METHODS

Animals

C57BL/6 and BALB/C-DO11.10 T cell receptor transgenic mice were purchased from Jackson Laboratory (Bar Harbor, Maine, USA) and housed under specific pathogen free conditions at the University of Leeds. Sentinel mice were routinely screened for common mouse pathogens and none was detected.

Isolation and culture of CEC and lamina propria T cells

We have previously described the isolation and culture of CEC and lamina propria T cells.²⁰ CEC purity was assessed by staining for cytokeratin and CD45. Isolated CEC routinely comprise crypts, enterocytes, and goblet cells. Under our normal culture conditions, clusters of crypt-like cells, or organoids, attach to the Matrigel substrate and form semi-confluent monolayers of defined polarity with the epithelial basolateral surface in contact with the Matrigel. However, in coculture with CD4 T cells, organoids remained intact and in suspension, allowing CD4 T cells to interact with both the basal and apical epithelial surfaces. CEC were retrieved from culture by digestion with dispase:collagenase IV (1 mg/ml; Sigma, Poole, Dorset, UK) for 5–10 minutes, after which cells were washed twice in Dulbecco’s modified Eagle’s medium containing 20% fetal bovine serum (FBS).

CEC:CD4 T cell coculture

Splenic CD4 T cells were prepared by depleting Mφ by plastic adherence, followed by negative immunomagnetic selection (Miltenyi Biotec, Bisley, UK) to produce a cell suspension containing >98% CD4 T cells. Colonic lamina propria CD4 cells were isolated as described previously.²⁰ For proliferation assays, T cells were labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Cambridge Bioscience, Cambridge, UK) immediately prior to adding to CEC cultures. CEC organoids (approximately 2×10⁵ cells/ml) were cultured for 6–24 hours before addition of whole spleen cells or CD4 T cells (2×10⁶/ml) in the presence or absence of ConA (5 μg/ml; Sigma), mitogenic anti-CD3 antibody (1452C11), anti-CD3 and anti-CD28 (37.51) antibodies, or OVA (10 μM; Sigma). As a positive control, CD4 cells were cultured with adherent splenic Mφ (2×10⁵cells/ml) in Matrigel coated tissue culture plates (Becton Dickinson Biosciences, Oxford, UK) under the same conditions as CEC:CD4 T cell cocultures. Lamina propria T cell:CEC cocultures were performed as described above in the presence of mitogenic anti-CD3 antibody. In some experiments, 200 U/ml recombinant murine interleukin 2 (IL-2; TOTAM Biologicals, Cambridgeshire, UK), goat anti- transforming growth factor β1 (TGF-β1) antibodies, or goat IgG (R&D Systems, Abingdon, UK) were added to CEC:T cell cocultures. T cells were harvested after 72 hours and analysed by antibody staining and flow cytometry.

Antigen specific CD4 T cell responses

Antigen primed CD4 T cells were obtained by culturing DO-11 CD4 T cells of which >90% expressed the OVA specific TcR (KJ1-26⁺) for five days in the presence of 10 μM OVA and 20 U/ml IL-2 in complete RPMI medium (10% v/v FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine) at a final concentration of 2×10⁶ cells/ml. Primed CD4 T cells were then isolated by positive immunomagnetic selection and cultured with CEC or Mφ in the presence or absence of OVA (10 μM) for 72 hours prior to analysis. Freshly isolated non-stimulated DO11 CD4 cells were used as a source of naïve T cells in CEC or Mφ cocultures. As an additional control, FITC-OVA pulsed CEC were cultured with the lacZ-inducible OVA/I-A^b specific CD4 hybridoma KZO, as described elsewhere.²⁰

Restimulation of CD4 T cells after coculture with CEC

CEC and CD4 T cells were cocultured in the presence of mitogenic anti-CD3 antibody. After 72 hours, CD4 cells were isolated by positive immunomagnetic selection and restimulated with anti-CD3 antibody for 72 hours, or with 10 nM phorbol-12-myristate 13-acetate (PMA) and 500 nM ionomycin (CN Biosciences, Nottingham, UK) for 24 hours, as described previously.¹⁷ T cell activation was assessed by cell cycle analysis and expression of activation markers. The ability of OVA primed CD4 T cells to respond to antigen after coculture with CEC was also assessed. CD4 T cells were isolated from initial CEC:T cell cocultures by Percoll density gradient centrifugation, washed, and plated onto antigen pulsed Mφ for 72 hours prior to analysis.

