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The Journal of Immunology, 2007, 178: 2908-2915.
Copyright © 2007 by The American Association of Immunologists, Inc.

Modulation of CD103 Expression on Human Colon Carcinoma-Specific CTL1

Khoon-Lin Ling2,*, Nicolas Dulphy2,*, Pru Bahl*, Mariolina Salio*, Kevin Maskell*, Juan Piris{dagger}, Bryan F. Warren{dagger}, Bruce D. George{ddagger}, Neil J. Mortensen{ddagger} and Vincenzo Cerundolo3,*

* Tumor Immunology Group, Human Immunology Unit, Weatherall Institute of Molecular Medicine, {dagger} Department of Cellular Pathology, and {ddagger} Department of Colorectal Surgery, John Radcliffe Hospital, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Recent results have shown a correlation between survival and frequency of tumor-infiltrating T cells in colorectal cancer patients. However, the mechanisms controlling the ability of human T lymphocytes to infiltrate colon carcinoma remain unclear. Although, it is known that expression of the integrin CD103{alpha}E/beta7 by intraepithelial lymphocytes controls the retention of lymphocytes in epithelial layers, very little is known about the expression of intestinal homing receptors in human T lymphocytes. In particular, it remains unknown whether expression of CD103/beta7 by human colon cancer-specific T lymphocytes is controlled by recognition of tumor Ags and is imprinted during T cell priming, facilitating its expression during memory T cell activation. In this study, we demonstrate that expression of CD103/beta7 in human colon carcinoma-specific CTL is synergistically enhanced by the simultaneous TGF-beta1 stimulation and Ag recognition. These results were confirmed by using a panel of human CTL clones. Finally, we show that priming of naive CD8+ T cells in the presence of TGF-beta1 ensures up-regulation of CD103/beta7 in recall responses, at concentrations of TGF-beta1 significantly lower than those required by memory T cells primed in the absence of TGF-beta1. These results indicate a role of TGF-beta1 during T cell priming in modulating expression of CD103/beta7 and controlling retention of human memory CD8+ T cells into tumor epithelium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The role of T lymphocytes in controlling the growth of colon carcinoma has been described recently by comparing the percentage of tumor-infiltrating CD3+ lymphocytes and patients’ survival (1, 2). These results have highlighted the importance of analyzing the frequency of tumor-infiltrating T lymphocytes as a prognostic marker in colon cancer patients. However, the trafficking properties of colon cancer-specific T cells remain unclear. Understanding these mechanisms is of importance to clarify the events that control recirculation of colon cancer-specific T cells and to design vaccination strategies capable of maximizing the ability of T cells to infiltrate colon carcinoma.

The local microenvironment plays an important role in providing direct signals to responding T cells and in controlling expression of tissue-specific homing receptors. Dendritic cells (DC)4 in organized mucosal lymphoid tissues preferentially induce the expression of defined integrins and chemokine receptors on T cells, which results in T cell homing to the intestinal lamina propria (3, 4). Although experiments have been conducted in mouse models, very little is known about the mechanisms that control the expression of tissue-specific homing receptors in human T lymphocytes.

Integrins are heterodimeric glycoproteins that play a prominent role in the trafficking of lymphocytes (5). Experiments conducted in mouse models have elegantly demonstrated that the integrin CD49d/beta7 (also named {alpha}4beta7) and the chemokine receptor CCR9 help lymphocytes home to the small intestinal mucosa (3, 4). The ligand for CD49d/beta7 is the mucosal addressin cell adhesion molecule 1, a vascular addressin present on postcapillary venules in the intestinal lamina propria (6), whereas the chemokine receptor CCR9 recognizes CCL25, a chemokine produced in the small intestines. The integrin CD103/beta7 (hereafter referred to as CD103) plays an important role in the localization of lymphocytes in the intraepithelial compartment (7). More recently, it has been shown that CD103 expression on DC can influence the balance between effector and T regulatory cells in the intestine (8). CD103 is found on >90% of intraepithelial lymphocytes and on only 0.5–3% of peripheral blood lymphocytes (PBL) (9). The only known ligand for CD103 is E-cadherin (10, 11), which is expressed on epithelial cells. Although T cells migrating to murine intestines are CD103– and CD49d/beta7+ (12), CD103 is induced only after their entry into the mucosa (13, 14). The factor known to up-regulate CD103 is TGF-beta1 (15), a cytokine produced by many cells, including both normal and malignant intestinal epithelial cells (16, 17, 18). The importance of CD103 in controlling homing and retention of CD8+ T cells into the epithelial layers has recently been confirmed by demonstrating that CD103+ CTL, unlike CD103– CTL, play an important role in the destruction during graft-vs-host disease (GVHD) of gut epithelial cells and the epithelial components of renal and pancreatic allografts (19, 20).

