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The Journal of Immunology, 2007, 179, 372 -381
Copyright © 2007 by The American Association of Immunologists, Inc.
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Gliadin Regulates the NK-Dendritic Cell Cross-Talk by HLA-E Surface Stabilization1

Giuseppe Terrazzano2,*, Michela Sica*, Carmen Gianfrani{dagger}, Giuseppe Mazzarella{dagger}, Francesco Maurano{dagger}, Beatrice De Giulio{dagger}, Sophie de Saint-Mezard{ddagger}, Delia Zanzi§, Luigi Maiuri, Marco Londei, Bana Jabri{ddagger}, Riccardo Troncone§, Salvatore Auricchio§, Serafino Zappacosta* and Ennio Carbone*,||,#,**

* Department of Cellular and Molecular Biology and Pathology, University Federico II, Naples, Italy; {dagger} Institute of Food Science and Technology, Consiglio Nazionale delle Ricerche, Avellino, Italy; {ddagger} Department of Pathology, University of Chicago, Chicago, IL 60607; § Department of Pediatrics and European Laboratory for the Investigation of Food-Induced Diseases, University Federico II, Naples, Italy; Institute of Child Health, University College London, London, United Kingdom; || Department of Experimental and Clinical Medicine University "Magna Graecia," Catanzaro, Italy; # Research Center for Studies of Integrative Recognition in the Immune System, Karolinska Institutet, Stockholm, Sweden; and ** Microbiology and Tumorbiology Centre, Karolinska Institutet, Stockholm, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We analyzed the autologous NK cell interaction with gliadin-presenting dendritic cells. Gliadin is the known Ag priming the celiac disease (CD) pathogenesis. We demonstrate that gliadin prevents immature dendritic cells (iDCs) elimination by NK cells. Furthermore, cooperation between human NK cells-iDCs and T cells increases IFN-{gamma} production of anti-gliadin immune response. Gliadin fractions were analyzed for their capability to stabilize HLA-E molecules. The {alpha} and {omega} fractions conferred the protection from NK cell lysis to iDCs and increased their HLA-E expression. Gliadin pancreatic enzyme digest and a peptide derived from gliadin {alpha} increased HLA-E levels on murine RMA-S/HLA-E-transfected cells. Analysis of HLA-E expression in the small intestinal mucosa of gluten-containing diet celiac patients and organ culture experiments confirmed the in vitro data.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Celiac disease (CD)3 is a small intestinal enteropathy triggered by the ingestion of gliadin in susceptible individuals (1). Genetic, environmental, and immunological factors play a central role in CD pathogenesis (1). The majority of patients are HLA-DQ2 positive, while a small group express DQ8. Both DQ2 and DQ8 molecules are involved in the presentation of gliadin peptides to mucosa infiltrating T cells (2). Gliadin intake in celiac patients correlates with the presence of serum autoantibodies against tissue transglutaminase. Although CD has been classified recently among the autoimmune pathologies (3), the mechanisms of its autoimmune pathogenesis still appear to be poorly understood. Innate immunity preceding the adaptive response may be involved in the CD pathogenesis (4).

NK cells are found in the murine intestinal epithelium and in the lamina propria (5, 6) and NK T cells in the gut (7). Intestinal NK cells have cytotoxic (5) and IFN-{gamma} production activity (6). The expansion of intraepithelial lymphocytes expressing CD94, a typical NK cell receptor (8), and of a subset of gut epithelium CD3–CD56+ lymphocytes (9) has been described in CD. IL-15, a cytokine that controls NK cell homeostasis (10), is up-regulated in atrophic mucosa where most of the epithelial changes occur in the active stage of the disease (11). The p31–43 gliadin peptide triggers IL-15 production in the gut (12). IL-15, together with NKG2D expression, induces CTL activity contributing to the tissue destruction in CD (12).

NK cells represent a distinct lymphocyte population of innate immunity characterized by cytotoxicity against tumors, virus-infected cells, and parasites (13). IFN-{gamma} and TNF-{alpha} are secreted by NK cells in response to cytokines produced by macrophages and dendritic cells (DCs) (14, 15). Moreover, IFN-{gamma} and TNF-{alpha} production by NK cells could promote DC maturation degree (16, 17). IFN-{gamma} promotes Th1 differentiation (18) and is expressed in small intestinal mucosa biopsy from celiac patients (19). This cytokine could induce both the cytotoxic intraepithelial lymphocytes and macrophage activation, fostering the damage of intestinal mucosal extracellular matrix (20). The interaction between NK cells and target cells is modulated by several receptor-ligand interactions (21, 22, 23). NK cells express several MHC class I (MHC-I) recognizing receptors, mainly classified into two families: the killer Ig-like receptors (KIRs) and the lectin-like receptors (22). NK cell cytotoxicity is triggered by non-MHC-I receptors such as CD16, CD69, 2B4, NKG2D, NKp30, NKp44, NKp46, and NKp80 (22).

DCs play a key role in immune responses (24). Immature DCs (iDCs) exert Ag capture and processing ability in peripheral tissue (25). Inflammatory cytokines and microbial components (26) trigger DC migration into lymphoid tissues and promote their maturation. In this process, DCs acquire the ability to stimulate T cells (27). DCs, present in the gut (28), in Peyer’s patches (29), and in lamina propria (30), have been described to have a key role in activating naive T cells in GALT (31).

NK cells are involved in several autoimmune diseases (32), and their cross-talk with iDCs leads to different outcomes (16): 1) NK cells recognize and eliminate immature but not mature DCs (33); 2) reciprocal activation (17, 34); and 3) NK cells comigrate with DCs in the lymph nodes (35).

Because NK cells are present in the GALT (36) and the gut Ag-loaded DCs migrate from GALT to mesenteric lymph nodes (37), the interaction between NK cells and mature DCs could occur in these environments and could contribute to the priming of immune response and to Th1 polarization (18, 35, 38, 39).

