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Expansion of CD8+ T cells with regulatory function after interaction with intestinal epithelial cells
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Expansion of CD8ⴙ T Cells With Regulatory Function After Interaction With Intestinal Epithelial Cells MATTHIEU ALLEZ, JENS BRIMNES, IRIS DOTAN, and LLOYD MAYER Immunobiology Center, Mount Sinai School of Medicine, New York, New York
Background & Aims: Regulatory T cells play a role in the control of immune responses in the intestinal mucosa and their absence may predispose to inflammatory bowel disease (IBD). We have previously shown that T cells activated by intestinal epithelial cells (IECs) are suppressive in function. Our goal was to characterize the phenotype and function of T cells proliferating after interaction with IECs. Methods: Irradiated human IECs, isolated from normal resection specimens, were cultured with carboxy fluorescein succinimidyl ester (CFSE) labeled T cells. Flow cytometric analysis of T cells was performed at days 5–10. CD8ⴙ T cells proliferating in culture with IECs were sorted and added to suppressive assays. Results: The precursor frequency of T cells proliferating in response to IECs ranged from 0.3%– 0.9%. Several subpopulations were shown to proliferate (CD8ⴙCD28ⴚ/CD8ⴙCD28ⴙ/CD4ⴙCD25ⴙ), but one population (CD8ⴙCD28ⴚCD101ⴙCD103ⴙ) appeared to be dependent on contact with the CD8 ligand gp180. After sorting, culture in the presence of interleukin (IL)-7 and IL-15 allowed for the generation of cell lines. IECactivated CD8ⴙ T cells, but not nonactivated CD8ⴙ T cells, were suppressive in function. Suppression belonged to the CD101ⴙCD103ⴙ subset of IEC-activated CD8ⴙ T cells and appeared to require cell contact. CD8ⴙ lamina propria T cells also showed suppressive function, suggesting the presence of CD8ⴙ regulatory T cells in the mucosa. Conclusions: IECs are able to induce the proliferation of a small fraction of CD8ⴙ peripheral T cells. The CD8ⴙCD28ⴚ subset of IEC-activated CD8ⴙ T cells, which express CD101 and CD103, interacts with IECs through gp180 and has regulatory function.
he mucosal immune system in the intestine is characterized by chronic low-grade inflammation that appears to relate to the presence of luminal bacteria.1 The level of inflammation is relatively constant and controlled, and when pathogenic bacteria or viruses induce gastroenteritis or colitis, there is a rapid return to the level of inflammation seen before the insult once the organism is cleared. The situation is clearly different in inflammatory bowel disease (IBD). Inflammation is brought to a higher level and may persist for several
T
weeks to months. Even in the remission phase of the disease inflammation is at a higher level than in normal controls, with a slight increase in activated lymphocytes in the lamina propria. The mechanisms responsible for controlled inflammation remain poorly defined and probably involve multiple cell types and pathways. There is accumulating evidence that regulatory T cells play a dominant role in the control of the intestinal immune response, particularly in animal models of IBD.2 Direct evidence for active regulation by a subpopulation of CD4⫹ T cells comes from the finding that colitis induced in severe combined immunodeficient mice by transfer of CD45RBhi CD4⫹ T cells could be prevented by cotransfer of the reciprocal CD45RBlo population.3,4 Other subsets of CD4⫹ regulatory T cells have been described in different systems. TR 1 CD4⫹ regulatory T cells, defined by their ability to produce high levels of interleukin (IL)-10 and transforming growth factor (TGF), have been identified in a human system.5 Such cells might play a role in the protection against colitis in the CD45RBhi–severe combined immunodeficient transfer model. CD4⫹ T helper 3 cells, which produce TGF and a lesser amount of IL-10, have been identified in models of oral tolerance.6,7 More recently, several groups have defined a new subset of CD4⫹ regulatory T cells that coexpress CD25.8 –11 These different subsets of CD4⫹ regulatory T cells suppress immune responses via cell contact (CD4⫹CD25⫹) and/or production of IL-10 and TGF (TR1/Th3 CD4⫹ regulatory). In fact, most of the current literature on the role of regulatory T cells in mucosal immunity is focused on CD4⫹ T cells. However, it has been shown that CD8⫹ regulatory T cells Abbreviations used in this paper: ELISA, enzyme-linked immunosorbent assay; IEC, intestinal epithelial cell; CFSE, carboxy fluorescein succinimidyl ester; IL, interleukin; LP, lamina propria; mAb, monoclonal antibody; MLR, mixed lymphocyte reaction; PBMC, peripheral blood mononuclear cells; PHA, phytohemagglutinin; TCR, T-cell receptor. © 2002 by the American Gastroenterological Association 0016-5085/02/$35.00 doi:10.1053/gast.2002.36588
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play a role in peripheral tolerance and control of autoimmunity both in mice and humans.12–17 Intestinal epithelial cells (IECs) may play a role in the activation and expansion of regulatory T cells. We have reported that IECs can process and present antigen to T cells, inducing CD8⫹ T-cell expansion.18 T cells harvested from IEC:T-cell cocultures were suppressive, suggesting that regulatory subsets were activated by IECs. We have shown that gp180, a CEA family member, which is expressed on IECs and is a CD8 ligand, plays a major role in the expansion of IEC-activated T cells.19,20 IECs from IBD patients are defective in the expression of this molecule and fail to expand CD8⫹ T cells.21 Also, T cells activated by IEC from IBD patients are not suppressive.22 Altogether, these data suggest that normal IECs activate and expand CD8⫹ T cells with regulatory function, and that the lack of activation of this regulatory population may play a role in the pathogenesis of IBD. In this study, we used carboxyfluorescein diacetate succinimidyl ester (CFSE) to analyze and characterize the T-cell proliferation induced by the interaction with IECs. We show that IECs are able to activate and induce the expansion of a small subset of peripheral CD8⫹ T cells. gp180 plays an important role in the activation of a CD8⫹ CD28⫺ subset, which expresses CD101 and CD103 and has suppressive function.
Materials and Methods Preparation of Human Intestinal Epithelial Cells and Lamina Propria Mononuclear Cells Surgical specimens from patients undergoing bowel resection for cancer (at least 10 cm away from tumor) at the Mount Sinai Medical Center were used as a source of IECs. IECs were isolated by a method described previously.18 Resected surgical specimens were washed extensively with phosphate-buffered saline (PBS). The mucosa was stripped off from the submucosa, minced into small pieces, and placed in 1 mmol/L dithiothreitol (Sigma Chemical Co., St. Louis, MO) for 10 minutes at room temperature to remove mucus. The pieces were washed in PBS and incubated twice in dispase (3 mg/mL in RPMI) for 30 minutes at 37°C, vortexed every 5 minutes. The cell suspension was collected and centrifuged on a Percoll (Pharmacia, Piscataway, NJ) density gradient. Enterocytes are located at the 0%–30% layer interface. Cells were washed with PBS and resuspended in serum-free medium (Aim V, 50 u/mL penicillin, 50 g/mL streptomycin, 2 mmol/L glutamine, all from Gibco BRL, Grand Island, NY). The purified IECs were greater than 95% viable and contained less than 2% IELs. Preparations of IECs were irradiated (3000 rads) before culture with T cells. The remaining tissue after dispase treatment was incubated with Collagenase (1 mg/mL) and DNAse I (5 g/mL) for 1–1.5 hours at 37°C with constant
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shaking. Lamina propria mononuclear cells were collected from the supernatant, centrifuged through a 2-layer percoll gradient, and harvested from the 35%– 65% interface.
Isolation of Peripheral Blood T Cells Heparinized venous blood was collected from normal donors, diluted 1:3 with PBS, layered on a Ficoll-hypaque density gradient, and centrifuged for 30 minutes at 1800 rpm. The peripheral blood mononuclear cells (PBMCs) were collected from the interface and washed 3 times with PBS. Cells were resuspended in RPMI and the cell density was adjusted to 5 ⫻ 106 cells/mL. T cells were separated by using a rosetting technique using neuraminidase-treated sheep red blood cells and Ficoll-hypaque density gradient centrifugation. Non–T cells were collected from the interface, washed with PBS, and resuspended in culture medium (Aim V, 50 u/mL penicillin, 50 g/mL streptomycin, 2 mmol/L glutamine). Rosetted T cells were treated with 0.75% ammonium chloride on ice for 5–10 minutes to lyse sheep red blood cells. The T-cell suspension was then washed with PBS. In some experiments, CD8⫹ T cells were negatively selected from blood by using the RosetteSep procedure (StemCell Technologies Inc, Vancouver, British Colombia, Canada) according to the manufacturer’s instructions. In brief, tetrameric antibody complexes directed against glycophorin A on red blood cells and against cell surface markers (CD4, CD16, CD19, CD36, CD56) were added to blood (50 L/mL), and incubated at room temperature. After 20 minutes, samples were diluted with equal volume of PBS/2% fetal bovine serum, layered on Ficollhypaque density gradient, and centrifuged for 20 minutes. The interface was collected, washed with PBS, and resuspended in Aim V. Purity of CD8⫹ T-cell suspensions was greater than 98% with less than 1% CD4⫹ T-cell contamination, as determined by flow cytometric analysis. In some experiments, CD101⫹ and CD103⫹ cells were depleted from the peripheral blood before the culture with IECs. T cells were incubated with anti-CD101 (18F7) and anti-CD103 (28C12), washed, and incubated with goat anti-mouse immunoglobulin (Ig) Dynabeads (Oslo, Norway). Depletion of positive cells was achieved according to the manufacturer’s instructions. This procedure was repeated once. After depletion, less than 0.2% of T cells were CD101⫹ or CD103⫹.
Membrane Labeling of T Cells With Carboxy Fluorescein Succinimidyl Ester T cells were resuspended in PBS at 5 ⫻ 106/mL for staining. CFSE (Molecular Probes, Eugene, OR) in the form of a 5-mmol/L stock solution in dimethyl sulfoxide was added at the final concentration of 1 mmol/L for 10 minutes at 37°C. T cells were then washed twice in RPMI.
Cocultures of Peripheral T Cells and Intestinal Epithelial Cells Mixed cell culture was performed as described previously by using irradiated IECs (3000 rad) as stimulator cells
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and allogeneic T cells as responder cells. CFSE-labeled T cells and IECs were cocultured at 1 ⫻ 106/mL and 0.5 ⫻ 106/mL, respectively, in culture medium at 37°C in a 5% CO2 humidified incubator for 5–10 days. These cells are referred to as IEC-activated T cells. At the same time, as controls, T cells were cultured alone (referred to as nonactivated T cells–negative control) or with allogeneic non–T cells (mixed lymphocyte reaction [MLR]–positive control).
Experiments With Blocking Antibodies In some experiments, IECs were preincubated for 1 hour at 4°C with an anti-gp180 monoclonal antibody (B9) or an IgG1 control (626.1, anti-CD40 antibody was used as an isotype control; CD40 is not expressed on IECs). The suspension of IECs was washed and irradiated before addition to the culture with T cells. Blocking experiments with monoclonal antibody (mAb) B9 were also performed in MLR cultures (control); allogeneic non–T cells were preincubated with mAb B9 or an isotype control as described with IECs.
Flow Cytometric Analysis Labeling of T cells with CFSE was performed before cocultures. After coculture (days 5–10), T cells were resuspended in PBS and incubated for 30 minutes with antibodies. The following antibodies were used: anti-CD3, anti-CD4, and anti-CD8 (conjugated with either PE, PerCP, or APC), antiCD28 (conjugated with either PE or APC), anti-CD94, antiCD56, anti-CD25 (conjugated with PE), and relevant isotype controls. These were purchased from Becton-Dickinson (San Diego, CA). Anti-CD152 (CTLA4) conjugated with PE was purchased from Pharmingen (San Diego, CA). Anti-CD101 (18F7) and anti-CD103 (28C12) were a gift from Drs. Michael Brenner and Gary Russell (Brigham and Womens Hospital, Boston, MA). Four-color analysis was performed by using Cell Quest software (Becton Dickinson).