It has been shown that mouse DC resident in the mesenteric lymph nodes (MLN) and the Peyer’s patches are responsible for inducing CD49d/beta7 and CCR9 on naive lymphocytes by secreting retinoic acid (21). Von Andrian and colleagues (22) have shown recently that tissue tropic effector CD8+ T cells that had been imprinted previously with the ability to migrate to a certain organ remain plastic to further influences by DC and can change their tissue-trafficking abilities. Although these results shed light on how the ability to traffic to the murine small intestine is imprinted on T cells, very little is known about the mechanisms which control homing of human T cells into the gut epithelium. In particular, it remains unclear whether Ag recognition contributes with TGF-beta1 to the up-regulation of CD103 and whether the ability of memory T cells to up-regulate CD103 in the presence of TGF-beta1 is controlled by events during T cell priming.

Because colon carcinomas are epithelial in origin and express E-cadherin, we analyzed CD103 expression in human colon carcinoma-specific T cells and assessed whether expression of CD103 segregated with their ability to recognize specifically autologous colon carcinoma cells. We then extended these results to a panel of Ag-specific CTL clones to determine whether factors, in addition to TGF-beta1 in the tumor stroma, might regulate CD103 expression. The results of our experiments demonstrated that, Ag recognition together with TGF-beta1 stimulation, synergistically enhance the proportion of T cells expressing CD103. The presence of TGF-beta1 during Ag-specific T cell priming ensures that memory CD8+ T cells express CD103 during subsequent Ag stimulations at concentrations of TGF-beta1 significantly lower than concentrations required by memory T cells primed in the absence of TGF-beta1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Patients and samples

Blood, colon tumor, MLN, and normal colonic tissues were obtained from patients undergoing surgery for primary colorectal carcinoma (n = 20, mean age 71.3 years, range 50–76 years; 9 females and 11 males). Blood was collected from patients before surgery. Normal colonic tissue was obtained from a macroscopically normal part of the excised colon, at least 10 cm from the tumor margin. None of patients had previously received radiotherapy or chemotherapy. Blood samples from six normal donors were used as controls. The study was approved by the Central Oxford Region Ethics Committee.

Immunohistochemistry

Immunohistochemistry was performed on frozen sections. The sections were fixed in acetone, washed in PBS, and blocked with goat serum before 50 µl of primary Ab (diluted to 5 µg/ml) were added for 30 min at room temperature. The sections were then washed in PBS and incubated with peroxidase-conjugated goat-anti-mouse IgG (Sigma-Aldrich) for 30 min. Bound peroxidase activity was developed using an 3-amino-9-ethylcarbazole kit (Vector Laboratories). The sections were counterstained in hematoxylin.

Abs and tetramers

The following Abs were used to stain ex vivo single-cell suspension or cell lines and in immunohistochemistry analysis: CD3-allophycocyanin (clone UCHT1), CD8-PerCP (SK1), CD8-allophycocyanin (RPA-T8), beta7 integrin-R-PE (FIB504), CD103 integrin-PE (Ber-ACT8), CD45RO-FITC (UCHL1), and CD45RA-R-PE (HI100) were produced by BD Pharmingen. CD103 integrin-FITC (LF61) and CD49d integrin-FITC (44H6) were produced by Serotec.

Tetrameric HLA-A*0201/peptide complexes were synthesized as described previously (23, 24) with EBV BMLF1259–267 (25) and Melan A26–35. Cells were stained with tetramers for 20 min at 37°C and then washed in PBS/1% FCS at 37°C, before incubating with anti-CD103-FITC and anti-CD8-allophycocyanin for 30 min on ice. Cells were washed twice in ice-cold PBS/1% FCS.

Flow cytometric analysis was done on a FACSCalibur (BD Biosciences), and data were analyzed using the CellQuest software (BD Biosciences). Lymphocytes were gated according to forward light scatter/side scatter profile, and dead cells were excluded by staining with propidium iodide (Sigma-Aldrich).

