Function of DQ2 and DQ8 as HLA susceptibility molecules in celiac disease

MINIREVIEW

Function of DQ2 and DQ8 as HLA Susceptibility Molecules in Celiac Disease Knut E. A. Lundin, Henrik A. Gjertsen, Helge Scott, Ludvig M. Sollid, and Erik Thorsby

ABBREVIATIONS APC antigen-presenting cell B-LCL B-lymphoblastoid cell line CD celiac disease mAb monoclonal antibody

TCC TCL TCR

T-cell clone T-cell line T-cell receptor

INTRODUCTION Celiac disease (CD) is a malabsorptive disorder of the small intestine. The disease is a unique model for studies of HLA-associated diseases since (a) the disease shows a stringent HLA association; (b) the disease-inducing agent (wheat gluten and related antigens from other cereals) is known; (c) activated, gluten-specific CD4 + T cells in the lamina propria of the small intestine most likely play an important immunopathogenic role; and (d) the target organ is accessible for obtaining biopsy specimens. Here we report studies on the mechanism behind the HLA association.

H L A A S S O C I A T I O N IN CELIAC DISEASE A strong association to the DQ(otl*0501, ]31"0201) heterodimer, i.e., DQ2, has been found in all populations studied [ 1]. This DQ heterodimer may be encoded by the DQAI*0501 and DQBI*0201 genes in cis position on DR3DQ2 haplotypes, or by the same two genes in trans position by DR5DQ7/DR7DQ2 heterozygotes [2]. This DQ heterodimer is carried by 90% or more of CD patients. However, approximately 2%-10% of CD patients (approximately 20% of CD patients from Bolo-

From the Institute of Transplantation Immunology (K.E.A.L., H.A.G. , L.M.S. , E. T. ) and the Institute of Pathology (H. S. ), National Hospital, Oslo, Norway. Address reprint requests to Dr. E. Thorsby, Institute of Transplantation Immunology, Nanonal Hospital, Oslo, Norway. 24 0198-8859/94/$7.00

gna, Italy; see Sollid and Thorsby [ 1]) do not carry DQ2. Most of them instead carry different variants of DR4 [ 1] and share the DQ(o~1"0301, ~1"0302) heterodimer, i.e., DQ8 [4, 5]. Thus, CD appears to be primarily associated with DQ2 and to a lesser extent with DQ8.

I S O L A T I O N OF GLUTEN-SPECIFIC T CELLS FROM T H E SMALL I N T E S T I N E The celiac lesion is characterized by a strong immunologic activation [1]. Treatment of the patients with a gluten-free diet, however, is usually followed by morphologic normalization and disease remission. Stimulation ex vivo of small intestinal biopsy specimens with a peptic/tryptic digest of gluten induces rapid activation (CD25 expression) of T cells in the lamina propria of treated CD patients, but not of the non-CD patients [6]. We isolated such activated T cells by an immunomagnetic method (beads coated with anti CD25 monoclonal antibody; mAb) and propagated them in the absence of further stimulation with gluten. T-cell lines (TCLs) were established from six of eight CD patients, and in four of these gluten-specific T-cell responses were observed (weak responses in one case; see Lundin et al. [7, 8]).

DQ2-RESTRICTED, GLUTEN-SPECIFIC T CELLS Gluten-specific T cells were isolated from small intestinal biopsy specimens of three patients carrying the Human Immunology 41, 24-27 (1994) © American Society for Histocompa~ibility and Immunogenetics, 1994

Minireview

Studies of the ability of DQ2 and DQ8 to bind various gluten-derived peptides are under way. Several important questions remain to be answered. First, what is the mechanism behind the tissue injury? The cytokine profiles of the DQ2- and DQ-8-restricted gluten-specific T cells from the small intestinal mucosa of CD patients have recently been shown to be dominated by Interferon-y (Nilsen et al. submitted for publication). Secondly, why is it only peptides from gluten, and not those derived from many other ingested proteins, that in susceptible individuals may cause the immunopathology leading to CD? Thirdly, if DQ2 and DQ8 mainly bind potential immunopathogenic peptides from gluten in the intestinal mucosa, why is it that only a minor proportion of individuals carrying DQ2 or DQ8 ever develop CD? These and other questions are common or relevant for most autoimmune disorders. As mentioned, CD is a good model for further studies because the primary HLA associations have been established and the triggering antigen is known.