Lines of Intestinal Epithelial Cell–Activated CD8ⴙ T Cells Sorting of CD3⫹ CD8⫹ cells from IEC:T-cell cocultures (proliferating, CFSE low; vs. nonproliferating, CFSE high) and T cells cultured alone was performed at days 8 –10 by using a Cytomation Moflo (Fort Collins, CO). These sorted populations were stimulated just after sorting, and every 3 weeks, by using phytohemagglutinin (PHA; 1 g/mL), IL-2 (100 u/mL), IL-7 (10 ng/mL), IL-15 (20 ng/mL), and feeder cells (irradiated allogeneic PBMCs) in Aim V medium. Preliminary studies using various cytokine combinations had shown that IL-7 and IL-15 were critically important to the development of long-term lines in these cocultures.
Suppressor Assays We studied the effect of IEC-activated CD8⫹ T cells (lines or sorted from short-term cocultures), nonactivated CD8⫹ T cells (lines or sorted from short-term cocultures), and lamina propria CD3⫹ CD8⫹ and CD3⫹ CD8⫺ T cells on pokeweed-stimulated PBMCs. These different populations
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were sorted by using a Cytomation Moflo. The suppressive function of subsets of IEC-activated CD8⫹ T cells was also studied, particularly CD101⫹/CD103⫹ and CD101⫺/ CD103⫺ IEC-activated CD8⫹ T cells. PBMCs were either unstimulated or stimulated with pokeweed nitrogen (1 g/ mL). IEC-activated or nonactivated CD8⫹ T cells were added to PBMCs before pokeweed mitogen stimulation at a ratio of 1:1 and 1:5 (1 CD8⫹ T cell to 5 PBMCs). Supernatants were harvested at day 7, and IgG secretion was measured by enzyme-linked immunosorbant assay (ELISA). We also studied the effect of IEC-activated and nonactivated CD8⫹ T cells on unrelated MLR cultures (T cells labeled with CFSE and allogeneic irradiated non–T cells cultured in Aim V at a 1:1 ratio). CD8⫹ T cells were added to the MLR at a ratio of 1:5 (1 CD8⫹ T cell to 5 T/non–T cells). The proliferation of CD4 cells in MLR was used as a readout. In some experiments, the MLR and CD8⫹ T cells were separated by a transwell (Falcon cell culture insert, 0.4 m, from Becton Dickinson).
Results Intestinal Epithelial Cells Activate Different Subsets of T Cells That Represent Less Than 1% of the Pool of Peripheral Blood T Cells To further study the interaction between IECs and lymphocytes, we used CFSE to assess the proliferation of T cells in IEC:T-cell cocultures. After 5 days of coculture, proliferating T cells (CD3⫹ CFSE low) represented 3.5%–35% of all T cells in more than 10 independent experiments (Figure 1). In contrast, nonactivated T cells (T cells cultured alone) did not divide. Analysis of the CFSE staining showed that the majority of these proliferating cells had undergone more than 5 divisions. By using the calculation described by Lyons,23 we found that only a small proportion of peripheral T cells (range 0.3%– 0.9%) start to divide and proliferate after contact with IECs (Figure 1). Thus, the precursor frequency in PB of responder T cells in these cocultures is less than 1%. Intestinal Epithelial Cells Preferentially Activate CD8ⴙ T Cells Previous studies have shown the preferential expansion of CD8⫹ T cells in IEC:T-cell cocultures, compared with mixed lymphocyte reaction cultures in which a preferential expansion of CD4⫹ T cells is observed. As previously observed, the CD4:CD8 ratio decreases in IEC:T-cell cocultures, and, as expected, this CD8⫹ Tcell expansion correlates with a higher proportion of proliferating cells among CD8⫹ T cells than among CD4⫹ T cells (Figure 2). The higher proliferation of CD8⫹ T cells in IEC:T-cell cocultures contrasts with the
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should be noted that, in all IEC:T-cell cocultures, there was a significant proliferation of CD4⫹ T cells as well; these IEC-activated CD4⫹ T cells were mainly CD28⫹ and CD25⫹ (data not shown). Phenotype of the Intestinal Epithelial Cell Activated CD8ⴙ T Cells Four-color fluorescence-activated cell sorter analysis was performed on CFSE-stained T cells cultured with or without (nonactivated T cells) IECs. The phenotype of the proliferating (CFSE low) and of the nonproliferating (CFSE high) T cells in IEC:T-cell cocultures was studied, and compared with the phenotype of nonactivated T cells (T cells cultured alone). Interestingly, we found that 34% ⫾ 12% (mean ⫾ SE, 5 experiments) of proliferating CD8⫹ T cells (CFSE low) are CD28⫺ (Figure 3, Table 1). This proportion of CD28⫺ cells in proliferating CD8⫹ T cells contrasts with those of nonproliferating CD8⫹ T cells (CFSE high), which are mainly CD28⫹ (Figure 4). IEC-activated CD8⫹ T cells express mainly the ␣ T-cell receptor (TCR); only 1%–2% express the ␥␦ TCR (data not shown).
Figure 1. Quantitative analysis of T-cell proliferation in an IEC:T-cell coculture and calculation of the proportion of the initial population that has responded by dividing. T cells were labeled with CFSE before addition to the culture with irradiated IECs. Cells were stained at day 5 with anti-CD3 PE. The analysis was gated on CD3⫹ cells. (A) The percentage of dividing cells (cells that have undergone more than one division) is shown. The proportion of the original population induced into cell division after contact with IECs can be determined by using the method reported by Lyons.23 (B) The number of events was measured in each cell division cohort peak. To calculate the percentage of original cells from which they arose, the percentage of events in a given cycle, n, is divided by 2n. The number resulting from this division is referred to as the undivided cohort number. The sum of these undivided cohort numbers (from division 2 to more than 6) represents the percentage of cells that started to divide after interaction with IECs (0.6% in this experiment). This is representative of 10 experiments.
higher proliferation of CD4⫹ T cells in a mixed lymphocyte reaction (Figure 2). The proliferation of these CD8⫹ T cells was not dependent on the CD4⫹ T cells; indeed, CD8⫹ T cells were still able to proliferate in IEC:CD8⫹ T-cell cocultures after depletion of CD4 T cells by using the RosetteSep procedure (Figure 3). It
Figure 2. Comparison of proliferation of CD8⫹ vs. CD4⫹ T cells in IEC:T-cell cocultures and in MLRs. Proliferation analysis at day 7 of CFSE-labeled T cells cultured (A) alone, (B) with IECs, and (C) with non–T cells. Cells were stained with anti-CD3 PE and anti-CD8 PerCP. Analysis was gated on CD3⫹ cells. Percentages represent the number of cells in each quadrant. The percentage of CD8⫹ and CD4⫹ T cells that were induced to divide from the initial population was calculated as described in Figure 1. It was 0.9% and 0.2% in the IEC:T-cell coculture and 0.6% and 0.7 % in the MLR culture, respectively. This is representative of 5 independent experiments.
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Figure 3. CD4⫹ T cells are not necessary for the CD8⫹ T-cell proliferation in IEC:T-cell cocultures. CD8⫹ T cells were negatively selected by using the RosetteSep procedure, CFSE labeled, and cultured (A) alone or (B) with IECs. Analysis was gated on lymphocytes by forwardscatter and side-scatter; the percentage of CD8⫹ proliferating T cells is shown. (C) Purity of CD8⫹ T cell suspension is shown. This is representative of 3 independent experiments.
Intestinal Epithelial Cell-Activated CD8ⴙ T Cells Express Mucosal Markers Table 1 shows the frequencies of different markers on the surface of CD8⫹ nonproliferating (CFSEhi) and proliferating (CFSElo) after interaction with IECs. To determine whether the peripheral T cells that expand in culture with IEC share common features with mucosal Table 1. Phenotype of Proliferating and Nonproliferating CD8⫹ T Cells in IEC:T-cell Cocultures
CD 28⫺ CD 101⫹ CD 103⫹ CD94⫹ CD56⫹ CD152⫹
CD8 proliferating CFSE lo
CD8 nonproliferating CFSE hi
34 ⫾ 12% 47 ⫾ 18% 40 ⫾ 16% 13 ⫾ 6% 6.7 ⫾ 2.1% 4.2 ⫾ 2.6%
7 ⫾ 5% 2.4 ⫾ 1.3% 2 ⫾ 1.4% 2.2 ⫾ 1.3% 0.7 ⫾ 0.3% 0.9 ⫾ 0.4%
NOTE. Data collected from 5 experiments.
Figure 4. Expansion of a CD8⫹ CD28⫺ subset in IEC:T-cell cocultures. Analysis at day 7 of CFSE-labeled T cells cultured with IECs. Cells were stained with anti-CD3 PE, anti-CD8 PerCP, and anti-CD28 APC. Analysis was gated on CD3⫹ cells. CD28 expression is shown on CD8⫹ (A) proliferating and (B) nonproliferating cells, and on CD8⫺ (C) proliferating and (D) nonproliferating T cells in an IEC:T-cell coculture. The percentage of CD28⫺ cells is indicated. This is representative of 5 independent experiments.
lymphocytes, we assessed the expression of mucosal markers (CD101 and CD103) on IEC-activated T cells. CD101 and CD103 (␣E7) were expressed on 47% ⫾ 18% and 40% ⫾ 16% of IEC-activated CD8⫹ T cells, respectively (Figure 5, Table 1). The expression of these markers on nonproliferating CD8⫹ T cells in IEC:T-cell cocultures was similar to that of nonactivated CD8⫹ T cells, CD101 and CD103 being present on 1%–5%, and 0.5%–3% of nonactivated T cells, respectively (Table 1). Because subsets of lamina propria lymphocytes and intraepithelial lymphocytes express CD94, we assessed the expression of CD94 on IEC-activated CD8⫹ T cells. CD94 was expressed on a small subset of CD8⫹ proliferating T cells (Table 1). Interestingly, the proliferation of this subset was not inhibited in the presence of mAb B9. CTLA4 was expressed on less than 5% of IECactivated CD8⫹ T cells. The CD4⫹ T cells proliferating after interaction with IECs did not express CD101 and CD103, and were
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pressed when IECs were pretreated with mAb B9, compared with the proliferation of CD8⫹ T cells when IECs were preincubated with control IgG1 (Figure 6A). As expected, the proliferation of CD4⫹ T cells was not decreased (data not shown). The inhibitory effect of B9 on CD8⫹ T-cell proliferation was more evident on the CD8⫹ CD28⫺ subset, particularly those cells expressing CD101 and CD103 (Figure 6B). The proliferation of CD8⫹ CD28⫹ cells was not decreased. As shown in Figure 2, the predominant cell type proliferating in a conventional MLR was a CD4⫹ T cell; these cells mainly expressed CD28 (84% ⫾ 9%, 4 experiments), and 20% ⫾ 3% and 46% ⫾ 14% (4 experiments) expressed CD101 and CD103, respectively. More importantly, the proliferation of CD8⫹ T cells expressing these markers in MLRs was never decreased in the presence of mAb B9 (Figure 6C).