Lymphocyte extraction and culture

PBL were isolated by Ficoll density gradient centrifugation. Freshly isolated tissue was collected in wash medium (RPMI 1640 medium supplemented with penicillin/streptomycin). The tissue was washed four times with wash medium before being cut into small pieces using blades. The pieces were digested in 2% FCS RPMI 1640 medium containing 1 mg/ml type IV collagenase (Worthington Biochemical) for 2 h at room temperature. Half of the sample was used for ex vivo FACS staining, and the other half was put into culture medium (RPMI 1640 medium supplemented with 2 mM L-glutamine, penicillin/streptomycin and 5% pooled human serum AB+) supplemented with recombinant human IL-2 (rhIL-2) 150 U/ml. The lymphocyte yield was 0.4–0.8 x 106 cells and 0.75–3.5 x 106 cells per gram of tissue from normal colon and from colon cancer, respectively. After 5–7 days, they were stimulated with purified phytohemagglutinin 20 µg/ml (Murex) and irradiated (50 Gy) allogeneic feeder cells. Tumor-infiltrating lymphocyte (TIL) cell lines were fed with 150 UI/ml rhIL-2 twice a week.

Mixed lymphocyte tumor culture (MLTC) was performed as follows: autologous tumor cells were irradiated (150 Gy) and cultured with PBL or TIL at a ratio of 1 tumor cell to 10 lymphocytes in RPMI 1640 medium supplemented with 5% human serum and IL-2 (150 U/ml). New batches of irradiated tumor cells were added to the lymphocyte culture at 2-wk intervals.

CD8+ T cell clones, specific for HLA-A*0201-binding EBV BMLF1259–267 (2G7) and Her2/neu369–377 (3F6) peptides, were generated from normal donors as described previously (23) and were maintained in medium supplemented with IL-2 (150 U/ml). In some of the experiments on CD103 expression, 2.5 x 105 T cells were pulsed with the peptide of interest at 1 µM for 1 h. The peptide was then washed off and the cells added to 106 unpulsed T cells of the same clone. In other experiments, T cells were stimulated with plate-bound anti-CD3 (OKT3, 10 µg/ml). These experiments were done in the presence or absence of rhTGF-beta1 (2 ng/ml; R&D Systems).

51Cr release cytotoxicity assay

Effector CTL are coincubated with 51Cr-loaded autologous tumor or EBV transformed B cells over 5 h. Maximally lysed cells (in 10% Triton X-100) and 51Cr-loaded target cells without effectors (spontaneous release of 51Cr) acted as reference points. Specific lysis of targets was calculated as follows: (sample – spontaneous release/maximum – spontaneous release) %.

ELISPOT assays

ELISPOT assays were performed as described previously (26). Spots were counted using an automated ELISPOT reader (Autoimmun-Diagnostika). Results were calculated by subtracting the mean background spot count from the mean spot count for each peptide or tumor cell line. Blocking assays were done with target cells that had been incubated with an Ab against MHC class I (w6/32, 30 µg/ml; American Type Culture Collection).

TGF-beta1 measurement by ELISA

TGF-beta1 levels were assayed using the TGF-beta1 Emax Immunoassay kit (Promega) according to the manufacturer’s recommendations. Samples were obtained from 48-h culture supernatants of 105 tumor cells. Levels of total and active TGF-beta1 were determined using a previously described protocol (27).

Generation of monocyte-derived DC and T cell priming

Monocytes were isolated from PBL from a healthy HLA-A2+ donor by positive sorting using anti-CD14-conjugated magnetic beads (Miltenyi Biotec). These were cultured in RPMI 1640 medium 10% FCS supplemented with 1000 U/ml IL-4 and 50 ng/ml GM-CSF (Leucomax; Norvatis) for 5 days. Cells were matured with 1 µg/ml LPS (Salmonella abortus equi; Sigma-Aldrich) over 36 h. DC were determined to be mature by showing an up-regulation of CD80, CD83, and CD86 expression by FACS analysis.

Mature DC were pulsed with Melan A26–35 ELAGIGILTV peptide at 1 µM for 2 h at 37°C in serum-free medium. The peptide was then washed off, and the DC were incubated with autologous PBL at a 1:10 ratio for 12 days in RPMI 1640 medium 5% human serum. No IL-2 was added for the first 4 days. IL-2 was then used at 20 U/ml for 5 days then at 500 U/ml for the last 3 days. Priming took place in the absence or presence of TGF-beta1 2 ng/ml and all-trans-retinoic acid 10 nM (Sigma-Aldrich).