Flow cytometry

Cells were analysed by flow cytometry on a FACScan flow cytometer using CELLQuest software (Becton Dickinson). Cells were stained with fluorochrome and/or biotin conjugated antibodies to CD45, B7.1 (CD80), B7.2 (CD86), ICAM-1 (CD54), CD40, KJ1-26, CD25, CD69, H-2D^b, CD45RB, CD62L, CD44, CD8α, CD4, NK1.1, GR-1, F4/80 (TCS Biologicals, Towcester, UK), Fas (CD95), CTLA-4 (CD152), I-A/I-E (Becton Dickinson), a pan-reactive anti-cytokeratin-FITC antisera (Sigma), CD3 (1452C11; ATCC, Rockville, Maryland, USA), CD28 (clone 37.51; Dr M Glennie, Southampton, UK), goat antihuman latency associated peptide of TGF-β1 (LAP), chicken antihuman TGF-β, and chicken IgY (Stratech, Cambridgeshire, UK). Isotype matched antibodies of irrelevant specificity were used to determine the level of non-specific staining and frequency of cells stained with test antibodies.²⁰ Apoptotic cells were identified by labelling cells with FITC-annexin V and propidium iodide. For intracellular staining, cells were surface stained, fixed in 0.1% paraformaldehyde, and permeablised with 0.1% saponin/1% milk protein prior to staining. For intracellular cytokine staining, freshly isolated cells were incubated with Brefeldin A (10 mg/ml) for five hours prior to staining, as described above. For cell cycle analysis, propidium iodide was added immediately prior to acquisition. Cytokine multiplex bead assay kits (Becton Dickinson) were used to measure levels of interferon γ (IFN-γ), tumour necrosis factor α (TNF-α), interleukin (IL)-5, IL-4, and IL-10 in CEC:T and Mφ:T cell conditioned media.

Statistics

Data are presented as means (SEM). Statistical significance was evaluated using a Mann-Whitney test.

RESULTS

CEC inhibit T cell activation by mitogens

In the presence of Mφ, colonic lamina propria (LP) and splenic CD4 T cells underwent blast formation and acquired the large granular morphology characteristic of activated cells. A significant proportion of splenic and lamina propria CD4 T cells were in S and G2 phases of the cell cycle (fig 1D, E) and proliferating as determined by CFSE staining of which splenic T cells had the response of greatest magnitude. Expression of activation associated surface antigens CD69 and CD25 (fig 1A, C) was induced or upregulated (CD95 (Fas), CD44, and CD45RB; fig 1B) on CD4 T cells.

In contrast, the response of both LP (fig 1C, E) and splenic CD4 cells (fig 1A, B, D, F, and G) to activation in the presence of CEC was inhibited. The majority of CD4 cells were small and quiescent, with >90% remaining in the G1 phase of the cell cycle (fig 1D, E). Few of the CD4 T cells in CEC coculture proliferated (data not shown) and the majority were CD25^−/lo (p<0.001; fig 1A, G) and expressed lower levels of CD45RB (p<0.001; fig 1B) and CD95 (p<0.01) compared with CD4 T cells cultured with Mφ. CEC also affected T cell cytokine production, and concentrations of IL-10, IL-5, IL-4, IFN-γ, and TNF-α were considerably reduced in the supernatants of CEC:CD4 T cell cocultures compared with Mφ:CD4 T cell cocultures (fig 1F, G). Using the CBA analysis system (detection limit of 10–20 pg), it was not possible to detect the presence of any of these cytokines in supernatants from CEC cultured in complete media alone (data not shown). Using intracellular staining with anticytokine antibodies, freshly isolated CD4 T cells expressed no detectable IFN-γ, TNF-α, IL-2, IL-4, or IL-10 (data not shown).