Figure 5. IEC-activated CD8⫹ T cells express CD101 and CD103. Analysis at day 7 of CFSE-labeled T cells cultured with IECs. Cells were incubated with anti-CD101 (18F7) or anti-CD103 (28C12), washed with PBS, stained with PE-conjugated goat anti-mouse antibody, washed and stained with anti-CD8 PerCP and anti-CD3 APC. Analysis was gated on CD3⫹ cells. The figure shows CD101 and CD103 expression of CD8⫹ proliferating (A and B, respectively) and CD8⫹ nonproliferating (C and D, respectively) T cells in IEC:T-cell cocultures. The percentage of CD101⫹ and CD103⫹ cells is indicated. This is representative of 5 independent experiments.
mainly CD25⫹ CD28⫹ (data not shown). Thus, there are at least 3 populations of T cells that proliferate after interaction with IECs: CD8⫹ CD28⫺ (also CD101⫹ and CD103⫹), CD8⫹ CD28⫹ and CD4⫹ CD25⫹ CD28⫹. CD8⫹ CD28⫹ T cells activated in these cocultures may be stimulated by costimulatory molecules other than CD80/86 (not expressed by normal IECs) such as B7h or B7h1. The Monoclonal Antibody Anti-gp180 (B9) Suppresses the Proliferation of the CD8ⴙ CD28ⴚ CD101ⴙ, CD103ⴙ Subset The capacity of IEC to induce CD8⫹ T-cell activation appears to be linked to the binding of CD8 molecules by gp180. It was previously shown that pretreatment of IEC with mAb B9 (anti-gp180) inhibited their capacity to induce T-cell proliferation and p56lck activation in T cells.15 To study whether gp180 is involved in the activation of this subpopulation of IECactivated CD8⫹ T cells, we compared the proliferation of CD8⫹ T cells in culture with IECs pretreated with mAb B9, or with an isotype-matched control IgG1 (626.1). The proliferation of CD8⫹ T cells was sup-
Lines of Intestinal Epithelial Cell Activated CD8ⴙ T Cells To study IEC-activated T cells in more depth, we needed to develop the capacity to generate long-term lines. Lines were derived from IEC-activated CD8⫹ T cells (sorting of CD3⫹ CD8⫹ CFSE low cells in IEC: T-cell cocultures), and expanded by stimulation with PHA, IL-2, IL-7, IL-15, and feeder cells every 3 weeks. Other lines were derived from nonactivated CD8 cells (sorting of CD3⫹ CD8⫹ cells cultured alone). The phenotype of these lines was studied after several weeks of culture. In these long-term cultures, both CD8⫹ T-cell lines (IEC activated and nonactivated) were CD28⫺. Also, there was no difference in CD101 expression, which was present on 30%– 40% of both IECactivated and nonactivated CD8⫹ T-cell lines (data not shown). In contrast, CD103 was highly expressed on IEC-activated CD8⫹ T-cell lines but not by nonactivated CD8⫹ T-cell lines (Figure 7). Intestinal Epithelial Cell-Activated CD8ⴙ T Cells Are Suppressive in Function To study the function of IEC-activated CD8⫹ T cells, we performed different assays (MLR, T-dependent Ig secretion), to which we added variable numbers of IEC-activated or nonactivated CD8⫹ T-cell lines. In contrast to nonactivated CD8⫹ T-cell lines, which were not suppressive, lines of IEC-activated CD8⫹ T cells suppressed T-dependent Ig secretion by B cells (Figure 8A). Lines of IEC-activated CD8⫹ T-cell lines were also able to suppress CD4⫹ T-cell proliferation in an unrelated MLR (Figure 8B). To ensure that this was not an artifact related to the use of cell lines, IEC-activated CD8⫹ T cells were sorted from short-term cultures.
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These suppressed the proliferation of CD4⫹ T cells in MLR (data not shown) as well. In contrast, nonactivated CD8⫹ T cells did not significantly suppress CD4 proliferation. Suppression was not observed when IEC-activated CD8⫹ T cells were separated from the MLR with a transwell (data not shown), suggesting that the suppression mediated by IEC-activated CD8⫹ T cells requires cell contact. To determine whether the suppressive function belongs to the subset of IEC-activated CD8⫹ T cells expressing CD101 and CD103, we sorted both CD101⫹/CD103⫹ and CD101⫺/CD103⫺ CD8⫹ T cells proliferating (CFSElo) in IEC:T-cell cocultures and added them to suppressive assays. In contrast to CD101⫺/CD103⫺ CD8⫹ T cells, CD101⫹/ CD103⫹ IEC-activated CD8⫹ T cells suppressed Ig secretion (Figure 9). However, depletion of CD101⫹/ CD103⫹ cells from peripheral blood T cells before the culture did not decrease the expression of CD101 and CD103 on IEC-activated CD8⫹ T cells (data not shown), suggesting that precursor cells of this subset do not express these markers before the interaction with IECs. To determine whether similar T-cell populations exist in vivo we isolated CD3⫹ CD8⫹ and CD3⫹ CD8⫺ LP cells. In contrast to unfractionated lamina propria (LP) mononuclear cells and CD3⫹ CD8⫺, CD3⫹ CD8⫹ LP T cells were able to suppress Ig secretion by PBMCs stimulated with pokeweed mitogen (Figure 10). Interestingly, preliminary results suggested that CD8⫹ CD28⫺ LP T cells may be more suppressive than CD8⫹ CD28⫹ LP T cells (data not shown).
Discussion By using in vitro IEC:T-cell cocultures, we show that only a small fraction of peripheral T cells respond to IEC. IECs express several antigen-presenting molecules, such as classical class I and II molecules, nonclassical class Ib molecules (CD1d, human leukocyte antigen E,
Figure 6. (A) The proliferation of CD8⫹ T cells is decreased when IECs are preincubated with anti-gp180 mAb B9. IECs were preincubated with mAb B9 or with an isotype control, and washed before addition to the coculture with T cells. Analysis of CFSE-labeled T cells was performed at days 5 and 7. Cells were stained with anti-CD3 PE and anti-CD8 PerCP. Analysis was gated on CD3⫹ CD8⫹ cells; the % of proliferating CD8⫹ T cells is indicated. (B) Preincubation of IECs with anti-gp180 mAb (B9) decreases the proliferation of CD28⫺, CD101⫹, and CD103⫹ CD8⫹ T cells. Cells were prepared at day 7 as in Figure 5A and stained with anti-CD3 PE, anti-CD8 PerCP and anti-CD28 APC, or with anti-CD101 and CD103 and GAM PE Ab, and
Š with anti-CD8 PerCP and anti-CD3 APC. The analysis was performed on proliferating CD8⫹ T cells, with a gate on CD3⫹ CD8⫹ CFSE low cells. The percentage of CD28⫾, CD101⫹, and CD103⫹ T cells among proliferating CD8⫹ T cells in cultures with IECs preincubated with an Ig control or with mAb B9 is shown. These are representative of 4 independent experiments. (C) Preincubation of non–T cells with mAb B9 does not decrease the proliferation of CD8⫹ T cells as well as CD28⫺ CD101⫹ CD103⫹ T cells in MLRs. Cells were prepared at day 7 as in Figures 6A and 6B. The analysis was gated on CD8⫹ T cells; the percentage of CD28⫺/CD28⫹, CD101⫹, and CD103⫹ among CD8⫹ proliferating T cells after preincubation with (A) isotype control or (B) mAb B9 is shown. This is representative of 3 independent experiments.
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Figure 7. Lines of IEC-activated CD8⫹ T cells express CD103. CFSE-labeled T cells were cultured alone or with IECs. CD3⫹ CD8⫹ T cells cultured (A) alone or (B) proliferating in culture with IECs were sorted at day 10 (boxed in area). These CD8 cells were expanded by using IL-2/-7/-15, PHA, and feeder cells, and restimulated every 3 weeks. The phenotype of these CD8⫹ Tcell lines was studied after 3 cycles of stimulation. CD103 expression of (A) nonactivated and (B) IEC-activated CD8⫹ Tcell lines is shown.
Figure 8. IEC-activated CD8⫹ T-cell lines are suppressive. (A) IECactivated and nonactivated CD8⫹ T-cell lines were added to PBMCs stimulated with pokeweed mitogen (1 g/mL) at a ratio of 1:5 (1 CD8⫹ T cell to 5 PBMCs). Ig secretion, measured by ELISA, was significantly reduced in the presence of IEC-activated CD8⫹ T-cell lines. In contrast, nonactivated CD8⫹ T-cell lines had no effect. (B) IEC-activated CD8⫹ T-cell lines were placed directly in the same well as an unrelated MLR (in which T cells were CFSE labeled before culture with irradiated allogeneic non–T cells) at a ratio of 1:5. Proliferation analysis was gated on CD3⫹ CD4⫹ cells. The percentage of CD4 cells that underwent more than 2 divisions is shown. The proliferation of CD4 cells was significantly reduced in the presence of IEC-activated CD8⫹ T-cell lines. This is representative of 4 independent experiments.
MICA/B, FcRn), and costimulatory molecules (gp180, B7h24). As expected, we found that IECs activated different subsets of T cells. Each of them may interact with a different combination of antigen-presenting and costimulatory molecules. The approach used in this study, the use of CFSE, shows the heterogeneity of T cells responding to IECs. The role of specific molecules involved in the interaction between IEC and subsets of T cells can be shown, as seen in this study for the role of gp180 in the activation of a CD8⫹ CD28⫺ subset. However, blocking with mAb B9 was not complete, suggesting that either the blocking was not completely efficient or, more likely, that there are other subsets activated through other pathways. The CD8⫹ CD28⫺ subset of IEC-activated T cells expresses CD101 and CD103, both considered to be mucosal markers. CD101 is expressed on most T lymphocytes in the intestinal mucosa, and only at low levels on a small proportion of peripheral T lymphocytes.25 CD101 has costimulatory function, of special relevance for CD28⫺ cells. CD103, or ␣E7 integrin, is expressed on greater than 90% of CD8⫹ lamina propria and intraepithelial lymphocytes.26 This integrin mediates T-cell adhesion to epithelial cells via its interaction with E-cadherin,27 but is also implicated in gut-homing of T-cell populations via the intestinal endothelium.28 CD103 is expressed on a small population of circulating, memory CD8⫹ T cells. This small population of circulating CD8⫹ T cells is dramatically expanded in cocultures with IECs. One could propose that CD103 is expressed on IEC-activated CD8⫹ cells as a result of nonspecific activation.29,30 However, as shown in Figure 6, lines of CD8⫹ T cells
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Figure 9. IEC-activated CD8⫹ T cells that express CD101 and/or CD103, in contrast to CD101⫺/CD103⫺ IEC-activated CD8⫹ T cells, are suppressive in function. CD101⫹/CD103⫹ and CD101⫺/ CD103⫺ CD8⫹ T cells proliferating (CFSElo) in an IEC:T-cell coculture, as well as nonactivated CD8⫹ T cells, were sorted and added to PBMCs stimulated with pokeweed mitogen (1 g/mL) at a ratio of 1:10 (1 CD8⫹ T cells to 10 PBMCs). Ig secretion, measured by ELISA, was significantly reduced in the presence of IEC-activated CD8⫹ T cells expressing CD101 and/or CD103 (P ⬍ 0.007). In contrast, CD101⫺/CD103⫺ IEC-activated CD8⫹ T cells, as well as nonactivated CD8⫹ T cells, had no significant effect.