ACKNOWLEDGMENTS

Our studies are supported by grants from the Research Council of Norway and by Pronova AS. REFERENCES 1. Sollid LM, Thorsby E: HLA susceptibility genes in celiac disease: genetic mapping and role in pathogenesis. Gastroenterology 105:910, 19932. Sollid LM, Markussen G, Ek J, Gjerde H, Vartdal F, Thorsby E: Evidence for a primary association of celiac disease to a particular HLA-DQ ot/[3 heterodimer. J Exp Med 169:345, 1989. 3. Mantovani V, Corazza GR, Bragliani M, Frisoni M, Zaniboni MG, Gasbarrani G: Asp-57 negative HLA DQ[3 chain and DQAI*0501 allele are essential for the onset of DQw2-positive and DQw2-negative coeliac disease. Clin Exp Immunol 91:153, 1993.

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4. Spurkland A, Sollid LM, Polanco I, Vartdal F, Thorsby E: HLA-DR and -DQ genotypes of celiac disease patients serologically typed to be non-DR3 and non-DR5/7. Hum Immunol 35:188, 1992. 5. Tighe MR, Hall MA, Ashkenazi A, Siegler E, Lanchbury JS, Ciclitira PJ: Celiac disease among Ashkenazi Jews from Israel: a study of the HLA class II alleles and their association with disease susceptibility. Hum Immunol 38:270, 1993. 6. Halstensen TS, Scott H, Fausa O, Brandtzaeg P: Gluten stimulation of coeliac mucosa in vitro induces activation (CD25) of lamina propria CD4 + T cells and macrophages but no crypt cell hyperplasia. Scand J Immunol 39:581, 19937. Lundin KEA, Scott H, Hansen T, Paulsen G, Halstensen TS, Fausa O, Thorsby E, Sollid LM: Gliadin-specific, HLA-DQ(o~1"0501,[31"0201) restricted T cells isolated from the small intestinal mucosa o(celiac disease patients. J Exp Med 178:187, 1993. 8. Lundin KEA, Scott H, Fausa O, Thorsby E, Sollid LM: T cells from the small intestinal mucosa of a DR4,DQ7/ DR4,DQ8 celiac disease patient preferentially recognize gliadin when presented by DQ8. Hum Immunol 1994 (in press). 9. Gjertsen HA, Lundin KEA, Sollid LM, Eriksen JA, Thorsby E: T cells recognize a peptide derived from ot-ghadin presented by the celiac-disease-associated HLADQ(otl*0501,[31*0201) heterodimer. Hum Immunol 39:243, 1994. 10. Sturgess RP, Day P, Ellis HJ, Lundin KEA, Gjertsen HA, Kantakou M, Ciclitira PJ: Wheat peptide challenge in coeliac disease. Lancet 343:758, 1994. 11. Sj6str6m H, Friis SU, Noren O, Anthonsen D: Purification and characterization of antigenic gliadins in coeliac disease. Clin Chim Acta 207:227: 1992. 12. Gjertsen HA, Sollid LM, EkJ, Thorsby E, Lundin KEA: T cells from the peripheral blood of celiac disease patients recognize gluten antigens when presented by HLA-DR, -DQ, or-DP molecules. ScandJ Immunol 39:567, 1994.

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K . E . A . Lundin et al.