In the research reported here, we explore the potential role that gliadin plays in regulating NK cells-DCs cross-talk and its role in regulating T cell response.


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

A total of 24 celiac patients (mean age of 14.2 years) was enrolled for this study. All patients gave their informed consensus, according to the ethical regulations of the University of Naples "Federico II." Patients were diagnosed according to the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition criteria (40); all of them had been on gluten-free diet for at least 2 years and were serum negative for anti-endomysial Abs.

Culture of NK polyclonal lines, iDCs, and gliadin treatments

PBMCs and NK cells were isolated from celiac patients or from healthy donors as reported previously (33). NK cell purity was always >95%. All NK polyclonal cell lines were CD94/NKG2A positive in a percentage ranging between 70 and 80% of total CD3–CD56+ lymphocytes.

iDCs were generated as described originally (26). iDC population was found to be 85% pure expressing high levels of CD1a, with no detectable amount of CD14 by FACS staining. iDCs were incubated for 18 h with 50 µg/ml peptic-tryptic digest (PT) of gliadin (PT-gliadin), 50 µg/ml PT of beta-lactoglobulin, lacto-albumin, and casein or with medium alone. Alternatively, the iDCs were treated with 50 µg/ml single-purified {alpha}-, beta-, {gamma}-, or {omega}-gliadin fractions in the same experimental conditions. The PT gliadin was prepared from gliadin extracted from San Pastore cultivars (41). Gliadin or gluten was digest with pepsine (50:1) in 5% formic acid for 4 h at 37°C while stirring, then the fraction of pepsine polypeptides was exposed to a sequential digestion with trypsin, chymotrypsin, and elastasis (50:1) in 50 mM ammonium bicarbonate for 4 h at 37°C. Digest preparations were collected in RPMI 1640 (Sigma-Aldrich) to obtain a complete pepsine-trypsin-chymotrypsin-elastasis digest (PTCE).

Fractionation of gliadin was performed as reported originally (41). {alpha} Gliadin was obtained as a unique fraction of all {alpha} gliadin subgroups. The amount of protein was determined according to the Bradford method (42). PT proteins and gliadin fractions were assayed for endotoxin LPS by using a detection kit (BioWhittaker) and found LPS levels to be <0.5 EU/ml (43).

Generation of gliadin-specific, intestinal T cell lines and evaluation of DC phenotype in mononuclear cells from mucosal explants

Mucosal explants from two DQ2-positive CD patients (female, untreated, 24 years old; male, treated, 20 years old) were digested with collagenase A at 1 mg/ml for 1 h at 37°C. Cells were plated in 24-well plates at 2–3 x 105/ml and stimulated with 1 x 106 irradiated autologous PBMCs and 50 µg/ml PT-gliadin. Cultures were fed with 10 ng/ml IL-15. At days 7 and 21, gliadin-restricted intestinal T cell lines (g-iTCLs) were restimulated with irradiated autologous PBMCs and PT-gliadin-tissue transglutaminase as described above and thereafter with cyclic (14 days) restimulation with a feeder-cell mixture containing PHA (0.5 µg/ml) and IL-2 (100 U/ml). Ten to 14 days after the last restimulation when cells were in complete resting phase, g-iTCLs were assayed for responsiveness to gliadin by IFN-{gamma}-ELISPOT assay.

After the digestion of mucosal explants by collagenase (1 mg/ml for 1 h at 37°C), the mononuclear cells obtained were stained by immunofluorescence and analyzed by FACS to evaluate DC phenotype.

IFN-{gamma}-ELISPOT assay

Ninety-six nitrocellulose-backed plates (MAHAS4510; Millipore) were coated with 10 µg/ml anti-human IFN-{gamma} mAb (BD Pharmingen) as described elsewhere (44). g-iTCLs (0.3 x 105) were plated in the presence of 0.6 x 105 irradiated autologous DCs (corresponding to a ratio g-iTCLs:DC of 1:2), which had been pulsed overnight with PT-gliadin or PT-beta-lactoglobulin at 50 µg/ml. Ag-pulsed-DCs were incubated 6 h in presence of 0.3 x 105 autologous NK cells, corresponding to a ratio of DCs:NK cells 2:1 before adding responder g-iTCLs in a final volume of 200 µl. After 36 h, plates were incubated for 2 h with biotinylated anti-IFN-{gamma} mAb, followed by streptavidin-HRP incubation (BD Pharmingen) for 1 h. IFN-{gamma}-Spot Count/well were counted by immunospot image analyzer (A.EL.VIS). To inhibit the IFN-{gamma} secretion by NK cells, 1 x 106 autologous NK cells were pretreated for a 37°C overnight incubation with 10 µg/ml Golgi protein-trafficking blocker brefeldin A (BFA) (Sigma-Aldrich).

Monoclonal Abs, immunofluorescence, and flow cytometry

Anti-HLA mAb (W6/32class I) was purchased from DakoCytomation. Anti-BDCA3 (AD5-14H12) and anti-BDCA2 (AC144) were purchased from Miltenyi Biotec. Anti-CD14 (M5E2), -CD1a (HI149), -CD83 (HB15), -CD86 (IT2.2), -CD80 (BB1), -CD40 (5C3), -CD3 (UCHT1), -CD56 (B159) -CD94 (Kp43), -CD11c (B-ly6), FITC-, CyChrome-, and PE-labeled mAbs and isotype-matched labeled mAb controls were purchased from BD Pharmingen and used as described previously (33). HLA-E expression was analyzed using two anti-HLA-E mAbs: the MEM-E/08 (IgG1) from EXBIO and the 3D12 (IgG1), a gift from Dr. D. Gerarhty (Fred Hutchinson Cancer Research Center, Seattle, WA). Anti-NKG2A/NKG2C (P25) mAb was a gift from Dr. L. Moretta (Istituto Gaslini, Genova, Italy).

