand, since they are able to avidly adhere to an E-cadherin–coated surface. This suggests that the αEβ7 integrin can be positively regulated within the tumor microenvironment, such as after TCR engagement and/or chemokine production, and may contribute to the induction of an efficient antitumor immune response in situ.

The activation of ERK and PKC are well known to be key signals for lytic granule exocytosis (26, 43–45). It has been reported that the triggering of β1, β2, and β3 fails to activate ERK in CTLs, but integrin ligation synergizes with the TCR stimulation upstream of MAPK to enhance CD8+ T-cell activation (27, 28). The cytotoxicity of the T-cell clones included in this study is decreased by the inhibitors of PI3K/ERK and PLC/PKC suggesting that both signaling pathways are involved in CD103-dependent TCR-mediated tumor cell lysis. Remarkably, the cross-linking of CD103 on CTL clones with immobilized E-cadherin-Fc is sufficient to induce the phosphorylation of ERK1/2 and PLCγ1, which is enhanced upon TCR stimulation with anti-CD3 mAb. Thus, in addition to facilitating T-cell adhesion to epithelial target cells, CD103 ligation promotes CTL activation not only by increasing target cell contact but also by initiating intracellular signals through the activation of PI3K/ERK and PLC/PKC pathways, leading to the amplification of signals from the TCR/CD3 complex.

The activation of T-cell cytotoxicity requires a combination of signals for MTOC and cytolytic granule polarization and subsequent degranulation. Here, we show that tumor-specific αEβ7+ CTL clones and freshly isolated CD8+/CD103+/granzyme B+ TIL, which are abundantly present within lung tumors [(6) and in the present study], form stable conjugates with E-cadherin–coupled beads. Therefore, adhesion by a TCR-independent pathway involving CD103 and E-cadherin may precede or concurrently occur with Ag recognition on epithelial targets. This could lower the threshold for T-cell activation and help in polarizing T-lymphocyte interactions toward the relevant tumor cell. More importantly, our results indicate that, similar to LFA-1, the ligation of CD103 is sufficient to trigger the polarization of cytotoxic granules in CTL clones and freshly isolated CD8+ TIL but does not lead to exocytosis. Thus, like in NK cells, polarization and exocytosis signals can be uncoupled in T cells. Indeed, the engagement of LFA-1 on NK cells by its ligand ICAM-1 induces cytotoxic granule polarization but not degranulation (46). In contrast, it has been described that the LFA-1/ICAM-1 interaction does not lead to the granule redistribution and recruitment of the MTOC toward the APC without TCR engagement in the CTL (29, 47, 48). This discrepancy may be due to the cellular models used in these studies and to the affinity/avidity of the TCR for the specific pMHC-I complex. Our data also show that inhibitors of PLCγ, but not of PI3K and ERK1/2, abrogate CD103-dependent granule polarization. Accordingly, it has been reported that PLCγ is necessary for MTOC polarization (49). Thus, CD103 engagement by its ligand E-cadherin plays a unique role in anti-epithelial tumor CTL function by initiating cytotoxic granule polarization in a PLCγ-dependent pathway.

Although TCR engagement can lead to the formation of stable conjugates between the Ag-specific CD103 PBL clone and autologous tumor cells, it does not trigger granule polarization (6). In addition, E-cadherin knockdown in tumor cells with specific siRNA abrogates cytotoxic granule relocalization in conjugates formed with the CD103+ TIL clone (6). We show here that the engagement of TCR with suboptimal doses of anti-CD3 mAb combined with the ligation of CD103 with immobilized rE-cadherin-Fc led to granule polarization and subsequent degranulation. This suggests that degranulation, but not polarization, can be induced by TCR and that CD103 synergizes with TCR stimuli to promote the activation of CTL function by triggering cytotoxic granule relocalization. It also suggests that CD103 and TCR initiate distinct intracellular signals that complement each other to elicit the effective killing of epithelial cancer cells by tumor-infiltrating CD8+ T cells, which frequently display low avidity for the pMHC-I complex. Nevertheless, even though TILs frequently express CD103, they are often unable to eradicate established epithelial tumors. This may be due to a low frequency of tumor-specific T-cell precursors, which may result in an insufficient expansion of the reactive CTLs in comparison with cancer cell growth.

Overall, our data show that CD103 can act at multiple levels to promote a local antitumor immune response. First, CD103 is directly involved in epithelial tumor recognition by CTLs. Second, the adhesion of CD103 on TIL to E-cadherin on target cells provides costimulatory signals that promote effector functions of activated CTL. Third, CD103 is directly involved in MTOC and cytotoxic granule polarization at the contact site with tumor cells. Finally, the release of cytotoxic granules requires synergistic activation through the engagement of both TCR and CD103. Our results also indicate that the CD103/E-cadherin interaction is involved in a cascade of signaling events that share, at least in part, common mechanisms with TCR in triggering CTL activity. These findings are consistent with the general role of CD103 in facilitating immune responses within epithelial tissues and provide new insights into mechanisms of cross-talk between αEβ7 integrin and TCR in promoting CTL functions. The capacity of CD8+/CD103+ T cells to bind to E-cadherin on epithelial cells and thus to trigger cytotoxic granule polarization may be an important feature not only of the antitumor immune response but also of the pathogenesis of specific tissue damage observed in acute allograft destruction (9) and in autoimmune and inflammatory diseases (11, 12). With regard to intratumoral immunity, we have previously demonstrated that TCR engagement within a TGF-β1–rich tumor microenvironment induces CD103 expression on tumor-specific T cells (6, 20). In addition, it has been reported that the loss of E-cadherin is often associated with increased invasion and metastasis in several solid cancers (50, 51). This may be due, at least in part, to an alteration in the capacity of tumor-specific T cells to adhere to and kill epithelial targets, which often lack ICAM. It may allow tumor cells to escape from the immune system and suggests a mechanism for the immune selection of cancer cells with a reduced expression of E-cadherin. Thus, the tumor microenvironment should be considered not only for its immunosuppressive activities but also for its potential beneficial effects in reshaping tumor immunity at least in the early stages of cancer development.

No potential conflicts of interest were disclosed.

We thank Dr. G Bismuth for helpful discussion. We also thank Y. Lecluse and O. Duc for their help with cell sorting and confocal microscopy.

This work was supported by grants from INSERM, Association pour la Recherche sur le Cancer, Institut National du Cancer, Agence National de la Recherche, Ligue contre le Cancer and Cancéropôle Ile de France.

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.

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