cultured alone that had been stimulated for 2 months with PHA, IL-2/-7/-15 and feeder cells did not express CD103. In contrast, lines of IEC-activated CD8⫹ T cells, which were expanded in parallel under the same conditions, highly expressed CD103. The only difference between these lines was that the latter had interacted with IEC. We think that CD103 expression on IECactivated CD8⫹ T cells more likely reflects the expansion of a mucosal lymphocyte lineage from the PB T-cell pool. It has been shown that TGF can induce the expression of CD103.31 In our experiments, CD103 was only increased on proliferating CD8⫹ T cells, but not on nonproliferating CD8⫹ T cells, in IEC:T-cell cultures. Thus, TGF is not likely to be responsible for these findings. The CD8⫹ CD28⫺ subset of IEC-activated T cells appears important for several reasons. First, mucosal markers (CD101 and CD103) are expressed on this subset. Second, as shown by blocking experiments with mAb B9, gp180 plays a major role in the interaction of this subset with IECs. Previous studies from our laboratory highlighted the potential role of this carcinoembryonic antigen family member in the activation of a regulatory subset. The role of gp180 in the activation of CD8⫹ CD28⫺ T cells suggest that it activates regulatory T cells. Furthermore, IEC-activated CD8⫹ T cells expressing CD101 and/or CD103, in contrast to CD101⫺/CD103⫺ IEC-activated CD8⫹ T cells, were suppressive in function. Interestingly, a subset of major histocompatibility complex class I–restricted CD8⫹
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CD28⫺ suppressor T cells, which act on antigen-presenting cells, rendering them tolerogenic to helper T cells, has been described in humans.17 In this study, CD8⫹ CD28⫹ T cells, unlike CD8⫹ CD28⫺ T cells, were not suppressive. The ability of CD8⫹ LP T cells to suppress Ig secretion by PBMCs stimulated with pokeweed mitogen suggests the presence of CD8⫹ with regulatory function in the lamina propria (Figure 10). Preliminary results also suggest that CD8⫹ CD28⫺ LP T cells may be more suppressive than CD8⫹ CD28⫹ LP T cells. Third, we have previously shown the potential role of CD1d in the interaction between IECs and T cells.32,33 We have shown that gp180 and CD1d associate on the surface of IECs and interact with both the CD8 molecule and the T-cell antigen receptor.19,34 Altogether these data suggest that the CD8⫹ CD28⫺ subset of IEC-activated T cells may recognize antigens presented by CD1d. CD1d is a nonpolymorphic major histocompatibility complex class I–like molecule whose expression is mainly localized to the epithelial cells of the gastrointestinal tract.35 CD1d is expressed both on the apical and the basolateral surfaces of IECs and may play an important role in the interaction with mucosal T cells. Based on in vitro studies of the antigens presented by human and mouse CD1d, CD1d expressed on IECs likely presents a glycolipid molecule possibly from the cell wall of bacteria or host cells.35 Our hypothesis is that IECactivated CD8⫹ regulatory T cells, and potentially the CD8⫹ CD28⫺ CD103⫹ subset, recognize Ag presented by CD1d on IECs. We have recently shown a skewing of the TCR repertoire of IEC-activated CD8⫹ T cells, and we have identified a subset of CD8⫹ T cells
Figure 10. LP CD3⫹ CD8⫹ T cells, in contrast to unfractionated and CD3⫹ CD8⫺ LP cells, can suppress Ig secretion by PBMCs stimulated with pokeweed mitogen. LP cells, LP CD3⫹ CD8⫹ and LP CD3⫹ CD8⫺ cells (sorted by using a Cytomation Moflo as described earlier), were added to PBMCs stimulated with pokeweed mitogen (1 g/mL) at a ratio of 1:4 (1 LP cell to 4 PBMCs). Ig secretion, measured by ELISA, was significantly reduced in the presence of CD3⫹ CD8⫹ LP T cells (P ⬍ 0.03). In contrast, unfractionated and CD3⫹ CD8⫺ LP cells had no significant effect.
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defined by specific VB and VA chains that interact with the gp180/CD1d complex (Allez et al., September 2002, unpublished data). Some Ag (such as bacterial products) processed from the lumen by IECs could activate these CD8⫹ regulatory T cells through IEC:T-cell interactions. It has been proposed that some bacterial strains in the intestinal flora play a beneficial role in the maintenance of physiologic, controlled inflammation. One of the potential mechanisms used by these good bacteria could be the activation of CD8⫹ regulatory T cells. Interestingly, depletion of CD101⫹ and CD103⫹ T cells from peripheral blood T cells before the culture did not decrease the expression of these markers on the CD8⫹ T cells proliferating in IEC:T-cell cocultures. We were not able to define the phenotype of the precursor cells that expand after interaction with IECs through gp180; at least they do not seem to express CD101 and CD103 on their surface before this interaction. We had previously been unable to generate T-cell lines from IEC-activated T cells by using cytokines (IL-2, IL-4) and PHA, which allow for CTL or helper T cell clonal outgrowth. The use of IL-7 and IL-15, cytokines produced by IECs allowed us to establish long-term lines that maintained both functional and phenotypic characteristics. These conditions permitted us to initiate studies defining the mechanism of IEC-activated CD8⫹ Tcell mediated suppression and also provided valuable reagents for the study of the TCR repertoire in these cells. Until now, we have failed to identify the mechanism by which these CD8⫹ T cells suppress. Experiments using transwell cultures (IEC-activated CD8 cells in the upper chamber and conventional MLR in the lower chamber) failed to reproduce the suppression seen when IEC-activated CD8⫹ cells were added directly to the cultures (data not shown), and suggest the requirement for cell contact. In preliminary studies, supernatants from normal IEC:T-cell cocultures failed to suppress MLR cultures (unpublished results) and preliminary data on the cytokines produced by these cells suggest that they produce IFN␥ but not TGF or IL-10. These results speak to the requirement for cell contact, and eventually to the role of immunoregulatory molecules on IEC-activated CD8⫹ T cells. Recent studies in mice suggest that CTLA4 may be involved in the suppressive function of regulatory CD4⫹ T cells. Indeed, CTLA4 is expressed on CD4⫹CD25⫹ regulatory T cells, and appears to play an important role in their suppressive function.8 Regulatory T cells that control intestinal inflammation in the mouse (CD4⫹ CD45RBlo) constitutively express CTLA4, and signals through CTLA4 are required for the function of these
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cells in vivo.10 CTLA4, which is weakly expressed on IEC-activated CD8⫹ T cells (Table 1), is probably not implicated in the suppressive effects of these cells. The ability of IEC-activated CD8⫹ T cells to kill various targets has been assessed in initial studies.18 No killing was shown by using K562 (NK killing), anti-CD3 coated P388D1 cells (redirected lysis), IEC lines (HT29, DLD1), and activated T cells (mitogen activated). In light of these data, it is unlikely that IEC-activated CD8⫹ T cells suppress via cytotoxicity. The new approach we used in this study, and our capacity to generate lines of IEC-activated CD8⫹ T cells, should help us to further identify the mechanisms of suppression used by IEC-activated CD8⫹ T cells. In conclusion, we further show in this study that IECs have the capacity to activate and expand CD8⫹ regulatory T cells. IECs interact with a number of T-cell subpopulations but appear to selectively activate a CD8⫹ CD28⫺ subset, which expresses CD101 and CD103 and has regulatory function. The molecule gp180, a CEA family member expressed in association with CD1d on the basolateral surface of IECs, plays a major role in the interaction with this subset. The defective expression of gp180 in IBD, which correlates with the lack of activation of CD8⫹ T cells, suggests that this subset is not activated in the mucosa of IBD patients. Precursor cells of this subset may be present in IBD patients but would be unable to become activated owing to an aberrant microenvironment. Better identification of the suppressor mechanism used by this subset and identification of growth requirements of such cells could open new avenues for therapeutic intervention in IBD patients.
References 1. Brandtzaeg P, Valnes K, Scott H, Rognum TO, Bjerke K, Baklien K. The human gastrointestinal secretory immune system in health and disease. Scand J Gastroenterol 1985;114:17–38. 2. Elson CO, Cong Y, Brandwein S, Weaver CT, McCabe RP, Mahler M, Sundberg JP, Leiter EH. Experimental models to study molecular mechanisms underlying intestinal inflammation. Ann N Y Acad Sci 1998;859:85–95. 3. Powrie F, Leach MW, Mauze S, Caddle LB, Coffman RL. Phenotypically distinct subsets of CD4⫹ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice. Int Immunol 1993;5:1461–1471. 4. Powrie F, Correa-Oliveira R, Mauze S, Coffman RL. Regulatory interactions between CD45RBhigh and CD45RBlow CD4⫹ T cells are important for the balance between protective and pathogenic cell-mediated immunity. J Exp Med 1994;179:589 – 600. 5. Groux H, O’Gara A, Bigler M, Rouleau M, Antonenko S, de Vries JE, Roncarolo MG. A CD4⫹ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 1997;389:737– 742. 6. Hafler DA, Kent SC, Pietrusewicz MJ, Khoury SJ, Weiner HL, Fukaura H. Oral administration of myelin induces antigen-specific
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TGF- 1 secreting T cells in patients with multiple sclerosis. Ann N Y Acad Sci 1997;835:120 –131. Inobe J, Slavin AJ, Komagata Y, Chen Y, Liu L, Weiner HL. IL-4 is a differentiation factor for transforming growth factor- secreting Th3 cells and oral administration of IL-4 enhances oral tolerance in experimental allergic encephalomyelitis. Eur J Immunol 1998; 28:2780 –2790. Takahashi T, Tagami T, Yamazaki S, Uede T, Shimizu J, Sakaguchi N, Mak TW, Sakaguchi S. Immunologic self-tolerance maintained by CD25(⫹)CD4(⫹) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med 2000;192:303–310. Thornton AM, Shevach EM. CD4⫹CD25⫹ immunoregulatory T cells suppress polyclonal activation in vitro by inhibiting interleukin 2 production. J Exp Med 1998;188:287–296. Read S, Malmstrom V, Powrie F. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(⫹)CD4(⫹) regulatory cells that control intestinal inflammation. J Exp Med 2000;192:295–302. Zhang X, Izikson L, Liu L, Weiner HL. Activation of CD25(⫹)CD4(⫹) regulatory T cells by oral antigen administration. J Immunol 2001;167:4245– 4253. Jiang H, Zhang SI, Pernis B. Role of CD8⫹ T cells in murine experimental allergic encephalomyelitis. Science 1992;256: 1213–1215. Tada Y, Ho A, Koh DR, Malk TW. Collagen-induced arthritis in CD4- or CD8-deficient mice: CD8⫹ T cells play a role in initiation and regulate recovery phase of collagen-induced arthritis. J Immunol 1996;156:4520 – 4526. Zhang L, Shannon J, Sheldon J, The HS, Mak TW, Miller RG. Role of infused CD8⫹ cells in the induction of peripheral tolerance. J Immunol 1994;152:2222–2228. Balashov KE, Khoury SJ, Hafler DA, Weiner HL. Inhibition of T cell responses by activated human CD8⫹ T cells is mediated by interferon-␥ and is defective in chronic progressive multiple sclerosis. J Clin Invest 1995;95:2711. Filaci G, Bacilieri S, Fravega M, Monetti M, Contini P, Ghio M, Setti M, Puppo F, Indiveri F. Impairment of CD8⫹ T suppressor cell function in patients with active systemic lupus erythematous. J Immunol 2001;166:6452– 6457. Liu Z, Tugulea S, Cortesini R, Suciu-Foca N. Specific suppression of T helper alloreactivity by allo-MHC class I-restricted CD8⫹ CD28- T cells. Int Immunol 1998;10:775–783. Mayer L, Shlien R. Evidence for function of Ia molecules on gut epithelial cells in man. J Exp Med 1987;166:1471–1483. Li Y, Yio XY, Mayer L. Human intestinal epithelial cell-induced CD8⫹ T cell activation is mediated through CD8 and the activation of CD8-associated p56lck. J Exp Med 1995;182:1079 – 1088. Yio XY, Mayer L. Characterization of a 180-kDa intestinal epithelial cell membrane glycoprotein, gp180. A candidate molecule mediating T cell-epithelial cell interactions. J Biol Chem 1997; 272:12786 –12792. Toy L, Yio XY, Lin A, Honig S, Mayer L. Defective expression of gp180, a novel CD8 ligand on intestinal epithelial cells, in inflammatory bowel disease. J Clin Invest 1997;100:2062–2071. Mayer L, Eisenhardt D. Lack of induction of suppressor T cells by intestinal epithelial cells from patients with inflammatory bowel disease. J Clin Invest 1990;86:1255–1260. Lyons AB. Analysing cell division in vivo and in vitro using flow
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cytometric measurement of CFSE dye dilution. J Immunol Methods 2000;243:147–154. Beuria P, Panja A, Kraus T, Perera LP, Sperber K. Human intestinal epithelial cells constitutively express the B7h costimulatory molecule (abstr). Gastroenterology 2001;120:A1643. Russel GJ, Parker CM, Arvind S, Mizoguchi E, Ebert E, Bhan AK, Brenner MB. P126 (CDw101), a costimulatory molecule preferentially expressed on mucosal T lymphocytes. J Immunol 1996; 157:3366 –3374. Farstad IN, Halstensen TS, Lien B, Kilshaw PJ, Lazarovits AI. Distribution of beta 7 integrins in human intestinal mucosa and organized gut-associated lymphoid tissue. Immunology 1996;89: 227–237. Cepek KL, Shaw SK, Parker CM, Russell GJ, Morrow JS, Rimm DL, Brenner MB. Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and alpha E beta 7 integrin. Nature 1994;372:190 –193. Strauch UG, Mueller RC, Li XY, Cernadas M, Higgins JM, Binion DG, Parker CM. Integrin alpha E (CD103) beta 7 mediates adhesion to intestinal microvascular endothelial cell lines via an Ecadherin-independent interaction. J Immunol 2001;166:3506 – 3514. Brew R, West DC, Burthem J, Christmas SE. Expression of the human mucosal lymphocyte antigen, HML-1, by T cells activated with mitogen or specific antigen in vitro. Scand J Immunol 1995; 41:553–562. Schieferdecker HL, Ullrich R, Weiss-Breckwoldt AN, Schwarting R, Stein H, Riecken EO, Zeitz M. The HML-1 antigen of intestinal lymphocytes is an activation antigen. J Immunol 1990;144: 2541–2549. Robinson PW, Green SJ, Carter C, Coadwell J, Kilshaw PJ. Studies on transcriptional regulation of the mucosal T-cell integrin ␣E7 (CD103). Immunology 2001;103:146 –154. Panja A, Barone A, Mayer L. Stimulation of lamina propria lymphocytes by intestinal epithelial cells: evidence for recognition of nonclassical restriction elements. J Exp Med 1994;179:943– 950. Panja A, Blumberg RS, Balk SP, Mayer L. CD1d is involved in T cell-intestinal epithelial cell interactions. J Exp Med 1993;178: 1115–1119. Campbell NA, Kim HS, Blumberg RS, Mayer L. The nonclassical class i molecule CD1d associates with the novel CD8 ligand gp180 on intestinal epithelial cells. J Biol Chem 1999;274: 26259 –26265. Blumberg RS, Colgan SP, Balk SP. CD1d: outside-in antigen presentation in the intestinal epithelium? [editorial, comment] Clin Exp Immunol 1997;109:223–225.