Detection of intracellular IFN-{gamma} and TNF-{alpha} by flow cytometry

PT-gliadin- or PT-beta-lactoglobulin- or medium-treated iDCs were mixed at a ratio of 1:1 with IL-2-activated NK effectors (2 x 105) and incubated in a sterile 96-well plate (Falcon) for 6 h at 37°C in 10% FCS-RPMI 1640 medium. BFA (Sigma-Aldrich) was added at 5 µg/ml during the incubation to induce cytokine intracellular retention, while it was not added in wells used to produce cytokine enriched supernatants. At the end of incubation, the 96-well plates were centrifuged at 800 x g/5 min, the medium supernatants were recovered and the cells used to perform intracellular cytokine FACS staining. Alternatively, after the 6 h of incubation, 2 x 105 g-iTCLs (stimulated as described in IFN-{gamma}-ELISPOT assay paragraph) were added to the DC-NK cells culture to perform intracellular cytokine FACS staining for an additional 36 h of incubation. For the cytokine-enriched supernatant transfer assay, 100 µl of medium supernatant from NK cell/medium-DCs or NK cell/PT-gliadin-DCs or NK cell/PT-beta-lactoglobulin-DC coculture were added to 2 x 105 g-iTCLs (stimulated as described in IFN-{gamma}-ELISPOT assay paragraph) to perform intracellular cytokine FACS staining after a 36-h incubation.

A cytokine staining kit (Caltag Laboratories) and FACS analysis were used to evaluate intracellular cytokine production in NK cells and g-iTCLs. IFN-{gamma} and TNF-{alpha} production was stained by anti IFN-{gamma} (4SB3) and anti-TNF-{alpha} (MAbeta11) mAbs (BD Pharmingen).

Cytotoxicity assay

Cytotoxicity was measured in a conventional 4-h 51Cr release assay. Forty-eight-hour IL-2-activated polyclonal NK cell lines were used as effectors. iDC targets were labeled with Na251CrO4 (Amersham Life Sciences) (100 µCi/1 x 106 cells), and the percentage of specific lysis was calculated as ((experimental release – spontaneous release)/(max release – spontaneous release)) x 100. The spontaneous release never exceeded 20%. Blocking of receptors on NK cells was made by two anti-NKG2A mAbs (Z270 and Z199 clones), two anti-CD94 (XA185 and HP3B1) mAbs (1 µg/ml), anti-CD158a (KIR2DL1, p58.1), or anti-CD158b (KIR2DL2, p58.2). Z199, Z270, XA185, p58.1, and p58.2 mAbs were gifts from Dr. L. Moretta (Istituto Gaslini). HP3B1 was a gift from M. Lopez-Botet (University Pompeu Fabra, Barcelona, Spain). As control, we used the 345.134 mAb (1 µg/ml), recognizing a glycoprotein widely expressed on human leukocytes, that was a gift from Dr. S. Ferrone (Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY).

HLA-E peptide design and stabilization studies

Gliadin chains were analyzed using Gene Jockey I software (Biosoft). Gliadin polypeptide sequences were matched to canonical HLA-E-binding peptide sequence VMAPRTLLL and compared with sequences generated by exchanging canonical amino acid moieties with other aa compatible with HLA-E binding and CD94 recognition (45).

The gliadin-derived peptide SQQPYLQLQ was used comparatively with positive control peptide VMAPRTLLL in HLA-E molecule stabilization experiments by using the murine HLA-E transfected RMA-S cell line (a gift from Dr. C. Mingari, Istituto Gaslini). HLA-A2-restricted influenza A virus peptide (FLU-M1 58–66, GILGFVFTL) was used as negative control. Peptides were synthesized by PRIMM. HLA-E+ RMA-S cell line was incubated overnight at 37°C in presence of the synthetic peptides at the concentration of 200 µM. After incubation, the cells were recovered and analyzed by FACS for the expression of HLA-E.

Immunohistochemistry and confocal microscopy analysis

Biopsy specimens from distal jejunum of 10 untreated CD patients (12–35 years old) were obtained during upper gastrointestinal endoscopies. Diagnosis was based on typical mucosal lesions with crypt cell hyperplasia and villous atrophy. All CD patients were positive for anti-endomysial Abs. Duodenal biopsies were also obtained from 8 treated CD patients (21–46 years old) and from 8 controls (23–54 years old) all having normal mucosal architecture with long villi and short crypts and all negative for endomysial Abs.

Tissue sections were prepared as described previously (46). Anti-HLA-E mAb (MEM-02; 4 µg/ml) was added at 4°C overnight. Primary Ab (4 µg/ml) was omitted in sections used as negative control samples. Slides were incubated with anti-mouse mAb conjugated to HRP (1/50 dilution; DakoCytomation) for 40 min at room temperature (RT). Immune-reactive cells were visualized by diaminobenzidine and hematoxylin (Sigma-Aldrich). Isotype control sections were prepared replacing anti-HLA-E mAb with purified mouse IgG1 mAb (Serotec). The number of cells expressing HLA-E was evaluated within a total area of 1 mm2 of lamina propria. The counts were double-blind analyzed independently by two observers.

In experiments to detect NK cells (CD94+CD3–) and HLA-A, -B, and -C Ags (G46-2.6 clone from BD Pharmingen), immunofluorescence combined with confocal microscopy was used. Cryosection were fixed in acetone and incubated for 1 h at RT with a mixture of mouse mAbs anti-CD94 human (1/25 dilution; Immunotech) and rabbit polyclonal anti-CD3 human (1/100 dilution; DakoCytomation), followed by a mixture of horse anti-mouse FITC conjugated (1/200 dilution; Vector Laboratories) and swine anti-rabbit tetramethylrhodamine isothiocyanate conjugated (1/300 dilution; DakoCytomation). These Abs were applied for 45 min in the dark, followed by 30 min of incubation with ToPro-3 (Molecular Probes, Invitrogen) for counterstaining the nuclei. Finally, the sections were mounted in PBS:glycerol (1/1 dilution). To detect HLA-A, -B, and -C Ags, the cryosections were fixed in acetone and incubated for 1 h at room temperature with anti HLA-A, -B, and -C mAb (1/300), followed by 30 min of horse anti-mouse FITC-conjugated incubation. CD94+ and CD3+ immunofluorescence cells were imaged with a Leica SP confocal microscope.