Recall assay

PBL that had been primed previously with Melan A26–35 and rested for 4 wk were used. A total of 0.5 x 106 T cells was stimulated with Melan A26–35 (1 µM) for 7 or 10 days. Cells from the same line that were not stimulated with peptide were used as controls. Cells were fed with 150 IU/ml rhIL-2 twice a week. Cultures with TGF-beta1 were fed with the cytokine every other day at concentrations of 2, 0.5, and 0.2 ng/ml.

Intracellular cytokine staining

Intracellular cytokine staining for IFN-{gamma} was done as described previously (28). Briefly, lymphocytes were stimulated with 20 µM peptide for a total of 6 h. Brefeldin A was added in the second hour of stimulation at a final concentration of 5 µg/ml (Sigma-Aldrich). TGF-beta1 at 2 ng/ml was added in some assays. The lymphocytes were washed and stained for CD8 and CD103 before they were fixed with 2% paraformaldehyde and permeabilized using FACS permeabilizing solution (BD Pharmingen). IFN-{gamma} was detected using FITC-conjugated IFN-{gamma} Ab (BD Pharmingen). Lymphocytes that had been either stimulated with PMA (10–6 M; Sigma-Aldrich)/ionomycin (1 µg/ml; Sigma-Aldrich) or not stimulated at all were used as positive and negative controls, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Expression of CD103 by colon carcinoma-specific CD8+ T cells

To study the mechanisms that control CD103 expression in colon carcinoma-specific T cells, a human tumor cell line was established from a colon carcinoma patient, (hereafter referred to as OX-CO-1), and has been used to stimulate in vitro autologous PBL. After two rounds of stimulation, we obtained a colon carcinoma-specific T cell line that was capable of killing in vitro the autologous tumor cells and producing IFN-{gamma} (Fig. 1, A and B). As a control, we showed that OX-CO-1-specific T cells failed to recognize autologous B cells (Fig. 1A) and the NK target K562 cells (data not shown). Although a small proportion (1.5%) of the patient’s CD8+ PBL expressed CD103 in ex vivo FACS staining (data not shown), a significant increase of CD103+CD8+ T cells (28%) was observed in the colon carcinoma-specific T cell line (Fig. 1C). Consistent with the observation that a large proportion of OX-CO-1-reactive T cells were CD8 positive, we showed that incubation of OX-CO-1 cells with the anti-MHC class I Ab W6/32 inhibited their recognition by autologous CD103-positive OX-CO-1-specific T cells (data not shown). In contrast, incubation of OX-CO-1 cells with an anti-CD103-blocking Ab failed to inhibit recognition by autologous CD103-positive OX-CO-1-specific T cells (data not shown).


Figure 1
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  		FIGURE 1. Recognition of colon carcinoma line by autologous CD103+ T lymphocytes. A, Killing of the tumor cell line OX-CO-1 by an autologous MLTC line ({diamondsuit}). Autologous EBV-transformed B cell line was used as negative control ({blacksquare}). B, IFN-{gamma} ELISPOT assay demonstrating the ability of the tumor cell line OX-CO-1 to sensitize IFN-{gamma} secretion by autologous MLTC line. Numbers of MLTC cells per well are indicated. The highest E:T ratio was 1:1. C, FACS staining with anti-CD8 and anti-CD103 Abs of the MLTC line. D, T cells were sorted according to the expression of CD103 and ELISPOT assay was performed using CD103+ and CD103– fractions. Target cells are shown.

 
To assess whether CD103 expression segregated with tumor-reactive T cells, CD103+ cells were sorted and tested for their ability to specifically recognize OX-CO-1 (Fig. 1D). The results of these experiments demonstrated a striking correlation between the expression of CD103 and the presence of tumor-specific T cell responses. These results were confirmed using a second tumor line, OX-CO-3 (from a different colon carcinoma patient), and an autologous MLTC derived from TIL (Fig. 2).