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data not shown). The only difference in the first domain between DQ8 and DQ9 is an Ala to Asp substitution at residue 57 of the DQ~ chain, while DQ7 differs from DQ8 at four residues of the DQI3 chain. Thus, DQ7, 8, and 9 are very similar and could very well present the same or very similar gluten-derived peptides to DQ8restricted T cells, when a mixture of peptides from a peptic/tryptic digest of gluten is used as antigen. A N T I G E N SPECIFICITY OF T H E GLUTEN-SPECIFIC T CELLS Identification of the gluten-derived peptides being recognized by small intestinal CD4 + T cells is important. All of the TCLs were established using gluten (containing both glutenin and gliadins), but uniformly only recognized the gliadin moiety [7, 8]. Gliadins are considered to be the harmful antigens in CD [1]. Some few TCCs also recognized gluten-related antigen both from wheat and rye, bur not from barley. We also examined the reactivity of the TCCs toward a panel of synthetic peptides covering the N-terminal region of one particular ot-gliadin. One of these peprides is recognized by a DQ2-restricted TCC from the peripheral blood of a CD patient [9] and induces histologic changes of the mucosa of CD patients following in vivo challenge [10]. However, none of the gut-derived TCCs recognized any of these peptides. We have also tested the gut-derived TCCs against gliadin fractions and purified gliadins isolated from the wheat strain Kadett [11]. Of two gutderived DQ2-restricted TCCs, one recognized an epitope common to one ot-gliadin and two different 3,-gliadins, while the other TCC recognized only o~-gliadin. Three gut-derived DQ8-restricted TCCs recognized only the two "y-gliadins (Sj6str6m et al., manuscript in preparation). Thus, while more studies are necessary to establish their peptide fine specificity, heterogeneity in peptide

FIGURE 2 Glutenspecific responses of four TCCs (1.12, 2.2, 2.5, and 2.17) from patient CD282 (DR4;DQ7,8). Legends otherwise as for Fig. l.

specificity of gut-derived gliadin-specific T cells from CD patients is likely. GLUTEN-SPECIFIC T CELLS IN P E R I P H E R A L BLOOD In both CD patients on a gluten-free diet and healthy controls, responses to gluten of peripheral blood T cells were detected, the strongest responses being observed among some CD patients. In the patients, many of the gluten-reactive T cells were restricted by DQ2 or DQ8 molecules, but also several DR and some DP-restricted gluten-reactive T cells were found [12]. Thus, even though more DQ-restricted T cells were found in peripheral blood when gluten instead of other antigens (PPD, TT, etc.) were used for in vitro stimulation, the extensive preponderance of DQ2 or DQ8 restriction seen of gluten-specific T cells from the intestinal mucosa was not observed. The reason is unknown, but may be due to differences in gluten antigen processing and/or expression of DQ molecules of the APCs in peripheral blood compared with intestinal mucosa. CONCLUSIONS CD appears to be primarily associated with D Q 2 - - i . e . , DQ(o~ 1"0501, [31"020 l)---and less strongly with DQ8. Possible mechanisms include effects of DQ2 and DQ8 during thymic development of potential gluten-reactive T cells or preferential binding and presentation to T cells of some gluten-derived peptides by DQ2 and DQ8 in the intestinal mucosa. Since no predominant TCR usage of the gluten-reactive T cells from the intestinal mucosa was detected, and since most of these gluten-reactive T cells only recognize gluten-derived peptides when presented by DQ2 or DQ8, the latter mechanism appears most likely. However, more patients need to be studied.

Degenerate T-Cell Recognition

that a given class II protein can bind many different peptides. In addition, a lack of allele specificity has been shown for interaction between HLA-DR proteins and some peptides described as promiscuous [4-6]. Contrasting with the weak discrimination existing between MHC and peptides, the recognition of MHC-peptide complexes by TCRs is believed to be highly specific and is in most cases critically dependent on both peptide and MHC polymorphism. There are, however, exceptions to this rule as the existence of Ag-specific T-cell clones recognizing the same epitope in the context of several distinct DR molecules has been reported [4, 7]. In this study, we took advantage of such a T-cell clone that recognizes promiscuous peptide HA306-320 in the context of several distant DR molecules. We comparatively analyzed the structures, the Ag-binding abilities, and the Ag-presenting properties of various HLA-DR alleles. Results were interpreted to clarify the molecular basis for T-cell degenerate recognition. MATERIALS AND METHODS Antigens. Synthetic peptide HA306-329 from the influenza A/Texas/77 virus hemagglutinin (CPKYVKQNTLKLATGMRNVPEKQT) was purchased from Neosystem (Strasbourg, France). Peptide HA306-320 was synthesized by a solid-phase method using a multiple-synthesis apparatus [8]. It was analyzed by high-performance liquid chromatography and purified. For binding assays, peptide HA306-320 was biotinylated at the N terminus after protection of side chains [8].