As LP T cells are relatively refractory to stimulation through their TcR,²¹ making measurement of normal indices of T cell function difficult, splenic CD4 T cells were used in subsequent experiments.

CEC inhibit antigen specific CD4 T cell responses

To assess if CEC could prime antigen specific T cells, CD4 cells from DO11.10 TcR transgenic mice, in which CD4 T cells express a TcR specific for a peptide of OVA and H-2^d, were cultured with OVA pulsed DO11.10 CEC. CEC were unable to prime CD4 T cells, as judged by lack of expression of CD25 and CD69 and absence of blast cell formation (data not shown).

CEC were unable to activate antigen primed T cells in a recall response. OVA primed CD4 cells cultured with Mφ or CEC in the absence of antigen were quiescent, resided in the G1 phase of the cell cycle (fig 2C), and expressed low levels of CD25 (fig 2A), CD45RB (fig 2B), and Fas. Addition of antigen to cocultures of primed T cells and CEC did not induce T cell activation. CD4 cells remained quiescent, in the G1 phase of the cell cycle (fig 2C), with low expression of CD25 and CD45RB (fig 2A, B), and produced little or no IFN-γ, TNF-α, or IL-10 (fig 2D). In contrast, primed CD4 cells cultured with Mφ and antigen expressed high levels of CD25 (p<0.005) and CD45RB (p<0.005), remained in the cell cycle (fig 2A), and produced cytokines, including IL-10 (fig 2D). Of note, CEC, were able to present antigen to the OVA specific CD4 hybridoma line KZO⁶ (fig 2E).

CEC express costimulatory molecules

The possibility that the inability of CEC to activate CD4 T cells was attributable to the absence of costimulation was investigated (fig 3) and compared with freshly isolated splenic Mφ. All Mφ expressed CD54 with a high median fluorescence (MFI 149 (SD 22.47)) compared with 7.3% CEC (MFI 10.54 (SD 3.8)). Up to 12% of Mφ (MFI 10.62 (SD 0.66)) and 3% CEC (MFI 9.5 (SD 4)) expressed B7.1. Seventy four per cent Mφ expressed B7.2 (MFI 89.7 (SD 3.21)), with a variable proportion of CEC (from 20% to 50%) also expressing B7.2 (MFI 37.01 (SD 10)). Approximately 29% Mφ expressed CD40 (MFI 17.7 (SD 4)) compared with 4.5% CEC (MFI 12.7 (SD 7.1)). Thus CEC expressed CD40 and B7.1 at levels comparable with those of splenic Mφ whereas the level of expression (MF) of B7.2 and CD54 was higher on Mφ than CEC. Expression of B7.1 and B7.2 was confirmed by reverse transcription-polymerase chain reaction and Southern blot analysis of CEC mRNA (Baumgart and Carding, unpublished observations). Of note, expression of costimulatory molecules on CEC was retained in culture.

CEC are not inert and do not induce anergy in CD4 T cells

The effects of increasing the threshold of T cell activation by addition of exogenous IL-2 (200 U/ml) or cross linking of CD28 on T cells which are known to overcome anergy were investigated. Addition of either IL-2 or anti-CD28 antibodies failed to restore T cell activation in the presence of CEC (fig 4). In contrast, addition of anti-CD28 antibody or IL-2 to Mφ:CD4 T cell cocultures potentiated the T cell response, increasing the proportion of cells in the cell cycle and levels of CD25 expression (fig 4). Of note, CD4 T cell responses to mitogen were inhibited by CEC, even in the presence of professional APC including Mφ (fig 4E), demonstrating that CEC are not inert and are actively suppressing or preventing CD4 T cell activation.