Figure 2
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  		FIGURE 2. Tumor-specific response of mixed lymphocyte tumor culture line to OX-CO-3. Ability of a second MLTC derived from TIL stimulated with the autologous tumor line OX-CO-3 to recognize the autologous tumor line. The MLTC line specific for OX-CO-3 was sorted according to the expression of CD103. ELISPOT assay was conducted using CD103+ and CD103– fractions incubated with the autologous tumor line OX-CO-3. Autologous fibroblasts and K562 cells were used as negative controls.

 
Demonstration that expression of CD103 by CD8+ T lymphocytes correlated with their ability to recognize autologous colon carcinoma lines prompted us to assess the expression of CD103 in situ on colon cancer-infiltrating T lymphocytes. Although in mice CD103 can be found on the cell surface of both CD4 and CD8 T cells (8, 29), in human colon carcinoma samples, the majority of CD4+ T cells are CD103 negative (Figs. 3 and 4). We showed that CD8+ T lymphocytes are mainly localized in the tumor epithelium (Fig. 3C) and are CD103 and CD49d/beta7 positive (Fig. 3, E and F). In contrast, CD4+ cells, which are mainly found in the tumor stroma, rather than in the epithelial layer (Fig. 3B), are only CD49d/beta7 positive (Fig. 3F). To further analyze the expression of CD103 by tumor-infiltrating CD4+ and CD8+ T lymphocytes, ex vivo FACS staining was performed on colon carcinoma samples (Fig. 4). These results demonstrated that expression of CD103 is restricted to a proportion of CD8+ T lymphocytes (32.3%) (Fig. 4). These results confirm previously published data (9) and are consistent with the results shown in Fig. 1C.


Figure 3
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  		FIGURE 3. In situ analysis of CD103 expression by TIL. Immunohistochemical analysis of the expression of CD4, CD8, CD45RO, CD49d, and CD103 on TIL within colon carcinoma sections (magnification, x400). NA, necrotic area; LP, lamina propria; TE, tumor epithelium.

 

Figure 4
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  		FIGURE 4. CD103 and CD49d/beta7 expression in colon cancer-infiltrating lymphocytes. Ex vivo FACS analysis of the T cells in a colon carcinoma sample. Cell acquisitions were gated on CD3+CD8– cells or CD3+CD8+ subpopulations (for CD103 or CD49d/beta7 analysis). Percentages of CD8+ cells were calculated on the whole CD3+ population. Percentages of integrin-positive lymphocytes were calculated in the CD3+CD8– or CD3+CD8+ lymphocytes.

 
CD103 expression on T lymphocytes is enhanced by simultaneous TGF-beta1 stimulation and recognition of cognate peptide epitope

It has been shown previously that the effect of TGF-beta1 in enhancing CD103 expression is greater on phytohemagglutinin-stimulated T cells (30, 31). Because OX-CO-1 secretes active TGF-beta1 at a level of 0.17 ng/ml per 105 cells (data not shown), we reasoned that the correlation between CD103 expression and tumor reactivity observed in Fig. 1 may be consistent with simultaneous recognition of the cognate peptide epitope(s) expressed by OX-CO-1 tumor line and TGF-beta1.

To assess the role of TGF-beta1 and Ag recognition either alone or in combination to up-regulate CD103 expression, we set up an in vitro model using a Her2/neu369–377-specific CTL clone (23) (Fig. 5A). Although rhTGF-beta1 alone or cognate peptide alone induced CD103 expression on a small proportion of cells (ranging from 0.23 to 4.5%), simultaneous exposure of the Her2/neu369–377 T cell clone to both TGF-beta1 and Her2/neu369–377 peptide resulted in a significant increase of the proportion of CD103+ cells (86%). Similar results were obtained by culturing the Her2/neu369–377 CTL clone with the cognate peptide in a Transwell separated from the TGF-beta1-secreting OX-CO-1 tumor line (Fig. 5B). This last result demonstrated that the tumor cell line was able to produce enough TGF-beta to promote CD103 expression on T cells activated with their cognate Ag.