Immunohistochemistry analysis in organ cultures

HLA-E staining was performed as described previously (47). Briefly, staining with anti HLA-E (3D12) and Ig1 isotype-matched mAb control on 4-µm cryostat sections from small intestinal biopsies in OCT compound (Sakura Fine Technologies) and snap-frozen in liquid nitrogen were used. Binding was detected by using secondary anti-mouse Ab conjugated to HRP-labeled polymer (EnVision+system; DakoCytomation) and diaminobenzidine chromogen. Sections were counterstained with Gill’s 3 hematoxylin, and coverslip was attached by using SubX mounting medium (Surgipath; Medical). The organ culture system was generated as described previously (48).

Statistical analysis

Statistical analysis was performed by using GraphPad Prism software (GraphPad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
An unique fashion of gliadin pretreatment in modulating the NK cell susceptibility of iDCs

We tested celiac patient NK cell cytotoxicity against autologous iDCs incubated with PT-gliadin or with PT-beta-lactoglobulin, as PT-protein control, or with medium alone. iDCs were derived from patient 1 (Fig. 1, A and B), patient 2 (Fig. 1, C and D), a normal donor (Fig. 1, E and F), and tested against autologous (Fig. 1, A, C, and E) and allogeneic (Fig. 1, B, D, and F) polyclonal NK cell lines. A strong decrease of NK cell-mediated lysis susceptibility was observed specifically when the iDC target was pretreated with the PT-gliadin, if compared with medium alone incubation or with PT of other food derived proteins, such as beta-lactoglobulin, lacto-albumin, OVA, and casein (Fig. 1G).


Figure 1
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  		FIGURE 1. Gliadin confers a NK cell autologous and allogeneic resistant iDC phenotype in both patients and normal donors. Human IL-2-activated polyclonal NK cell lines were tested in a cytotoxicity assay against human autologous and allogeneic DC targets. DCs were derived from two patients (A–D) and from one normal donor (E and F) and tested against autologous (A, C, and E) and allogeneic (B, D, and F) polyclonal NK cell lines. DCs were pretreated with PT-gliadin ({square}) or with PT-beta-lactoglobulin ({blacktriangleup}) or with medium alone ({circ}). Gliadin, but not other food-derived proteins, inhibits DC recognition by NK cells (G). iDCs were pretreated with PT-gliadin ({square}) or with PT-beta-lactoglobulin ({blacktriangleup}), PT-lacto-albumin ({triangleup}), PT-OVA (•), PT casein ({blacksquare}), or with medium alone ({circ}). CD94/NKG2A blockade reverted the gliadin-dependent inhibition of the NK cell/iDC targets recognition (H). Autologous iDCs incubated with PT-gliadin ({triangleup}, {square}, and •) or with medium alone ({circ}). Before adding to PT-gliadin-treated DCs, NK cells were incubated with anti-CD94/NKG2A (Z270) ({triangleup}) or with control 345.134 mAb (•). The percentage of lysis and E:T ratio were reported on the abscissa and ordinate axes, respectively. Data are representative of one of four independent experiments.

 
Gliadin pretreatment conferred autologous and allogeneic NK cell lysis resistance to iDCs in patients as well as in healthy donors. The differences were statistically significant (p < 0.05). To perform statistic analysis of results, we excluded from this study fresh, not IL-2-stimulated, NK cells because of their low cytotoxicity against autologous iDCs (data not shown). When NK cells were preincubated with anti-NKG2A mAb (Z270), their killing activity against PT-gliadin pulsed autologous target cells was restored at level similar to medium alone DCs (Fig. 1H). As previously described (16, 33, 49, 50, 51), the anti-NKG2A mAb (Z270) pretreatment of NK effectors enhanced their killing against medium or PT-beta-lactoglobulin incubated DCs (data not shown). Similar results were observed by using another anti-NKG2A (Z199) mAb as well as with anti-CD94 mAbs (XA185 and HP3B1), while no effect was observed when NK cell treatment was performed by using mAbs against the CD158a or CD158b KIRs (data not shown) or by 345.134 control mAb. Therefore, it appears conceivable that part of the gliadin induced DC protection from NK cell recognition could be NKG2A dependent. Because HLA-E is the canonical CD94/NKG2A ligand (52), these experiments point to an involvement of HLA-E in the regulation of NK cell-dependent, PT-gliadin-treated iDC lysis.

Notably, the celiac NK cell killing activity against the K562 cell line was comparable to the cytotoxic activity exerted by NK cells from the normal donors (data not shown). These suggest that the natural cytotoxicity baseline levels are unaffected in celiac patients.

DC phenotype: gliadin and its fractions effect on HLA-E expression

DC maturation was analyzed and no significant differences in the expression of CD14, MHC-I, MHC class II, CD1a, CD40, CD83, CD86, CD11c, CD1c, and BDCA3 molecules were found in healthy donor or celiac patient-derived iDCs preincubated with PT-gliadin or with PT-beta-lactoglobulin if compared with medium alone incubation. DC phenotype evaluation was performed either in absence or in presence of NK cells in coculture (Fig. 2 and data not shown). Notably, DCs generated in vitro for our experiments share CD11c+CD86+BDCA3– phenotype (Fig. 2) with the recently described DCs that accumulate in duodenal sections from celiac-active stage patients (53).