Received December 18, 2001. Accepted July 18, 2002. Address requests for reprints to: Matthieu Allez, M.D., Immunobiology Center, Mount Sinai School of Medicine, 1425 Madison Avenue, Room 11-20, New York, New York 10029. e-mail: [email protected]; fax: (212) 987-5593. Supported by National Institutes of Health grants AI 23504, AI 24671, AI 44236, a Crohn’s and Colitis Foundation of America fellowship award (to M.A. and I.D.), Socie ´te ´ Nationale Franc¸aise de GastroEnte ´rologie (to M.A.), Institut de recherche des maladies de l’appareil digestif (to M.A.), Laboratoire Glaxo-Wellcome (to M.A.), and the Danish medical research agency (to J.B.).
Expansion of CD8ⴙ T Cells With Regulatory Function After Interaction With Intestinal Epithelial Cells MATTHIEU ALLEZ, JENS BRIMNES, IRIS DOTAN, and LLOYD MAYER Immunobiology Center, Mount Sinai School of Medicine, New York, New York
Background & Aims: Regulatory T cells play a role in the control of immune responses in the intestinal mucosa and their absence may predispose to inflammatory bowel disease (IBD). We have previously shown that T cells activated by intestinal epithelial cells (IECs) are suppressive in function. Our goal was to characterize the phenotype and function of T cells proliferating after interaction with IECs. Methods: Irradiated human IECs, isolated from normal resection specimens, were cultured with carboxy fluorescein succinimidyl ester (CFSE) labeled T cells. Flow cytometric analysis of T cells was performed at days 5–10. CD8ⴙ T cells proliferating in culture with IECs were sorted and added to suppressive assays. Results: The precursor frequency of T cells proliferating in response to IECs ranged from 0.3%– 0.9%. Several subpopulations were shown to proliferate (CD8ⴙCD28ⴚ/CD8ⴙCD28ⴙ/CD4ⴙCD25ⴙ), but one population (CD8ⴙCD28ⴚCD101ⴙCD103ⴙ) appeared to be dependent on contact with the CD8 ligand gp180. After sorting, culture in the presence of interleukin (IL)-7 and IL-15 allowed for the generation of cell lines. IECactivated CD8ⴙ T cells, but not nonactivated CD8ⴙ T cells, were suppressive in function. Suppression belonged to the CD101ⴙCD103ⴙ subset of IEC-activated CD8ⴙ T cells and appeared to require cell contact. CD8ⴙ lamina propria T cells also showed suppressive function, suggesting the presence of CD8ⴙ regulatory T cells in the mucosa. Conclusions: IECs are able to induce the proliferation of a small fraction of CD8ⴙ peripheral T cells. The CD8ⴙCD28ⴚ subset of IEC-activated CD8ⴙ T cells, which express CD101 and CD103, interacts with IECs through gp180 and has regulatory function.
he mucosal immune system in the intestine is characterized by chronic low-grade inflammation that appears to relate to the presence of luminal bacteria.1 The level of inflammation is relatively constant and controlled, and when pathogenic bacteria or viruses induce gastroenteritis or colitis, there is a rapid return to the level of inflammation seen before the insult once the organism is cleared. The situation is clearly different in inflammatory bowel disease (IBD). Inflammation is brought to a higher level and may persist for several
T
weeks to months. Even in the remission phase of the disease inflammation is at a higher level than in normal controls, with a slight increase in activated lymphocytes in the lamina propria. The mechanisms responsible for controlled inflammation remain poorly defined and probably involve multiple cell types and pathways. There is accumulating evidence that regulatory T cells play a dominant role in the control of the intestinal immune response, particularly in animal models of IBD.2 Direct evidence for active regulation by a subpopulation of CD4⫹ T cells comes from the finding that colitis induced in severe combined immunodeficient mice by transfer of CD45RBhi CD4⫹ T cells could be prevented by cotransfer of the reciprocal CD45RBlo population.3,4 Other subsets of CD4⫹ regulatory T cells have been described in different systems. TR 1 CD4⫹ regulatory T cells, defined by their ability to produce high levels of interleukin (IL)-10 and transforming growth factor (TGF), have been identified in a human system.5 Such cells might play a role in the protection against colitis in the CD45RBhi–severe combined immunodeficient transfer model. CD4⫹ T helper 3 cells, which produce TGF and a lesser amount of IL-10, have been identified in models of oral tolerance.6,7 More recently, several groups have defined a new subset of CD4⫹ regulatory T cells that coexpress CD25.8 –11 These different subsets of CD4⫹ regulatory T cells suppress immune responses via cell contact (CD4⫹CD25⫹) and/or production of IL-10 and TGF (TR1/Th3 CD4⫹ regulatory). In fact, most of the current literature on the role of regulatory T cells in mucosal immunity is focused on CD4⫹ T cells. However, it has been shown that CD8⫹ regulatory T cells Abbreviations used in this paper: ELISA, enzyme-linked immunosorbent assay; IEC, intestinal epithelial cell; CFSE, carboxy fluorescein succinimidyl ester; IL, interleukin; LP, lamina propria; mAb, monoclonal antibody; MLR, mixed lymphocyte reaction; PBMC, peripheral blood mononuclear cells; PHA, phytohemagglutinin; TCR, T-cell receptor. © 2002 by the American Gastroenterological Association 0016-5085/02/$35.00 doi:10.1053/gast.2002.36588
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play a role in peripheral tolerance and control of autoimmunity both in mice and humans.12–17 Intestinal epithelial cells (IECs) may play a role in the activation and expansion of regulatory T cells. We have reported that IECs can process and present antigen to T cells, inducing CD8⫹ T-cell expansion.18 T cells harvested from IEC:T-cell cocultures were suppressive, suggesting that regulatory subsets were activated by IECs. We have shown that gp180, a CEA family member, which is expressed on IECs and is a CD8 ligand, plays a major role in the expansion of IEC-activated T cells.19,20 IECs from IBD patients are defective in the expression of this molecule and fail to expand CD8⫹ T cells.21 Also, T cells activated by IEC from IBD patients are not suppressive.22 Altogether, these data suggest that normal IECs activate and expand CD8⫹ T cells with regulatory function, and that the lack of activation of this regulatory population may play a role in the pathogenesis of IBD. In this study, we used carboxyfluorescein diacetate succinimidyl ester (CFSE) to analyze and characterize the T-cell proliferation induced by the interaction with IECs. We show that IECs are able to activate and induce the expansion of a small subset of peripheral CD8⫹ T cells. gp180 plays an important role in the activation of a CD8⫹ CD28⫺ subset, which expresses CD101 and CD103 and has suppressive function.
Materials and Methods Preparation of Human Intestinal Epithelial Cells and Lamina Propria Mononuclear Cells Surgical specimens from patients undergoing bowel resection for cancer (at least 10 cm away from tumor) at the Mount Sinai Medical Center were used as a source of IECs. IECs were isolated by a method described previously.18 Resected surgical specimens were washed extensively with phosphate-buffered saline (PBS). The mucosa was stripped off from the submucosa, minced into small pieces, and placed in 1 mmol/L dithiothreitol (Sigma Chemical Co., St. Louis, MO) for 10 minutes at room temperature to remove mucus. The pieces were washed in PBS and incubated twice in dispase (3 mg/mL in RPMI) for 30 minutes at 37°C, vortexed every 5 minutes. The cell suspension was collected and centrifuged on a Percoll (Pharmacia, Piscataway, NJ) density gradient. Enterocytes are located at the 0%–30% layer interface. Cells were washed with PBS and resuspended in serum-free medium (Aim V, 50 u/mL penicillin, 50 g/mL streptomycin, 2 mmol/L glutamine, all from Gibco BRL, Grand Island, NY). The purified IECs were greater than 95% viable and contained less than 2% IELs. Preparations of IECs were irradiated (3000 rads) before culture with T cells. The remaining tissue after dispase treatment was incubated with Collagenase (1 mg/mL) and DNAse I (5 g/mL) for 1–1.5 hours at 37°C with constant
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shaking. Lamina propria mononuclear cells were collected from the supernatant, centrifuged through a 2-layer percoll gradient, and harvested from the 35%– 65% interface.