Figure 5
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  		FIGURE 5. Proportion of CD103-positive cells is synergistically enhanced by the simultaneous T cell stimulation with TGF-beta1 and cognate peptide epitope. A, The CD8+ CTL clone 3F6 specific for the Her2/neu369–377 epitope KIFGSLAFL was cultured alone (a), with TGF-beta1 at 2 ng/ml (b), pulsed with peptide at 1 µM (c), or was pulsed with peptide and then cultured with TGF-beta1 (d). The percentage of CD103+ cells is indicated in each panel. B, The CTL clone 3F6 was separated from the colon tumor cells line OX-CO-1 by a Transwell. Expression of CD103 was compared in the absence (a) or in the presence (b) of the Her2/neu369–377 peptide. The colon tumor cells acted as a source of TGF-beta1. A diagram of the experimental protocol is shown. C, CD103 expression is triggered in cis by the simultaneous TGF-beta1 secretion and TCR stimulation. Staining with anti-CD103 and HLA-A2/EBV BMLF1259–267 tetramer of EBV BMLF1259–267-specific CTL clone (a–d, g, and h) and OX-CO-1-specific MLTC (e–g and h) is shown. a–d, A CTL clone that recognizes the EBV BMLF1259–267 was cultured alone (a), with irradiated OX-CO-1 (b), which acted as a source of TGF-beta1, pulsed with peptide (c), or was pulsed with peptide and cultured with irradiated OX-CO-1 (d). e and f, The OX-CO-1-specific MLTC was cultured either alone (e) or with irradiated OX-CO-1 (f), which acted as both a source of Ag and of TGF-beta1. g and h, The EBV BMLF1259–267-specific clone, OX-CO-1-specific MLTC, and OX-CO-1 tumor cells were cultured together without (g), or with (h) EBV BMLF1259–267 peptide. Percentage of CD103-positive cells is indicated.

 
To further assess whether TGF-beta1 secreted by the colon carcinoma cells can induce CD103 expression on third-party T cells, we cocultured the OX-CO-1-specific MLTC line with an EBV BMLF1259–267 CTL clone in the presence or absence of OX-CO-1 (Fig. 5C, panel c). A small proportion of EBV BMLF1259–267 T cells express CD103 in the presence of either the cognate peptide alone (Fig. 5C) or OX-CO-1 tumor cells (Fig. 5C, panel b). However, consistent with results shown in Fig. 5A, coincubation of the EBV BMLF1259–267 T cells with OX-CO-1 tumor cells and the cognate peptide epitope resulted in a significant increase of the proportion of CD103+ cells (51%) (Fig. 5C, panel d).

The results of these experiments indicate that bystander secretion of TGF-beta1 by colon carcinoma cells can result in the expression of CD103 by third-party T cells in the presence of antigenic stimulation.

Role of TGF-beta1 in the expression of CD103 during priming of naive CD8+ T cells

It has been demonstrated that the ability of murine T cells to home into the gut is controlled by events during T cell priming (3, 4). It has been shown that in mice, secretion of retinoic acid by MLN-derived DC imprints on T cells the ability to express integrins CD49d/beta7 and the chemokine receptor CCR9, which in turn enable lymphocytes to home to the intestine (21). However, it remains unclear whether up-regulation of CD103 may also be controlled by events during T cell priming.

To assess whether exposure of naive T cells to TGF-beta1 during priming controls CD103 expression, we studied the expression of CD103 on in vitro-primed Ag-specific CD8+ T lymphocytes. We have demonstrated previously that Melan-A26–35-specific CTL precursors with functional and phenotypic markers of a naive population can be expanded from buffy coat of healthy volunteers with mature DC (28), providing the opportunity of characterizing the optimal conditions to expand in vitro tumor-specific CTL.

LPS-matured monocyte-derived DC were used to prime naive Melan A26–35-specific T cells from healthy donors in the presence or absence of TGF-beta1 (Fig. 6). CD103 expression on Melan A26–35-specific T cells was assessed 12 days after priming (Fig. 6A). The results of these experiments demonstrated that priming in the presence of TGF-beta1 ensures the expansion of a larger proportion of CD103-positive cells, compared with the proportion of CD103-positive cells primed in the absence of TGF-beta1 (Fig. 6A). No difference in the percentage of Melan A26–35 tetramer-positive T cells was observed in the cell populations primed in the presence or absence of TGF-beta1 (Fig. 6A), demonstrating that the presence of TGF-beta1 during T cell priming did not affect proliferation of Melan A26–35-specific T cells. Addition of retinoic acid during Melan-A26–35-specific T cell priming, either alone or in combination with TGF-beta1, did not affect CD103 expression on T cells, indicating differences between the mechanisms inducing CD103 expression in human cells and those controlling expression of CD49d/beta7 or CCR9 in mouse T cells (data not shown).