Figure 2
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  		FIGURE 2. The effect of gliadin on DC phenotype in NK cell-DC coculture. NK cell-DC coculture were harvested after 6 h of incubation to evaluate the expression of CD1a, CD86, CD11c, BDCA3, and HLA-I Ags on DCs in the different condition of DC treatments (as indicated); y-values indicate the percentage of positive cells, and means ± SD were reported. The DCs were gated basing on their high forward scatter (data not shown). Results from three independent experiments were summarized.

 
Therefore, we suggest that gliadin did not interfere with DC phenotype maturation in NK cells/DC cross-talk (Fig. 2). A significant (p < 0.05) higher expression of HLA-E was observed in celiac patient derived, PT-gliadin-treated iDCs compared with their PT-beta-lactoglobulin-treated counterpart (Fig. 3A) or with the other food protein-derived PT-digest treatments (data not shown).


Figure 3
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  		FIGURE 3. Gliadin up-regulates the HLA-E expression on DCs. FACS-staining profile for HLA-E molecule on DCs treated with medium alone, PT-gliadin, and PT-beta-lactoglobulin, as indicated (A). HLA-E expression detected with 3D12 mAb on celiac patient-derived DCs treated with {alpha}, {gamma}, and {omega} fractions or medium alone, as indicated (B). The mean fluorescence intensities (MFIs) were reported on the abscissas. The effect on DCs was observed in four independent experiments performed by using DCs from healthy donors, summarized as means ± SD (C) (*, p < 0.05). DCs were gated basing on their high forward scatter and stained with anti-CD14 and anti-CD1a mAbs. In this experiment, MEM08 anti-HLA-E Ab was used. Live gates were set such that only single CD14–CD1a+cells were collected for the subsequent analysis (data not shown).

 
These data suggest that the increasing of HLA-E on PT-gliadin-treated DCs was a gliadin-specific effect.

Among the described (54), four principal gliadin-fractions ({alpha}1, {alpha}2, {gamma}, and {omega}), we tested the effect of the most represented: the {alpha} (all {alpha} fractions, see Materials and Methods), {gamma} and {omega} fractions. Fig. 3B shows the overexpression of HLA-E on healthy donor and celiac patient-derived iDCs, treated with {alpha} and {omega} gliadins. This effect was comparable to result obtained with the whole PT-gliadin incubation, and it was statistically significant (p < 0.05) in four independent experiments (Fig. 3C). In contrast, {gamma} gliadin conferred a weaker expression of HLA-E on iDCs.

To investigate the possibility that gliadin fraction DC incubation could generate HLA E-binding peptides, we predict in silico the homology in the amino acid sequences of known HLA-E-binding peptides (VMAPRTLLL and VQQPYAILF) (45), {alpha}1-, {alpha}2-, {gamma}-, and {omega}- gliadin fractions and the OVA, by using Gene Jockey I software (Table I).


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  		Table I. In silico prediction of gliadin fraction HLA E-binding peptides

 
Notably, aa 52–60 of {alpha}2 chain contains a nonamer SQQPYLQLQ carrying glutamine at P2 and P9 positions, known to be an amino acidic substitution compatible with the HLA-E cleft loading (52), although, we cannot exclude that other peptides with potential HLA-E binding motif are present in the gliadin fractions, see Table I.

To verify the functional effect of gliadin fractions on NK cell lysis, we performed cytotoxicity assays using as target iDCs incubated with either the whole gliadin or with its fractions (Fig. 4A). A direct correlation was found between {alpha} and {omega} gliadin fractions in inducing high levels of HLA-E on presenting iDCs and their inhibitory effect on autologous NK cell cytotoxicity. In addition, blocking of CD94/NKG2A receptor, by using Z270 mAb, was able to revert the NK cell lysis inhibition against iDCs treated with {alpha} (Fig. 4B) and {omega} (data not shown) gliadin fractions.


Figure 4
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  		FIGURE 4. The purified gliadin fractions induce differential effects on DC susceptibility to NK cell lysis. IL-2-activated polyclonal NK cell lines were used as effectors in a cytotoxicity assay against human allogeneic DC targets (A) incubated with medium alone ({circ}) or PT-beta-lactoglobulin ({blacktriangleup}) or PT-gliadin ({square}) or with {alpha} (•), {gamma} ({blacksquare}), and {omega} ({triangleup}) fractions. CD94/NKG2A blocking on NK effectors reverted {alpha} fraction-dependent lysis inhibition on DC targets (B). Autologous iDCs incubated with {alpha} gliadin ({square}, {triangleup}, and •), with PT-beta-lactologlobulin ({blacktriangleup}) or with medium alone ({circ}). Before adding to {alpha} gliadin-incubated DCs, NK cells were pretreated with anti-CD94/NKG2A (Z270) ({triangleup}) or with control 345.134 mAb (•) or with medium alone ({circ}). The percentage of lysis and E:T ratio were reported on the abscissas and ordinate axes, respectively. Data are representative of one of four independent experiments. NK cells and DCs were obtained from healthy donors.

 
These data suggest that gliadin fractions, mainly {alpha} and {omega}, could contain peptides able to load HLA-E cleft and preventing the killing by autologous NK.

To verify the effect of gliadin-derived peptides to increase the HLA-E surface expression, HLA class I stabilization experiments using the RMA-S/HLA-E-transfected cell system were performed, as described elsewhere (55). We have compared the HLA-E stabilization capacity of canonical VMAPRTLLL peptide with gluten, gliadin pancreatic digest (PTCE), and the gliadin-derived peptide SQQPYLQLQ (Fig. 5). Gluten, PTCE, and SQQPYLQLQ peptide potently increase the HLA-E levels on the cell membrane. In addition, the up-regulation of HLA-E molecules did not occur in RMA-S/HLA-E line performing experiment at low temperature (26°C), confirming previously reported results by Borrego et al. (56) and data not shown. This result suggests a role for active metabolism, as at 37°C, in processing and uploading peptides into HLA-E.