Antigen-presenting cells. Antigen-presenting cells (APCs) were homozygous B-lymphoblastoid cell lines (B-LCLs) from the Tenth International Histocompatibility Workshop (New York, November 1987). B-LCL BOB was the kind gift of Dr. A. Urlacher (CRTS, Strasbourg, France). The cell line RM3 (class-II-negative mutant) used in binding assays was the kind gift of Dr. B. Benichou (CNRS UPR420, Villejuif, France). T-cell clone. The T-cell clone G S l l is issued from a DRll01/0401 donor whose DRB1 sequence was deduced from oligotyping analysis. This clone was obtained as previously described [9]. Briefly, peripheral blood mononuclear cells (PBMCs) were primed with peptide HA306-329 (20 ~g/ml). After 6 days, recombinant interleukin 2 (10 IU/ml) was added every 2 days until day 14. Then, repeated secondary antigenic stimulations were performed every week, using irradiated autologous PBMCs previously incubated with peptide HA306-329, until an oligoclonal Ag-specific T-cell line was obtained. This T-cell line was then cloned by a limiting-dilution procedure at 0.3 cell/well as previously described [9], and clone GS 11 was expanded by weekly

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stimulations with irradiated PBMCs, PHA (1 ~g/ml), and interleukin 2.

Chromium-release assay. Chromium-release tests (CRTs) were performed as described [9], 7-10 days after the clone's last stimulation. Briefly, B-LCLs used as target cells were labeled with 7.4 MBq Na251CrO4. Then, 5 × 103 labeled cells were seeded into V-bottomed microtiter plates together with the peptide (5 gg/ml) and the clone GS11 at an effector-target ratio of 10:1. After a 4 hours of incubation at 37°C, 100 ~l of supernatant was harvested from each well and counted. Tests were performed in triplicate. Results are expressed as described in the figure legends. For the cold-target inhibition assay, each target was pre-pulsed (or not) with the peptide for 1.5 hours. Cold targets were seeded together with the effector cells 30 minutes before labeled target. Binding assays. Binding assays were performed as previously described [9]. Briefly, 3.5 × 105 B-LCLs were incubated with biotinylated peptide HA306--320 (40 ~M) at 37°C for 4 hours. Cells were washed twice at 4°C, resuspended in 5 p~g/ml of streptavidin-fluorescein conjugate (fluorescein isothiocyanate, FITC) and incubated at 4°C for 30 minutes. After washings and resuspension of the cells, the cell surface fluorescence was measured by flow cytometry on a FACScan analyzer (Becton Dickinson, Mountain View, CA, USA). The mean fluorescence of 5000 stained cells was determined. Dead cells were excluded from the analysis by staining with propidium iodide (1 ~g/ml). The background noise value was assessed by measuring the fluorescence in the absence of biotinylated peptide and was subtracted from all measurements. RESULTS

HA306-320 peptide is recognized by T-cell clone GS11 in association with several different class II molecules. In a previous study [9], we showed that a proliferative and cytolytic CD4 ÷ T-cell clone, GS11, specifically recognized peptide HA306-320 presented by the autologous DRl101 molecule as demonstrated in blocking experiments using various anti-class-II monoclonal antibodies (mAbs) and by restriction-pattern analysis. This latter analysis showed that DR11 molecules substituted at positions 86 (DRl104), 86 ÷ 71 (DRl103), or 86 ÷ 71 ÷ 67 (DRl102) were much less efficient or inefficient in presenting peptide HA306-320 to GS11 [9]. In the present study, we analyzed the recognition of the same peptide by GS11 in the context of additional class II molecules by testing a large panel of HLA-phenotyped homozygous APCs in cytolysis assays. As shown in Fig. 1, several allogeneic APCs efficiently present peptide