Figure 6
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  		FIGURE 6. Priming of Melan A26–35-specific T cells in the presence of TGF-beta1 enhances the sensitivity of memory Melan A26–35-specific T cells to TGF-beta1-dependent CD103 up-regulation. A, CD103 expression of in vitro-primed Melan A26–35-specific T cells using peptide-pulsed matured DC in the absence (DC only) or in the presence of TGF-beta1 (DC + TGFbeta1) at 2 ng/ml. The dot plots show the percentage of CD8+ T cells that were stained with the Melan A26–35 tetramer. FACS analysis was conducted on day 12. B, In vitro-primed Melan A26–35-specific T cells were then rested for 28 days and CD103 expression monitored at different time points. C, Memory Melan A26–35-specific T cells were stimulated with Melan A26–35 peptide in the presence of different concentrations of TGF-beta1. Percentage of CD8+ T cells at day 7 that were Melan A26–35 tetramer positive is shown. D, The percentage of CD8+ Melan A26–35 tetramer-positive T cells that express CD103. Results from one of three experiments are shown. E, Memory Melan A26–35-specific T cells, primed without TGF-beta1 (DC only) or with TGF-beta1 for 48 h (DC + 48H TGFbeta1) or 12 days (DC + 12 days TGFbeta1) were restimulated with 1 µM peptide in the presence of different concentrations of TGF-beta1. Percentage of CD8+ Melan A26–35 tetramer-positive T cells that express CD103. Results from one of two experiments are shown.

 
Melan A26–35-specific T cells were rested in the absence of exogenous TGF-beta1 until CD103 expression had decreased to background levels (Fig. 6B). Memory T cells were then restimulated with Melan A26–35 peptide in the presence of different concentrations of TGF-beta1 (Fig. 6, C and D). No functional differences in the proliferation (Fig. 6C) and IFN-{gamma} secretion (Fig. 7) of Melan A26–35-specific T cells were observed in the presence or absence of TGF-beta1. However, memory Melan A26–35-specific T cells primed in the presence of TGF-beta1 required ~10-fold lower doses of TGF-beta1 to re-express CD103, compared with the ones required by memory Melan A26–35-specific T cells primed in the absence of TGF-beta1 (Fig. 6D). Time-course experiments confirmed these results, indicating that the presence of TGF-beta1 during priming of naive CD8+ T cells, even for a shorter period of time (Fig. 6E), controls the ability of memory T cells to respond to lower concentrations of TGF-beta1. These results underscore a mechanism that lowers the threshold of TGF-beta1 required for the expression of CD103 and facilitates retention of CD8+ T cells in epithelial layers.


Figure 7
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  		FIGURE 7. Melan A26–35-specific T cells that were primed and restimulated in TGF-beta1 express IFN-{gamma} in intracellular cytokine staining assays. A, Intracellular staining with IFN-{gamma}-specific Ab of Melan A26–35-specific T cells after 10 days restimulation with the Melan A26–35 peptide. The FACS plots shown are those for T cells that had been primed and restimulated without TGF-beta1, and T cells that had been primed in TGF-beta1 for 12 days and restimulated in TGF-beta1 at 2 ng/ml. Percentage of IFN-{gamma} secreted by Melan A26–35-specific T cells in the presence or absence of peptide is shown. B, Percentage of Melan A26–35-specific T cells used for the intracellular staining assay shown in A as measured by A2 tetramers containing the Melan A26–35 peptide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CD103 is expressed by a large proportion of CD8+ IEL, compared with <3% of CD8+ PBL (9). Unlike the integrin CD49d/beta7, which facilitates migration of lymphocytes into the mucosa lamina propria, CD103 is essential for the retention of T cells into the mucosa epithelium layer (19). Our results, demonstrating that TGF-beta1 stimulation and TCR engagement enhance the expression of CD103 on Ag-specific T cells, confirm and extend this model by highlighting a mechanism to retain in the epithelial layer T cells specific for Ags expressed by epithelial cells.