Figure 5
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  		FIGURE 5. Gluten and its pancreatic polypeptides digest and stabilize HLA-E molecules. HLA-E surface expression on HLA-E-transfected RMA-S cell line incubated with medium alone or with PTCE-gluten (A), PTCE-gliadin (B), SQQPYLQLQ peptide (C), VMAPRTLLL peptide (D), or GILGFVFTL as control peptide (E). Data are representative of one of three independent experiments.

 
Cytokine production in the interplay between gliadin-treated-DCs, NK cells, and gliadin-specific intestinal CD4+ T cells

No significant differences were found in the percentage of CD3–CD56+ NK cells in infiltrating lymphocytes between CD patients and healthy donor mucosa (mean ± SD): 1.7 ± 0.7% in untreated, 1.85 ± 0.27% in treated patients, and 1.67 ± 0.57% in control mucosa. In contrast, T lymphocytes (CD56–CD3+) were largely increased in acute CD compared with treated CD and healthy donor mucosa: 15.85 ± 10.59, 6.45 ± 4.4, and 10.3 ± 7.9%, respectively.

The gliadin-specific, T-dependent IFN-{gamma} response was analyzed using two different methods: ELISPOT, to preserve a more physiological conditions in absence of any treatment to induce cellular cytokine retention, and FACS intracellular staining, to detect the cytokine in the producing cell subsets.

Two CD3+CD4+CD69+CD94–, NKG2A–, and NKG2C– CD56– g-iTCLs were obtained from mucosal explants from HLA DQ2 patients (see Materials and Methods). IFN-{gamma} production by g-iTCLs was tested by ELISPOT (Fig. 6A) and by intracellular FACS staining (Fig. 6B) using autologous DCs pulsed with PT-gliadin-tissue transglutaminase or beta-lactoglobulin as control dietary protein. As expected, IFN-{gamma} production by the gliadin-restricted T cell lines were strongly significant when these cells were cocultured with gliadin-pulsed DCs compared with medium or beta-lactoglobulin-pulsed iDCs.


Figure 6
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  		FIGURE 6. Cytokine production in the interaction between NK cells, gliadin-treated DCs, and g-iTCLs. IFN-{gamma} responses were evaluated by ELISPOT assay (A) in the presence or absence of autologous, peripheral blood-derived NK cells. Medium ({square}) or PT-gliadin ({blacksquare})- or PT-beta-lactoglobulin (Figure 6)-pretreated autologous DCs were used in the coculture as indicated. Ratio g-iTCLs:APC:NK cells was of 1:2:1. DCs were incubated 6 h in the presence of untreated or BFA-treated NK cells, indicated as NK (BFA), before adding the responder g-iTCLs. Results were summarized as means ± SD of IFN-{gamma} spot count/well of four independent experiments. Statistical analysis is reported. IFN-{gamma} production by g-iTCLs was analyzed by intracellular FACS staining (B) in the autologous g-iTCLs/DC or g-iTCLs/DC/NK coculture at the different DC treatments, as indicated. IFN-{gamma} production by g-iTCLs was measured also after the addition of cytokine-enriched supernatants from NK cell/DC medium (SUP1) or NK cell/PT-gliadin DC (SUP2) or NK cell/PT-beta-lactoglobulin DC (SUP3) to g-iTCLs/DC coculture at the different DC treatments, as indicated (B). In the same experimental condition were analyzed the intracellular IFN-{gamma} (C) and TNF-{alpha} (D) staining in IL-2-activated polyclonal NK cells derived from a celiac patient incubated for 6 h with medium alone, PT-gliadin-, or PT-beta-lactoglobulin-treated autologous DCs, in absence or in presence of g-iTCLs, as indicated. In B, C, and D, y-axes values refer the percentage of cytokine-producing cells, as indicated. Live gates were set such that only CD3+CD56– g-iTCLs or CD3–CD56+ NK cells were collected for the subsequent analysis (data not shown). Statistical analysis is reported. Results from two independent experiments were summarized.

 
To address the possible regulatory effect of NK cells on IFN-{gamma} production by g-iTCLs, DCs were preincubated 6 h with autologous NK cells at g-iTCLs:APC:NK ratio of 1:2:1. A significant increased production was observed adding autologous NK cells in the PT-gliadin DC/g-iTCLs coculture, as scored using ELISPOT assays (Fig. 6A) and intracellular FACS staining (Fig. 6B).

The increasing of IFN-{gamma} production was significantly (p = 0.0450) prevented when NK cells were pretreated with a high dose (10 µg/ml) of Golgi protein trafficking blocker (BFA) to prevent their cytokines secretion (Fig. 6A).

Intriguingly, PT-gliadin treatment of DCs did not influence the NK cell-dependent IFN-{gamma} (Fig. 6, A and C) and TNF-{alpha} production (Fig. 6D) in the DC-NK cell coculture. In contrast, the addition of g-iTCLs to the NK cell/gliadin-treated DCs coculture prompted NK cells to an increased cytokine secretion (Fig. 6, C and D). In addition, the transfer of cytokine-enriched medium supernatant from coculture between NK cells and gliadin-pretreated DCs to the coculture between g-iTCLs and autologous gliadin-treated DCs did not enhanced the IFN-{gamma} production by g-iTCLs (Fig. 6B). Such data suggested that the "cell-to-cell contact" and the reciprocal cytokine-generated signal by NK cells/g-iTCLs interplay are crucial for the increased IFN-{gamma} response to gliadin.

As expected, in NK cell/DC/g-iTCLs coculture, we observed the presence of a higher number of the Ag-presenting, gliadin-treated DCs (ranging from 30 to 40% of surviving DCs) than medium- or PT-beta-lactolgobulin DCs (ranging from 15 to 25% of surviving DCs) (data not shown). We do not rule out that the rescue of PT-gliadin-DCs from NK cell killing could provide cell-to-cell contact and/or other soluble stimuli-inducing T and NK cells to cytokine secretion.