Isolation of Peripheral Blood T Cells Heparinized venous blood was collected from normal donors, diluted 1:3 with PBS, layered on a Ficoll-hypaque density gradient, and centrifuged for 30 minutes at 1800 rpm. The peripheral blood mononuclear cells (PBMCs) were collected from the interface and washed 3 times with PBS. Cells were resuspended in RPMI and the cell density was adjusted to 5 ⫻ 106 cells/mL. T cells were separated by using a rosetting technique using neuraminidase-treated sheep red blood cells and Ficoll-hypaque density gradient centrifugation. Non–T cells were collected from the interface, washed with PBS, and resuspended in culture medium (Aim V, 50 u/mL penicillin, 50 g/mL streptomycin, 2 mmol/L glutamine). Rosetted T cells were treated with 0.75% ammonium chloride on ice for 5–10 minutes to lyse sheep red blood cells. The T-cell suspension was then washed with PBS. In some experiments, CD8⫹ T cells were negatively selected from blood by using the RosetteSep procedure (StemCell Technologies Inc, Vancouver, British Colombia, Canada) according to the manufacturer’s instructions. In brief, tetrameric antibody complexes directed against glycophorin A on red blood cells and against cell surface markers (CD4, CD16, CD19, CD36, CD56) were added to blood (50 L/mL), and incubated at room temperature. After 20 minutes, samples were diluted with equal volume of PBS/2% fetal bovine serum, layered on Ficollhypaque density gradient, and centrifuged for 20 minutes. The interface was collected, washed with PBS, and resuspended in Aim V. Purity of CD8⫹ T-cell suspensions was greater than 98% with less than 1% CD4⫹ T-cell contamination, as determined by flow cytometric analysis. In some experiments, CD101⫹ and CD103⫹ cells were depleted from the peripheral blood before the culture with IECs. T cells were incubated with anti-CD101 (18F7) and anti-CD103 (28C12), washed, and incubated with goat anti-mouse immunoglobulin (Ig) Dynabeads (Oslo, Norway). Depletion of positive cells was achieved according to the manufacturer’s instructions. This procedure was repeated once. After depletion, less than 0.2% of T cells were CD101⫹ or CD103⫹.
Membrane Labeling of T Cells With Carboxy Fluorescein Succinimidyl Ester T cells were resuspended in PBS at 5 ⫻ 106/mL for staining. CFSE (Molecular Probes, Eugene, OR) in the form of a 5-mmol/L stock solution in dimethyl sulfoxide was added at the final concentration of 1 mmol/L for 10 minutes at 37°C. T cells were then washed twice in RPMI.
Cocultures of Peripheral T Cells and Intestinal Epithelial Cells Mixed cell culture was performed as described previously by using irradiated IECs (3000 rad) as stimulator cells
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and allogeneic T cells as responder cells. CFSE-labeled T cells and IECs were cocultured at 1 ⫻ 106/mL and 0.5 ⫻ 106/mL, respectively, in culture medium at 37°C in a 5% CO2 humidified incubator for 5–10 days. These cells are referred to as IEC-activated T cells. At the same time, as controls, T cells were cultured alone (referred to as nonactivated T cells–negative control) or with allogeneic non–T cells (mixed lymphocyte reaction [MLR]–positive control).
Experiments With Blocking Antibodies In some experiments, IECs were preincubated for 1 hour at 4°C with an anti-gp180 monoclonal antibody (B9) or an IgG1 control (626.1, anti-CD40 antibody was used as an isotype control; CD40 is not expressed on IECs). The suspension of IECs was washed and irradiated before addition to the culture with T cells. Blocking experiments with monoclonal antibody (mAb) B9 were also performed in MLR cultures (control); allogeneic non–T cells were preincubated with mAb B9 or an isotype control as described with IECs.
Flow Cytometric Analysis Labeling of T cells with CFSE was performed before cocultures. After coculture (days 5–10), T cells were resuspended in PBS and incubated for 30 minutes with antibodies. The following antibodies were used: anti-CD3, anti-CD4, and anti-CD8 (conjugated with either PE, PerCP, or APC), antiCD28 (conjugated with either PE or APC), anti-CD94, antiCD56, anti-CD25 (conjugated with PE), and relevant isotype controls. These were purchased from Becton-Dickinson (San Diego, CA). Anti-CD152 (CTLA4) conjugated with PE was purchased from Pharmingen (San Diego, CA). Anti-CD101 (18F7) and anti-CD103 (28C12) were a gift from Drs. Michael Brenner and Gary Russell (Brigham and Womens Hospital, Boston, MA). Four-color analysis was performed by using Cell Quest software (Becton Dickinson).
Lines of Intestinal Epithelial Cell–Activated CD8ⴙ T Cells Sorting of CD3⫹ CD8⫹ cells from IEC:T-cell cocultures (proliferating, CFSE low; vs. nonproliferating, CFSE high) and T cells cultured alone was performed at days 8 –10 by using a Cytomation Moflo (Fort Collins, CO). These sorted populations were stimulated just after sorting, and every 3 weeks, by using phytohemagglutinin (PHA; 1 g/mL), IL-2 (100 u/mL), IL-7 (10 ng/mL), IL-15 (20 ng/mL), and feeder cells (irradiated allogeneic PBMCs) in Aim V medium. Preliminary studies using various cytokine combinations had shown that IL-7 and IL-15 were critically important to the development of long-term lines in these cocultures.
Suppressor Assays We studied the effect of IEC-activated CD8⫹ T cells (lines or sorted from short-term cocultures), nonactivated CD8⫹ T cells (lines or sorted from short-term cocultures), and lamina propria CD3⫹ CD8⫹ and CD3⫹ CD8⫺ T cells on pokeweed-stimulated PBMCs. These different populations
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were sorted by using a Cytomation Moflo. The suppressive function of subsets of IEC-activated CD8⫹ T cells was also studied, particularly CD101⫹/CD103⫹ and CD101⫺/ CD103⫺ IEC-activated CD8⫹ T cells. PBMCs were either unstimulated or stimulated with pokeweed nitrogen (1 g/ mL). IEC-activated or nonactivated CD8⫹ T cells were added to PBMCs before pokeweed mitogen stimulation at a ratio of 1:1 and 1:5 (1 CD8⫹ T cell to 5 PBMCs). Supernatants were harvested at day 7, and IgG secretion was measured by enzyme-linked immunosorbant assay (ELISA). We also studied the effect of IEC-activated and nonactivated CD8⫹ T cells on unrelated MLR cultures (T cells labeled with CFSE and allogeneic irradiated non–T cells cultured in Aim V at a 1:1 ratio). CD8⫹ T cells were added to the MLR at a ratio of 1:5 (1 CD8⫹ T cell to 5 T/non–T cells). The proliferation of CD4 cells in MLR was used as a readout. In some experiments, the MLR and CD8⫹ T cells were separated by a transwell (Falcon cell culture insert, 0.4 m, from Becton Dickinson).
Results Intestinal Epithelial Cells Activate Different Subsets of T Cells That Represent Less Than 1% of the Pool of Peripheral Blood T Cells To further study the interaction between IECs and lymphocytes, we used CFSE to assess the proliferation of T cells in IEC:T-cell cocultures. After 5 days of coculture, proliferating T cells (CD3⫹ CFSE low) represented 3.5%–35% of all T cells in more than 10 independent experiments (Figure 1). In contrast, nonactivated T cells (T cells cultured alone) did not divide. Analysis of the CFSE staining showed that the majority of these proliferating cells had undergone more than 5 divisions. By using the calculation described by Lyons,23 we found that only a small proportion of peripheral T cells (range 0.3%– 0.9%) start to divide and proliferate after contact with IECs (Figure 1). Thus, the precursor frequency in PB of responder T cells in these cocultures is less than 1%. Intestinal Epithelial Cells Preferentially Activate CD8ⴙ T Cells Previous studies have shown the preferential expansion of CD8⫹ T cells in IEC:T-cell cocultures, compared with mixed lymphocyte reaction cultures in which a preferential expansion of CD4⫹ T cells is observed. As previously observed, the CD4:CD8 ratio decreases in IEC:T-cell cocultures, and, as expected, this CD8⫹ Tcell expansion correlates with a higher proportion of proliferating cells among CD8⫹ T cells than among CD4⫹ T cells (Figure 2). The higher proliferation of CD8⫹ T cells in IEC:T-cell cocultures contrasts with the
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should be noted that, in all IEC:T-cell cocultures, there was a significant proliferation of CD4⫹ T cells as well; these IEC-activated CD4⫹ T cells were mainly CD28⫹ and CD25⫹ (data not shown). Phenotype of the Intestinal Epithelial Cell Activated CD8ⴙ T Cells Four-color fluorescence-activated cell sorter analysis was performed on CFSE-stained T cells cultured with or without (nonactivated T cells) IECs. The phenotype of the proliferating (CFSE low) and of the nonproliferating (CFSE high) T cells in IEC:T-cell cocultures was studied, and compared with the phenotype of nonactivated T cells (T cells cultured alone). Interestingly, we found that 34% ⫾ 12% (mean ⫾ SE, 5 experiments) of proliferating CD8⫹ T cells (CFSE low) are CD28⫺ (Figure 3, Table 1). This proportion of CD28⫺ cells in proliferating CD8⫹ T cells contrasts with those of nonproliferating CD8⫹ T cells (CFSE high), which are mainly CD28⫹ (Figure 4). IEC-activated CD8⫹ T cells express mainly the ␣ T-cell receptor (TCR); only 1%–2% express the ␥␦ TCR (data not shown).
Figure 1. Quantitative analysis of T-cell proliferation in an IEC:T-cell coculture and calculation of the proportion of the initial population that has responded by dividing. T cells were labeled with CFSE before addition to the culture with irradiated IECs. Cells were stained at day 5 with anti-CD3 PE. The analysis was gated on CD3⫹ cells. (A) The percentage of dividing cells (cells that have undergone more than one division) is shown. The proportion of the original population induced into cell division after contact with IECs can be determined by using the method reported by Lyons.23 (B) The number of events was measured in each cell division cohort peak. To calculate the percentage of original cells from which they arose, the percentage of events in a given cycle, n, is divided by 2n. The number resulting from this division is referred to as the undivided cohort number. The sum of these undivided cohort numbers (from division 2 to more than 6) represents the percentage of cells that started to divide after interaction with IECs (0.6% in this experiment). This is representative of 10 experiments.
higher proliferation of CD4⫹ T cells in a mixed lymphocyte reaction (Figure 2). The proliferation of these CD8⫹ T cells was not dependent on the CD4⫹ T cells; indeed, CD8⫹ T cells were still able to proliferate in IEC:CD8⫹ T-cell cocultures after depletion of CD4 T cells by using the RosetteSep procedure (Figure 3). It
Figure 2. Comparison of proliferation of CD8⫹ vs. CD4⫹ T cells in IEC:T-cell cocultures and in MLRs. Proliferation analysis at day 7 of CFSE-labeled T cells cultured (A) alone, (B) with IECs, and (C) with non–T cells. Cells were stained with anti-CD3 PE and anti-CD8 PerCP. Analysis was gated on CD3⫹ cells. Percentages represent the number of cells in each quadrant. The percentage of CD8⫹ and CD4⫹ T cells that were induced to divide from the initial population was calculated as described in Figure 1. It was 0.9% and 0.2% in the IEC:T-cell coculture and 0.6% and 0.7 % in the MLR culture, respectively. This is representative of 5 independent experiments.
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Figure 3. CD4⫹ T cells are not necessary for the CD8⫹ T-cell proliferation in IEC:T-cell cocultures. CD8⫹ T cells were negatively selected by using the RosetteSep procedure, CFSE labeled, and cultured (A) alone or (B) with IECs. Analysis was gated on lymphocytes by forwardscatter and side-scatter; the percentage of CD8⫹ proliferating T cells is shown. (C) Purity of CD8⫹ T cell suspension is shown. This is representative of 3 independent experiments.