Recent papers in mice have clarified some of the mechanisms involved in the homing of murine T lymphocytes into the small intestinal mucosa after priming in either the MLN or the mucosa-associated lymphoid tissue (3, 4, 21). T cells primed in the alimentary tract express the integrin CD49d/beta7 and CCR9, the receptor for TECK/CCL25 (3, 4). The ability to express CD49d/beta7 and CCR9 on gut-homing lymphocytes is imprinted on T cells by DC via secretion of retinoic acid (21). Although it remains unclear whether retinoic acid is capable of controlling up-regulation of CD103 in mouse T lymphocytes, we did not observe an effect of retinoic acid on CD103 expression during priming of human naive T cells. In contrast, our results indicate that the presence of TGF-beta1 during priming of human Ag-specific CD8+ T cells lowers the threshold of TGF-beta1 required for the up-regulation of CD103 by memory CD8+ T cells. These results indicate that TGF-beta1 in the priming phase of CD8+ T cell responses enhances the ability of T cells to express CD103 during subsequent Ag stimulations. Consistent with this model, T cells specific for gut-derived Ags are primed in tissues which are known to express TGF-beta1, such as epithelial cells of the intestinal mucosa (16, 17), and MLN (32).

The mechanisms controlling the lower threshold of TGF-beta1 required for the expression of CD103 by memory T cells primed in the presence of TGF-beta1 remain unclear. One possibility is that the presence of TGF-beta1 during T cell priming may increase the density of TGF-beta receptors on primed lymphocytes, which would result in an increased sensitivity of these cells to TGF-beta1, when subsequently exposed to lower doses of TGF-beta1. Alternatively, priming in the presence of TGF-beta1 may influence the transcription of both the CD103 and beta7 genes, lowering the threshold of TGF-beta1 required to express these gene products.

Although TGF-beta1 is better known as an immunosuppressive cytokine (18), the observation that TGF-beta1-treated Melan-A26–35-specific T cells are capable of proliferating and releasing IFN-{gamma}, when treated with the cognate peptide, is consistent with previously published results (31, 33, 34). In vitro experiments have shown that the duration and intensity of stimulation (33), the presence of CD28 costimulation (31, 33) and the concentration of TGF-beta1 (31, 33, 34) modulate the effects of TGF-beta1 on lymphocytes. It has also been shown that lymphocytes stimulated in the presence of TGF-beta1 in the primary culture proliferated more efficiently and secreted more abundant IL-2 and IFN-{gamma} when restimulated in the presence of TGF-beta1, as compared with cells which had not previously exposed to TGF-beta1 (31, 32, 33). Furthermore, in mice, TGF-beta1-dependent expression of CD103 on effector T cells is essential for intestinal GVHD (19), and for the destruction of renal and pancreatic allografts (20, 35). T cells from CD103 knockout mice, and T cells from dominant-negative TGFbeta2R-transgenic mice, which are unable to express CD103, are less able to migrate into the epithelial components of intestines, renal allografts, or pancreatic allografts (19, 20). Such CD8+ effector T cells were much less capable of causing intestinal GVHD, and were not able to reject renal and pancreatic allografts. These results are consistent with a model in which T cells activated in the presence of TGF-beta1 retain some of their properties and acquire the ability to express CD103.

In conclusion, we have analyzed the expression of CD103 on human colon cancer-specific T cells and described the importance for the up-regulation of CD103 of both TGF-beta1 and TCR stimulation of Ag-specific T cells. The observation that CD8+ T cell priming in the presence of TGF-beta1 modulates the ability of CD8+ T cells to up-regulate CD103 is of importance to provide important insights into the mechanisms which control T cell retention in the intestinal epithelium and to optimize the design of vaccination strategies capable of expanding tumor-specific T cells capable of homing into colon carcinoma.


   Acknowledgment
 
We thank Dawn Shepherd for technical support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 This work was supported by the Cancer Research UK (Programme Grant C399), Medical Research Council and the U.S. Cancer Research Institute. K.-L.L. received a fellowship from the Singapore National Medical Research Council. Back

2 K.-L.L. and N.D. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Vincenzo Cerundolo, Weatherall Institute of Molecular Medicine, Oxford, OX3 9DS, U.K. E-mail address: vincenzo.cerundolo{at}molecular-medicine.oxford.ac.uk Back

4 Abbreviations used in this paper: DC, dendritic cell; GVDH, graft-vs-host disease; MLN, mesenteric lymph node; PBL, peripheral blood lymphocyte; rhIL-2, recombinant human IL-2; MLTC, mixed lymphocyte tumor culture; TIL, tumor-infiltrating lymphocyte. Back

Received for publication October 27, 2006. Accepted for publication December 15, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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