Mononuclear cells expressing high levels of HLA-E and CD3–CD94/NKG2A+ lymphocytes are present in the lamina propria from celiac patient

The number of mononuclear cells/mm2 expressing HLA-E was significantly higher (p < 0.01) in the lamina propria biopsy from untreated celiac patients (Fig. 7B) than from control (Fig. 7A) (mean ± SD: 153 ± 43 and 64 ± 39, respectively). No significant differences were found in biopsy specimens from treated CD patients (90 ± 53) when compared with healthy donors.


Figure 7
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  		FIGURE 7. Lamina propria from celiac patient shows the presence of mononuclear cells expressing high levels of HLA-E and of CD3–CD94/NKG2A+ lymphocytes. Mononuclear cells expressing HLA-E of both duodenal mucosa sections from healthy donor (A) and from celiac patient (B). Immunohistochemical analysis of HLA-E expression on enterocytes and in lamina propria of an active celiac patient (C) and a gluten-free diet patient stimulated overnight with medium (D) or gliadin (E). Magnification, x10. One representative experiment of three is shown. Immunofluorescence staining revealed similar levels of HLA-A, -B, and -C Ag expression in the basolateral membrane of epithelial and lamina propria mononuclear cells of duodenal mucosa of both CD patients (F) and normal control (G). In green the HLA-A, -B, and -C molecule staining by using G46-26 mAb, and in red the nuclei counterstaining by using ToPro-3 tracker (see Materials and Methods). One representative experiment of two is shown. H, CD3–CD94+ NK cells (red stained), CD3+CD94– T lymphocytes (green stained), and CD3+ CD94+ T cells (yellow stained) was detected by confocal microscopy of jejunal mucosa from an untreated celiac patient. One representative experiment of four is shown.

 
Immunohistochemical analysis demonstrated a very high HLA-E expression on enterocytes and monocytes in lamina propria of active celiac patient (Fig. 7C). In addition, overnight stimulation with gliadin of duodenal mucosa organ cultures highly up-regulated HLA-E expression in lamina propria mononuclear cells from a gluten-free diet patient (Fig. 7E); no HLA-E was detected after an overnight incubation with complete medium (Fig. 7D).

Because the induction of HLA-E could be dependent on the availability of leader sequences form HLA class I (45), we evaluated the expression of classical HLA-A, -B, and -C molecules in both untreated CD and normal duodenal section. Staining for HLA-A, -B, and -C molecules was intense on epithelial cells and in particular on basolateral membranes, as well as in lamina propria mononuclear cells (Fig. 7, F and G). Notably, none intensity difference in the jejunal mucosa was observed between untreated celiac patient (Fig. 7F) and control donor (Fig. 7G). This result is in agreement with previous data (57) and suggests a direct specific role for gliadin in the induction of high HLA-E expression level.

Confocal microscopy of jejunal mucosa from an untreated celiac patient (Fig. 7H) shows the simultaneous presence in the lamina propria of CD94/NKG2A+CD3– NK cells, CD94/NKG2A–CD3+ T lymphocytes, and CD94/NKG2A+CD3+ T cells. This result point to a potential role in vivo for CD94/NKG2A NK cells in the peripheral tissue control of surveillance regulation.

To evaluate the presence in the celiac gut of cells ascribing to DC phenotype and expressing HLA-E molecules, we stained for CD1a, CD1c, CD11c, CD14, BDCA3, and HLA-E molecules the mononuclear cells obtained from digestion of mucosal explants of untreated celiac patient (Fig. 8). We found the 3–4% of all mucosal explants mononuclear cells were CD45 CD1a double positive (Fig. 8A). In addition, CD45+/CD1a+ cells were also positive for HLA-E, CD1c, and CD11c and negative for CD14 and BDCA3 (Fig. 8B). To this regard, it is relevant that vast majority of ascribed duodenal mucosa myeloid DCs has been recently described as positive for CD11c, a typical myeloid DC marker (58), and negative for BDCA3, a marker differentially expressed on DC subsets (53, 59). These results could suggest the expression of HLA-E molecule on myeloid DC resident in intestinal mucosa of untreated CD patient.


Figure 8
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  		FIGURE 8. Celiac patient intestinal DC phenotype. Mononuclear cells from digestion of celiac patient duodenal mucosa explants were stained by FACS analysis with anti-CD45 and anti-CD1a mAbs. A live gate (R1 in A) was set to collect only CD45+CD1a+ mucosal explanted cells for the subsequent analysis of anti-HLA-E, -CD1c, -CD11c, -CD14, -BDCA3, and color isotype control mAb staining (B). One representative experiment of two is reported.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our data reveal gliadin peculiar biological features in interfering within the NK cell-DC cross-talk. We addressed the potential role for NK cell-DC cross-talk in the pathogenesis of CD, and we hypothesized that the CD94/NKG2A+ NK cell-dependent killing of iDCs could be part of the peripheral tolerance mechanisms preventing the immune system priming by not fully mature DCs (33).

In this regard, it is relevant that CD94/NKG2A+ NK cell subset could regulate the maturation of new DC precursors from the inflamed tissue to the lymphoid secondary organs (18), and it could prevent the aberrant priming activity of iDCs in the lymph nodes (35, 49, 50).

We suggest that gliadin could have a detrimental role in the regulation of NK cell-iDC cross-talk with consequences on the adaptive immunity activation. iDC gliadin treatment inhibits NK cell cytoxicity against iDCs via the interaction between CD94/NKG2A and HLA-E. Indeed, gliadin increases the HLA-E expression on iDCs and the blocking of CD94/NKG2A receptor on NK cells restores their killing against gliadin-treated iDCs.