Mechanisms of CEC mediated suppression of CD4 T cell activation

The production of suppressive cytokines or soluble factors, the action of counterregulatory molecules of costimulation (for example, CTLA-4), or insufficient levels of MHC molecules were excluded as mechanisms of CEC mediated suppression of CD4 T cell activation. CEC conditioned media was unable to inhibit mitogen activation of CD4 cells by Mφ, indicating that CEC mediated suppression was not due to the production of suppressive soluble factors, including IL-10 (figs 1G, 2D), and was cell contact dependent.

The possibility that CTLA-4 (CD152) was involved in CEC mediated suppression of T cell activation was excluded as CD4 cells in CEC cocultures were CTLA-4 negative although low levels of CTLA-4 were detected on the surface and cytoplasm of T cells from Mφ:CD4 T cell cocultures (data not shown). Cross linking of CD28 on T cells in the presence of CEC (fig 4C, D) failed to restore T cell function, consistent with no role for B7.2, the ligand for CTLA-4, in CEC mediated suppression of T cells.

Increasing the level of MHC II on CEC was also unable to overcome CEC mediated suppression of T cells and CD4 T cell responses to mitogens (cross linking CD3), which are not dependent on levels of MHC, were also inhibited in the presence of CEC (fig 1).

Although freshly isolated CEC expressed both cell surface associated TGF-β (fig 5A) and latency associated peptide of TGF-β (LAP; fig 5B), a neutralising antibody to TGF-β1–3 (20 μg/ml) had no effect on CEC mediated suppression of CD4 T cell activation (fig 5C). This antibody was however able to block TGF-β mediated inhibition of fibroblast proliferation (fig 5D). These experiments demonstrate that TGF-β is not involved in CEC mediated suppression of T cells, either in soluble or cell surface bound form.

Removal and restimulation of CD4 T cells from CEC cocultures renders them responsive

CEC do not irreversibly suppress T cell activation. CD4 T cells recovered from CEC or Mφ cocultures and cultured with freshly isolated Mφ were activated by PMA/ionomycin or anti-CD3 antibody (fig 6B). Indeed, CD4 T cells previously cultured with CEC responded more vigorously than those previously cultured with Mφ to pharmacological challenge, with a greater proportion of cells entering the cell cycle (fig 6A). Thus CEC did not induce death of CD4 T cells, which was independently confirmed by failure to detect increased apoptosis by annexin V and propidium iodide staining (data not shown).

Antigen specific suppression of CD4 T cell activation was also reversible. DO11 T cells recovered from CEC cocultures containing antigen (OVA) and recultured with freshly isolated Mφ and OVA were activated, as assessed by cell cycle analysis and CD25 expression (fig 6C, D). The response of T cells previously cultured with CEC was of a slightly greater magnitude than those cells previously cultured with Mφ. This may reflect activation induced cell death of T cells during the initial response to OVA in the presence of Mφ. This contrasts with CD4 T cells previously cultured with CEC which remain in a resting state and have not undergone any significant apoptosis, thus providing a larger pool of responder T cells when restimulated with fresh Mφ and OVA. Taken together, these data demonstrates that CEC do not permanently disable CD4 T cell function, with normal function being restored when CEC were removed.

DISCUSSION

Using a novel primary CEC culture system, we have shown that CEC, in contrast with splenic Mφ, suppress CD4 T cell activation. This inhibitory effect was dependent on the continual presence of CEC and was reversible. CEC mediated suppression of T cell responses may therefore play a role in maintaining non-responsiveness to enteric antigens. Although the experimental approach we have used is somewhat artificial, and fails to account for the cellular heterogeneity of the colonic mucosa and possible influence of other mucosal cells on CEC function, it provides an in vitro accessible system with which to dissect colonic mucosal immune responses. The observation that CEC suppress activation of both naive and primed splenic as well as colonic LP CD4 T cells suggests that the suppressive effects of CEC are not restricted to resident mucosal T cells but also to cells from other tissues that may migrate to the colon.