Intestinal Epithelial Cell-Activated CD8ⴙ T Cells Express Mucosal Markers Table 1 shows the frequencies of different markers on the surface of CD8⫹ nonproliferating (CFSEhi) and proliferating (CFSElo) after interaction with IECs. To determine whether the peripheral T cells that expand in culture with IEC share common features with mucosal Table 1. Phenotype of Proliferating and Nonproliferating CD8⫹ T Cells in IEC:T-cell Cocultures
CD 28⫺ CD 101⫹ CD 103⫹ CD94⫹ CD56⫹ CD152⫹
CD8 proliferating CFSE lo
CD8 nonproliferating CFSE hi
34 ⫾ 12% 47 ⫾ 18% 40 ⫾ 16% 13 ⫾ 6% 6.7 ⫾ 2.1% 4.2 ⫾ 2.6%
7 ⫾ 5% 2.4 ⫾ 1.3% 2 ⫾ 1.4% 2.2 ⫾ 1.3% 0.7 ⫾ 0.3% 0.9 ⫾ 0.4%
NOTE. Data collected from 5 experiments.
Figure 4. Expansion of a CD8⫹ CD28⫺ subset in IEC:T-cell cocultures. Analysis at day 7 of CFSE-labeled T cells cultured with IECs. Cells were stained with anti-CD3 PE, anti-CD8 PerCP, and anti-CD28 APC. Analysis was gated on CD3⫹ cells. CD28 expression is shown on CD8⫹ (A) proliferating and (B) nonproliferating cells, and on CD8⫺ (C) proliferating and (D) nonproliferating T cells in an IEC:T-cell coculture. The percentage of CD28⫺ cells is indicated. This is representative of 5 independent experiments.
lymphocytes, we assessed the expression of mucosal markers (CD101 and CD103) on IEC-activated T cells. CD101 and CD103 (␣E7) were expressed on 47% ⫾ 18% and 40% ⫾ 16% of IEC-activated CD8⫹ T cells, respectively (Figure 5, Table 1). The expression of these markers on nonproliferating CD8⫹ T cells in IEC:T-cell cocultures was similar to that of nonactivated CD8⫹ T cells, CD101 and CD103 being present on 1%–5%, and 0.5%–3% of nonactivated T cells, respectively (Table 1). Because subsets of lamina propria lymphocytes and intraepithelial lymphocytes express CD94, we assessed the expression of CD94 on IEC-activated CD8⫹ T cells. CD94 was expressed on a small subset of CD8⫹ proliferating T cells (Table 1). Interestingly, the proliferation of this subset was not inhibited in the presence of mAb B9. CTLA4 was expressed on less than 5% of IECactivated CD8⫹ T cells. The CD4⫹ T cells proliferating after interaction with IECs did not express CD101 and CD103, and were
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pressed when IECs were pretreated with mAb B9, compared with the proliferation of CD8⫹ T cells when IECs were preincubated with control IgG1 (Figure 6A). As expected, the proliferation of CD4⫹ T cells was not decreased (data not shown). The inhibitory effect of B9 on CD8⫹ T-cell proliferation was more evident on the CD8⫹ CD28⫺ subset, particularly those cells expressing CD101 and CD103 (Figure 6B). The proliferation of CD8⫹ CD28⫹ cells was not decreased. As shown in Figure 2, the predominant cell type proliferating in a conventional MLR was a CD4⫹ T cell; these cells mainly expressed CD28 (84% ⫾ 9%, 4 experiments), and 20% ⫾ 3% and 46% ⫾ 14% (4 experiments) expressed CD101 and CD103, respectively. More importantly, the proliferation of CD8⫹ T cells expressing these markers in MLRs was never decreased in the presence of mAb B9 (Figure 6C).
Figure 5. IEC-activated CD8⫹ T cells express CD101 and CD103. Analysis at day 7 of CFSE-labeled T cells cultured with IECs. Cells were incubated with anti-CD101 (18F7) or anti-CD103 (28C12), washed with PBS, stained with PE-conjugated goat anti-mouse antibody, washed and stained with anti-CD8 PerCP and anti-CD3 APC. Analysis was gated on CD3⫹ cells. The figure shows CD101 and CD103 expression of CD8⫹ proliferating (A and B, respectively) and CD8⫹ nonproliferating (C and D, respectively) T cells in IEC:T-cell cocultures. The percentage of CD101⫹ and CD103⫹ cells is indicated. This is representative of 5 independent experiments.
mainly CD25⫹ CD28⫹ (data not shown). Thus, there are at least 3 populations of T cells that proliferate after interaction with IECs: CD8⫹ CD28⫺ (also CD101⫹ and CD103⫹), CD8⫹ CD28⫹ and CD4⫹ CD25⫹ CD28⫹. CD8⫹ CD28⫹ T cells activated in these cocultures may be stimulated by costimulatory molecules other than CD80/86 (not expressed by normal IECs) such as B7h or B7h1. The Monoclonal Antibody Anti-gp180 (B9) Suppresses the Proliferation of the CD8ⴙ CD28ⴚ CD101ⴙ, CD103ⴙ Subset The capacity of IEC to induce CD8⫹ T-cell activation appears to be linked to the binding of CD8 molecules by gp180. It was previously shown that pretreatment of IEC with mAb B9 (anti-gp180) inhibited their capacity to induce T-cell proliferation and p56lck activation in T cells.15 To study whether gp180 is involved in the activation of this subpopulation of IECactivated CD8⫹ T cells, we compared the proliferation of CD8⫹ T cells in culture with IECs pretreated with mAb B9, or with an isotype-matched control IgG1 (626.1). The proliferation of CD8⫹ T cells was sup-
Lines of Intestinal Epithelial Cell Activated CD8ⴙ T Cells To study IEC-activated T cells in more depth, we needed to develop the capacity to generate long-term lines. Lines were derived from IEC-activated CD8⫹ T cells (sorting of CD3⫹ CD8⫹ CFSE low cells in IEC: T-cell cocultures), and expanded by stimulation with PHA, IL-2, IL-7, IL-15, and feeder cells every 3 weeks. Other lines were derived from nonactivated CD8 cells (sorting of CD3⫹ CD8⫹ cells cultured alone). The phenotype of these lines was studied after several weeks of culture. In these long-term cultures, both CD8⫹ T-cell lines (IEC activated and nonactivated) were CD28⫺. Also, there was no difference in CD101 expression, which was present on 30%– 40% of both IECactivated and nonactivated CD8⫹ T-cell lines (data not shown). In contrast, CD103 was highly expressed on IEC-activated CD8⫹ T-cell lines but not by nonactivated CD8⫹ T-cell lines (Figure 7). Intestinal Epithelial Cell-Activated CD8ⴙ T Cells Are Suppressive in Function To study the function of IEC-activated CD8⫹ T cells, we performed different assays (MLR, T-dependent Ig secretion), to which we added variable numbers of IEC-activated or nonactivated CD8⫹ T-cell lines. In contrast to nonactivated CD8⫹ T-cell lines, which were not suppressive, lines of IEC-activated CD8⫹ T cells suppressed T-dependent Ig secretion by B cells (Figure 8A). Lines of IEC-activated CD8⫹ T-cell lines were also able to suppress CD4⫹ T-cell proliferation in an unrelated MLR (Figure 8B). To ensure that this was not an artifact related to the use of cell lines, IEC-activated CD8⫹ T cells were sorted from short-term cultures.
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These suppressed the proliferation of CD4⫹ T cells in MLR (data not shown) as well. In contrast, nonactivated CD8⫹ T cells did not significantly suppress CD4 proliferation. Suppression was not observed when IEC-activated CD8⫹ T cells were separated from the MLR with a transwell (data not shown), suggesting that the suppression mediated by IEC-activated CD8⫹ T cells requires cell contact. To determine whether the suppressive function belongs to the subset of IEC-activated CD8⫹ T cells expressing CD101 and CD103, we sorted both CD101⫹/CD103⫹ and CD101⫺/CD103⫺ CD8⫹ T cells proliferating (CFSElo) in IEC:T-cell cocultures and added them to suppressive assays. In contrast to CD101⫺/CD103⫺ CD8⫹ T cells, CD101⫹/ CD103⫹ IEC-activated CD8⫹ T cells suppressed Ig secretion (Figure 9). However, depletion of CD101⫹/ CD103⫹ cells from peripheral blood T cells before the culture did not decrease the expression of CD101 and CD103 on IEC-activated CD8⫹ T cells (data not shown), suggesting that precursor cells of this subset do not express these markers before the interaction with IECs. To determine whether similar T-cell populations exist in vivo we isolated CD3⫹ CD8⫹ and CD3⫹ CD8⫺ LP cells. In contrast to unfractionated lamina propria (LP) mononuclear cells and CD3⫹ CD8⫺, CD3⫹ CD8⫹ LP T cells were able to suppress Ig secretion by PBMCs stimulated with pokeweed mitogen (Figure 10). Interestingly, preliminary results suggested that CD8⫹ CD28⫺ LP T cells may be more suppressive than CD8⫹ CD28⫹ LP T cells (data not shown).
Discussion By using in vitro IEC:T-cell cocultures, we show that only a small fraction of peripheral T cells respond to IEC. IECs express several antigen-presenting molecules, such as classical class I and II molecules, nonclassical class Ib molecules (CD1d, human leukocyte antigen E,
Figure 6. (A) The proliferation of CD8⫹ T cells is decreased when IECs are preincubated with anti-gp180 mAb B9. IECs were preincubated with mAb B9 or with an isotype control, and washed before addition to the coculture with T cells. Analysis of CFSE-labeled T cells was performed at days 5 and 7. Cells were stained with anti-CD3 PE and anti-CD8 PerCP. Analysis was gated on CD3⫹ CD8⫹ cells; the % of proliferating CD8⫹ T cells is indicated. (B) Preincubation of IECs with anti-gp180 mAb (B9) decreases the proliferation of CD28⫺, CD101⫹, and CD103⫹ CD8⫹ T cells. Cells were prepared at day 7 as in Figure 5A and stained with anti-CD3 PE, anti-CD8 PerCP and anti-CD28 APC, or with anti-CD101 and CD103 and GAM PE Ab, and
Š with anti-CD8 PerCP and anti-CD3 APC. The analysis was performed on proliferating CD8⫹ T cells, with a gate on CD3⫹ CD8⫹ CFSE low cells. The percentage of CD28⫾, CD101⫹, and CD103⫹ T cells among proliferating CD8⫹ T cells in cultures with IECs preincubated with an Ig control or with mAb B9 is shown. These are representative of 4 independent experiments. (C) Preincubation of non–T cells with mAb B9 does not decrease the proliferation of CD8⫹ T cells as well as CD28⫺ CD101⫹ CD103⫹ T cells in MLRs. Cells were prepared at day 7 as in Figures 6A and 6B. The analysis was gated on CD8⫹ T cells; the percentage of CD28⫺/CD28⫹, CD101⫹, and CD103⫹ among CD8⫹ proliferating T cells after preincubation with (A) isotype control or (B) mAb B9 is shown. This is representative of 3 independent experiments.
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Figure 7. Lines of IEC-activated CD8⫹ T cells express CD103. CFSE-labeled T cells were cultured alone or with IECs. CD3⫹ CD8⫹ T cells cultured (A) alone or (B) proliferating in culture with IECs were sorted at day 10 (boxed in area). These CD8 cells were expanded by using IL-2/-7/-15, PHA, and feeder cells, and restimulated every 3 weeks. The phenotype of these CD8⫹ Tcell lines was studied after 3 cycles of stimulation. CD103 expression of (A) nonactivated and (B) IEC-activated CD8⫹ Tcell lines is shown.