Autologous interaction between gliadin-presenting iDCs, NK, and celiac patient-derived, T-restricted lines potently increases the T-dependent IFN-{gamma} response to gliadin. This effect appears to be dependent on cytokine production by NK cells since a secretion-inhibiting drug (BFA) pretreatment of NK cells strongly reduces the IFN-{gamma} CD4+ T response to gliadin. It is worth noting that the direct interplay between NK and T cells appears to generate a cytokine environment able to induce reciprocal activation. Indeed, the addition of both T and NK cells in the coculture with PT-gliadin-pretreated DCs strongly enhanced T and NK cell-dependent cytokine secretion, whereas IFN-{gamma} and TNF-{alpha} by NK cells was unaltered in the interaction with PT-gliadin-DCs alone. In addition, the presence of NK cells strongly increased the anti-gliadin-specific IFN-{gamma} T cell response primed by gliadin-presenting DCs. We observed that the NK cell/g-iTCLs interplay could increase cytokines production by NK cells and could contribute to foster IFN-{gamma} production by g-iTCLs in triple interaction between NK cell/DCs/g-iTCLs. We do not exclude that other cytokines could contribute to this effect. In this context, it remains to be solved the nature of all NK cell and T cytokine secretion patterns when cocultured with gliadin-treated iDCs. Because the 20–30% of our NK cell preparations are CD94/NKG2A negative, we do not rule out that other NK cell subsets, expressing the activating receptors, could establish different relationship with gliadin-presenting DCs and therefore contribute in a different way to celiac pathogenesis.

Moreover, we demonstrated the presence of HLA-E-expressing mononuclear cells and CD3–CD94/NKG2A+ NK lymphocytes in lamina propria from celiac patient. In addition, we found a coexpression of CD11c, a marker recently used to identify intestinal myeloid DCs (53) and HLA-E in CD45+CD1a+CD14–BDCA3–BDCA2– mononuclear cells from gut mucosa of CD patients. Such evidence points to the in vivo role for HLA-E-mediated myeloid iDC-NK cell interaction in celiac disease, as previously described for interaction between NK cell like-CTL and HLA-E-expressing enterocytes (60).

Given the multichain composition of the gliadin, we analyzed also which fraction was responsible for NK cell protection and which for the HLA-E up-regulation. Both biological activities were found to cluster in {alpha}2 and {omega} fractions. Gene Jockey I software reveals a nonamers potentially able to bound HLA-E. We focused on SQQPYLQLQ peptide derived from {alpha}2 chain. Even if this peptide increased the HLA-E molecule levels, the presence of a tyrosine makes it potentially susceptible to pancreatic enzymatic degradation. In this regard, the strong ability of gliadin-pancreatic digests to induce HLA-E expression on RMA-S suggests that pancreatic enzyme digestion could generate a large unknown and heterogeneous peptide repertoire binding to HLA-E molecule.

It is conceivable to speculate that gliadin could switch NK cell-iDC cross-talk from a tolerogenic pathway, based on iDC elimination, to a pathogenic one in which gliadin drives iDC-NK cell interaction to restricted CD4+T cells activation; either by proinflammatory cytokines production or by gliadin-presenting iDC maturation. In this respect, other studies suggested that NK cell-DC interaction provide a regulatory feedback of immune response during infectious and neoplastic diseases (51, 61) and could have a role in the disease pathogenesis (62). In addition, gliadin up-regulates IL-15 production (63); its 31–43 peptide stimulates IL-15 production from macrophage and DCs (11); and IL-15 induces MICA expression on epithelial cells and NK cell activation (10, 64).

Moreover, we demonstrated that gliadin could increase the expression of HLA-E on iDCs, while it does not affect the DC maturation degree.

Gliadin could shape the innate response toward a proinflammatory condition and together with other genetic features trigger the CD pathogenesis.

Preincubation of NK cells with anti-CD94/NKG2A mAb restores their killing against autologous celiac PT-gliadin-treated DCs, indicating that the gliadin processing could produce peptides increasing HLA-E expression on the APC. We speculate that gliadin processing could occur in a DC compartment where MHC-I and MHC class II are converging (65).

Immunohystochemical study in the celiac patient mucosa confirms in vitro data and suggests that HLA-E is up-regulated on APCs during the active stage of the disease and after overnight intestinal mucosa incubation with gliadin. Moreover, an extended immunohistochemical analysis of the CD patient showed that EC express high HLA-E levels after gliadin uptake (data not shown).

It is conceivable that NK cell-gliadin presenting DC interactions can be considered as pathogenetic cofactor operating in vivo together with other immunological mechanisms, all contributing in giving the final pathological treat.


   Acknowledgments
 
Dedicated to the memory of Professor Serafino Zappacosta. We thank Dr. L. Sollid for critical reading of the manuscript and suggestions, Miguel Lopez-Botet for helpful discussion, Dr. Daniel Geraghty for providing the 3D12 anti-HLA-E Ab, Dr. Heather Bond for providing Gene Jockey I software, Dr. Carmen Palomba for the initial experimental support, C. Meccariello for technical help, and Tricia Reynolds for revising the English version of the manuscript.


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


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

1 This work was supported by grants from the Italian Association for Celiac Disease, the Italian Association for Cancer Research, and the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Giuseppe Terrazzano, Department of Cellular and Molecular Biology and Pathology, University Federico II, Naples, Italy. E-mail address: terrazza{at}unina.it Back

3 Abbreviations used in this paper: CD, celiac disease; BFA, brefeldin A; DC, dendritic cell; g-iTCL, gliadin-restricted intestinal T cell line; iDC, immature DC; KIR, killer Ig-like receptor; MHC-I, MHC class I; PT, peptic-tryptic digest; PTCE, pepsine-trypsin-chymotrypsin-elastasis digest; RT, room temperature. Back

Received for publication July 20, 2006. Accepted for publication April 16, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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E. J. Petrie, C. S. Clements, J. Lin, L. C. Sullivan, D. Johnson, T. Huyton, A. Heroux, H. L. Hoare, T. Beddoe, H. H. Reid, et al.
CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence
J. Exp. Med., March 17, 2008; 205(3): 725 - 735.
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