Previously we have shown that murine small intestinal epithelial cells and CEC can present soluble protein antigen to antigen specific CD4⁺ T cell hybridomas.¹⁰ These findings conflict with our current observations that murine CEC inhibit the responses of primary CD4 T cells. This apparent discrepancy may be explained by the fact that T cell hybridomas and primary T cells have different requirements for activation, with T cell hybridomas having a lower threshold for activation and are costimulation independent.²² Although it has been shown that CEC can process antigen and present soluble protein antigens,^1–6 primary T cells will not respond to antigen in the absence of costimulation. Our finding that murine CEC from two strains of mice constitutively expressed costimulatory molecules contrasts with a previous analysis of normal human CEC.¹⁹ This may reflect species differences or differences in the methods of detection as our analysis of murine CEC has shown that it was only possible to detect expression of B7.2 using a sensitive indirect staining procedure and was undetectable using direct methods (Cruickshank and Carding, unpublished observations).

The observation that CEC failed to promote mitogen or antigen specific responses despite expressing B7 suggests that their costimulatory molecules are not functional. The absence of CTLA-4 expression by T cells cultured with CEC excludes the involvement of this counter receptor. Also, the ability of CEC to suppress T cell activation in the presence of splenic Mφ or anti-CD28 antibodies is consistent with the interpretation that CEC are not inert but actively suppress CD4 T cell activation. CEC do not anergise CD4 T cells as T cells previously cultured with CEC responded normally to rechallenge with mitogens or antigens. Thus the continued presence of the epithelial cell is needed for the suppressive effect. Similar suppressive effects have been reported for small IEC.²³

In contrast with previous studies using non-viable (fixed) primary or established CEC lines,^24–26 we have been unable to demonstrate that CD4 T cells cultured with CEC become T regulatory or suppressor cells. Although CD4 T cells cultured with murine CEC resembled Treg cells in being CD45RB^lo they were also CD25^-/lo, did not secrete IL-10, and responded normally to rechallenge by a variety of stimuli. Our data suggest therefore that CEC themselves are regulatory cells.

We were unable to identify the mechanism(s) by which CEC suppress T cell activation. The observation that supernatants taken from growing cultures of CEC had no effect on T cell activation are consistent with the effect being cell and probably contact dependent. However, the possibility that suppression is mediated by a soluble factor that is only produced when T cells are present, or only acts when cells are in close proximity, cannot be excluded. Although a recent publication has ascribed a role for cell surface bound TGF-β in mediating the suppressive function of regulatory T cells,²⁷ our data show that TGF-β is not involved in CEC mediated suppression of CD4 T cell activation. Potential mechanisms include mucins and novel inhibitory costimulatory molecules. The mucin associated molecule MUC-1 has been reported as having suppressive or proapoptotic effects on T cell activation ^28,29 and murine CEC expressed MUC-1 (Cruickshank and Carding, unpublished data). The lack of suitable reagents however prevents us from determining what role, if any, it might play in CEC mediated suppression of CD4 T cells. The recent observations that novel members of the B7 family (B7x,³⁰ and the ligands for PD-1; B7-H1 (PD-L1) and PD-L2³¹) have inhibitory effects on T cell activation may also provide an alternative mechanism for CEC mediated suppression of T cell activation although it is not known if CEC express these negative receptors.

In conclusion, we have demonstrated that normal murine CEC actively suppress CD4 T cell responses and that this effect is reversible. We believe that CEC, as the major site of antigen contact in the colon, play a role in preventing the underlying T cells in the stroma from responding to enteric antigens, which can be presented by epithelial cells. Our findings also may explain how global non-responsiveness of mucosal T cells to the vast array of enteric antigens sampled by CEC is maintained. Dysregulation of CEC mediated suppression of T cell responses may also contribute to the uncontrolled inflammation seen in inflammatory bowel diseases.