Figure 8. IEC-activated CD8⫹ T-cell lines are suppressive. (A) IECactivated and nonactivated CD8⫹ T-cell lines were added to PBMCs stimulated with pokeweed mitogen (1 g/mL) at a ratio of 1:5 (1 CD8⫹ T cell to 5 PBMCs). Ig secretion, measured by ELISA, was significantly reduced in the presence of IEC-activated CD8⫹ T-cell lines. In contrast, nonactivated CD8⫹ T-cell lines had no effect. (B) IEC-activated CD8⫹ T-cell lines were placed directly in the same well as an unrelated MLR (in which T cells were CFSE labeled before culture with irradiated allogeneic non–T cells) at a ratio of 1:5. Proliferation analysis was gated on CD3⫹ CD4⫹ cells. The percentage of CD4 cells that underwent more than 2 divisions is shown. The proliferation of CD4 cells was significantly reduced in the presence of IEC-activated CD8⫹ T-cell lines. This is representative of 4 independent experiments.
MICA/B, FcRn), and costimulatory molecules (gp180, B7h24). As expected, we found that IECs activated different subsets of T cells. Each of them may interact with a different combination of antigen-presenting and costimulatory molecules. The approach used in this study, the use of CFSE, shows the heterogeneity of T cells responding to IECs. The role of specific molecules involved in the interaction between IEC and subsets of T cells can be shown, as seen in this study for the role of gp180 in the activation of a CD8⫹ CD28⫺ subset. However, blocking with mAb B9 was not complete, suggesting that either the blocking was not completely efficient or, more likely, that there are other subsets activated through other pathways. The CD8⫹ CD28⫺ subset of IEC-activated T cells expresses CD101 and CD103, both considered to be mucosal markers. CD101 is expressed on most T lymphocytes in the intestinal mucosa, and only at low levels on a small proportion of peripheral T lymphocytes.25 CD101 has costimulatory function, of special relevance for CD28⫺ cells. CD103, or ␣E7 integrin, is expressed on greater than 90% of CD8⫹ lamina propria and intraepithelial lymphocytes.26 This integrin mediates T-cell adhesion to epithelial cells via its interaction with E-cadherin,27 but is also implicated in gut-homing of T-cell populations via the intestinal endothelium.28 CD103 is expressed on a small population of circulating, memory CD8⫹ T cells. This small population of circulating CD8⫹ T cells is dramatically expanded in cocultures with IECs. One could propose that CD103 is expressed on IEC-activated CD8⫹ cells as a result of nonspecific activation.29,30 However, as shown in Figure 6, lines of CD8⫹ T cells
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Figure 9. IEC-activated CD8⫹ T cells that express CD101 and/or CD103, in contrast to CD101⫺/CD103⫺ IEC-activated CD8⫹ T cells, are suppressive in function. CD101⫹/CD103⫹ and CD101⫺/ CD103⫺ CD8⫹ T cells proliferating (CFSElo) in an IEC:T-cell coculture, as well as nonactivated CD8⫹ T cells, were sorted and added to PBMCs stimulated with pokeweed mitogen (1 g/mL) at a ratio of 1:10 (1 CD8⫹ T cells to 10 PBMCs). Ig secretion, measured by ELISA, was significantly reduced in the presence of IEC-activated CD8⫹ T cells expressing CD101 and/or CD103 (P ⬍ 0.007). In contrast, CD101⫺/CD103⫺ IEC-activated CD8⫹ T cells, as well as nonactivated CD8⫹ T cells, had no significant effect.
cultured alone that had been stimulated for 2 months with PHA, IL-2/-7/-15 and feeder cells did not express CD103. In contrast, lines of IEC-activated CD8⫹ T cells, which were expanded in parallel under the same conditions, highly expressed CD103. The only difference between these lines was that the latter had interacted with IEC. We think that CD103 expression on IECactivated CD8⫹ T cells more likely reflects the expansion of a mucosal lymphocyte lineage from the PB T-cell pool. It has been shown that TGF can induce the expression of CD103.31 In our experiments, CD103 was only increased on proliferating CD8⫹ T cells, but not on nonproliferating CD8⫹ T cells, in IEC:T-cell cultures. Thus, TGF is not likely to be responsible for these findings. The CD8⫹ CD28⫺ subset of IEC-activated T cells appears important for several reasons. First, mucosal markers (CD101 and CD103) are expressed on this subset. Second, as shown by blocking experiments with mAb B9, gp180 plays a major role in the interaction of this subset with IECs. Previous studies from our laboratory highlighted the potential role of this carcinoembryonic antigen family member in the activation of a regulatory subset. The role of gp180 in the activation of CD8⫹ CD28⫺ T cells suggest that it activates regulatory T cells. Furthermore, IEC-activated CD8⫹ T cells expressing CD101 and/or CD103, in contrast to CD101⫺/CD103⫺ IEC-activated CD8⫹ T cells, were suppressive in function. Interestingly, a subset of major histocompatibility complex class I–restricted CD8⫹
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CD28⫺ suppressor T cells, which act on antigen-presenting cells, rendering them tolerogenic to helper T cells, has been described in humans.17 In this study, CD8⫹ CD28⫹ T cells, unlike CD8⫹ CD28⫺ T cells, were not suppressive. The ability of CD8⫹ LP T cells to suppress Ig secretion by PBMCs stimulated with pokeweed mitogen suggests the presence of CD8⫹ with regulatory function in the lamina propria (Figure 10). Preliminary results also suggest that CD8⫹ CD28⫺ LP T cells may be more suppressive than CD8⫹ CD28⫹ LP T cells. Third, we have previously shown the potential role of CD1d in the interaction between IECs and T cells.32,33 We have shown that gp180 and CD1d associate on the surface of IECs and interact with both the CD8 molecule and the T-cell antigen receptor.19,34 Altogether these data suggest that the CD8⫹ CD28⫺ subset of IEC-activated T cells may recognize antigens presented by CD1d. CD1d is a nonpolymorphic major histocompatibility complex class I–like molecule whose expression is mainly localized to the epithelial cells of the gastrointestinal tract.35 CD1d is expressed both on the apical and the basolateral surfaces of IECs and may play an important role in the interaction with mucosal T cells. Based on in vitro studies of the antigens presented by human and mouse CD1d, CD1d expressed on IECs likely presents a glycolipid molecule possibly from the cell wall of bacteria or host cells.35 Our hypothesis is that IECactivated CD8⫹ regulatory T cells, and potentially the CD8⫹ CD28⫺ CD103⫹ subset, recognize Ag presented by CD1d on IECs. We have recently shown a skewing of the TCR repertoire of IEC-activated CD8⫹ T cells, and we have identified a subset of CD8⫹ T cells
Figure 10. LP CD3⫹ CD8⫹ T cells, in contrast to unfractionated and CD3⫹ CD8⫺ LP cells, can suppress Ig secretion by PBMCs stimulated with pokeweed mitogen. LP cells, LP CD3⫹ CD8⫹ and LP CD3⫹ CD8⫺ cells (sorted by using a Cytomation Moflo as described earlier), were added to PBMCs stimulated with pokeweed mitogen (1 g/mL) at a ratio of 1:4 (1 LP cell to 4 PBMCs). Ig secretion, measured by ELISA, was significantly reduced in the presence of CD3⫹ CD8⫹ LP T cells (P ⬍ 0.03). In contrast, unfractionated and CD3⫹ CD8⫺ LP cells had no significant effect.
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defined by specific VB and VA chains that interact with the gp180/CD1d complex (Allez et al., September 2002, unpublished data). Some Ag (such as bacterial products) processed from the lumen by IECs could activate these CD8⫹ regulatory T cells through IEC:T-cell interactions. It has been proposed that some bacterial strains in the intestinal flora play a beneficial role in the maintenance of physiologic, controlled inflammation. One of the potential mechanisms used by these good bacteria could be the activation of CD8⫹ regulatory T cells. Interestingly, depletion of CD101⫹ and CD103⫹ T cells from peripheral blood T cells before the culture did not decrease the expression of these markers on the CD8⫹ T cells proliferating in IEC:T-cell cocultures. We were not able to define the phenotype of the precursor cells that expand after interaction with IECs through gp180; at least they do not seem to express CD101 and CD103 on their surface before this interaction. We had previously been unable to generate T-cell lines from IEC-activated T cells by using cytokines (IL-2, IL-4) and PHA, which allow for CTL or helper T cell clonal outgrowth. The use of IL-7 and IL-15, cytokines produced by IECs allowed us to establish long-term lines that maintained both functional and phenotypic characteristics. These conditions permitted us to initiate studies defining the mechanism of IEC-activated CD8⫹ Tcell mediated suppression and also provided valuable reagents for the study of the TCR repertoire in these cells. Until now, we have failed to identify the mechanism by which these CD8⫹ T cells suppress. Experiments using transwell cultures (IEC-activated CD8 cells in the upper chamber and conventional MLR in the lower chamber) failed to reproduce the suppression seen when IEC-activated CD8⫹ cells were added directly to the cultures (data not shown), and suggest the requirement for cell contact. In preliminary studies, supernatants from normal IEC:T-cell cocultures failed to suppress MLR cultures (unpublished results) and preliminary data on the cytokines produced by these cells suggest that they produce IFN␥ but not TGF or IL-10. These results speak to the requirement for cell contact, and eventually to the role of immunoregulatory molecules on IEC-activated CD8⫹ T cells. Recent studies in mice suggest that CTLA4 may be involved in the suppressive function of regulatory CD4⫹ T cells. Indeed, CTLA4 is expressed on CD4⫹CD25⫹ regulatory T cells, and appears to play an important role in their suppressive function.8 Regulatory T cells that control intestinal inflammation in the mouse (CD4⫹ CD45RBlo) constitutively express CTLA4, and signals through CTLA4 are required for the function of these
ACTIVATION OF REGULATORY CD8ⴙ T CELLS BY IECS
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cells in vivo.10 CTLA4, which is weakly expressed on IEC-activated CD8⫹ T cells (Table 1), is probably not implicated in the suppressive effects of these cells. The ability of IEC-activated CD8⫹ T cells to kill various targets has been assessed in initial studies.18 No killing was shown by using K562 (NK killing), anti-CD3 coated P388D1 cells (redirected lysis), IEC lines (HT29, DLD1), and activated T cells (mitogen activated). In light of these data, it is unlikely that IEC-activated CD8⫹ T cells suppress via cytotoxicity. The new approach we used in this study, and our capacity to generate lines of IEC-activated CD8⫹ T cells, should help us to further identify the mechanisms of suppression used by IEC-activated CD8⫹ T cells. In conclusion, we further show in this study that IECs have the capacity to activate and expand CD8⫹ regulatory T cells. IECs interact with a number of T-cell subpopulations but appear to selectively activate a CD8⫹ CD28⫺ subset, which expresses CD101 and CD103 and has regulatory function. The molecule gp180, a CEA family member expressed in association with CD1d on the basolateral surface of IECs, plays a major role in the interaction with this subset. The defective expression of gp180 in IBD, which correlates with the lack of activation of CD8⫹ T cells, suggests that this subset is not activated in the mucosa of IBD patients. Precursor cells of this subset may be present in IBD patients but would be unable to become activated owing to an aberrant microenvironment. Better identification of the suppressor mechanism used by this subset and identification of growth requirements of such cells could open new avenues for therapeutic intervention in IBD patients.
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Received December 18, 2001. Accepted July 18, 2002. Address requests for reprints to: Matthieu Allez, M.D., Immunobiology Center, Mount Sinai School of Medicine, 1425 Madison Avenue, Room 11-20, New York, New York 10029. e-mail: [email protected]; fax: (212) 987-5593. Supported by National Institutes of Health grants AI 23504, AI 24671, AI 44236, a Crohn’s and Colitis Foundation of America fellowship award (to M.A. and I.D.), Socie ´te ´ Nationale Franc¸aise de GastroEnte ´rologie (to M.A.), Institut de recherche des maladies de l’appareil digestif (to M.A.), Laboratoire Glaxo-Wellcome (to M.A.), and the Danish medical research agency (to J.B.).