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In a population of 46 children with CD recruited in the Paris area of France, an excess of DRB1∗03 and DRB1∗07 alleles and of DR3/DR7, DR3/ DR3, and DR11(or12)/DR7 phenotypes was found (RRs of 6.3, 9.3, 24.6,15, and15.1, respectively), which is reminiscent of the markers of susceptibility observed in southern rather than in northern European celiac patients. More importantly, the highest association with CD was not found in individuals expressing the DQA1∗0501 -DQB1 ∗ 0201 heterodimer in single dosage (RR = 24.9) or in homozygous state, but in people co-expressing one copy of DQA1∗0501-DQB1∗0201 on one haplotype and a second copy of DQB1∗0201 on the second haplotype (RR = 35.7). This suggests that in our population either DQB1∗0201 or a gene closely linked to DQB1∗0201 influences the susceptibility to CD conferred by the DQA1∗0501-DQB1∗0201 heterodimer. Significant positive or negative RRs conferred by some TAP2 or DPB1 alleles were found. However, they were moderate compared to the RR conferred by the expression of a second copy of DQB1∗0201. Moreover, they were no longer significant when patients were compared with HLA-DR matched controls. This suggests that associations of CD with TAP2 and DPB1 alleles are secondary to linkage disequilibria and argues against the contribution of these alleles in resistance and/or susceptibility to CD. Thus the “raison d'etre” of a “DQB1∗0201 second haplotype effect” in susceptibility to CD remains to be elucidated.

Susceptibility to CD is strongly associated with particular HLA class II molecules. However, additive genetic factors are likely to be required for the development of the disease. The polymorphic TAP and LMP genes, located within the HLA class II region, are involved in the antigen presentation pathway and thus represent candidate susceptibility genes. HLA class II DRB1, DRB3, DQA1, DQB1, and DPB1 as well as TAP1, TAP2, and LMP2 polymorphism was studied in 80 Caucasian CD patients and 213 normal controls by DNA oligotyping. The DQB1*0201 allele was found in 96.3% of CD patients and provided the highest risk (RR = 50), whereas only 89% of CD patients carried the DQ alpha 501/beta 201 heterodimer (RR = 30). The participation of the DRB3 and DPB1 locus was ruled out as it was attributed to a linkage disequilibrium on the DR3 haplotype. TAP1 and LMP2 allelic distribution was not significantly different among CD patients and controls. The TAP2-C allele was completely absent from the CD population, while it was found in 22.5% of controls. Although linkage disequilibrium between TAP2 and class II loci clearly exists in some haplotypes, TAP could act as additional susceptibility genes.

HLA association with myasthenia gravis (MG) has been studied in a series of 114 patients using class I and class II genotyping after PCR amplification. Positive association was found with DR3, particularly in women (RR=2.6) and in early MG onset (RR=3.4). DRB1, DRB3, DQB1, DQA1 and B (B8 and B18_genotyping revealed that the association was predominantly with the B8 DRB1 ∗ 03 DRB3 ∗ 0101 DQB1 ∗ 0201 DQA1 ∗ 0501 ancestral haplotype. This haplotype frequency was also increased in patients with thymic hyperplasia (RR=3.5) and was greatly reduced in patients with thymona (RR=0.35). Sixteen out of 48 patients carrying this 8.1 ancestral haplotype showed absence of B8 ( n =4) or of DR3 ( n =12). HLA class II genotyping further revealed the existence of two other significant associations. MG was positively associated with the DQB1 ∗ 0604 allele (RR=3.4), particularly in patients with thymona (RR=5.7). Furthermore, the disease was negatively associated with DR1 in females (RR=0.32). These data suggest that MG is placed under the control of at least three distinct genes: (1) a class II predisposing gene in the 8.1 ancestral haplotype; (2) a thymona-associated class II allele on the DQB1 ∗ 0604 haplotype; and (3) a protective allele DR1.

HLA class II association with insulin-dependent diabetes mellitus (IDDM) is well established but is still difficult to map to a particular locus. Polymorphism of the genes coding for transporter associated with antigen processing (TAP1 and TAP2), and located in the HLA class II region, was studied in 167 IDDM patients (116 adult-onset and 51 childhood-onset patients) and 98 normal controls using oligotyping after genomic amplification. A dominant protective effect was observed for the TAP2*0201 allele [relative risk (RR) = 0.3, corrected probability (pc) < 0.001]. Conversely, susceptibility to IDDM was associated with apparent homozygosity for the TAP2*0101 allele (RR = 3.4, pc < 0.001). Protection was independent from but additive to the protection conferred by the DRB1*02 DQB1*0602 haplotype (RR = 0.06, pc < 0.05), and antagonistic to the DRB1*03 DQB1*0201 and DRB1*04 DQB1*0302 haplotypes predisposing effect (RR = 1.1, not significant), arguing in favor of an absence of linkage disequilibrium between TAP2 and HLA class II genes. This was assessed by chi 2 analysis. TAP1 allelic distribution was not different among diabetics and controls. A significant association was observed between the presence of TAP2*0101 and that of islet cell antibodies (p < 0.05). These data suggest that the TAP2 gene, which encodes protein required for delivery of antigen peptides to class I molecules in the endoplasmic reticulum, could modulate the autoimmune response leading to beta cell destruction. From a practical point of view, they make the combined screening of HLA class II and TAP2 loci a highly valuable tool in IDDM prediction.

Like other organ-specific autoimmune diseases, endocrine autoimmune diseases are genetically determined. The role of HLA remains central, essentially by permitting recognition of autoantigenic peptides and ultimately differentiation of helper or effector T cells. The association, only strong in the case of IDDM, is significant but much less clear cut for other diseases that are mostly associated with DR3, posing the question of other mechanisms than Ir gene control. The nature and role of non HLA genes are still elusive. However, regarding the data obtained in the spontaneous experimental models of the NOD mouse, the BB rat and the obese chicken it may be anticipated that such genes will be defined in man in a not too distant future. Undoubtedly, their knowledge will illuminate the common features and unique pecularities of all these diseases and hopefully provide, together with HLA genes, the basis for new therapeutic approaches that will no longer be based only on substitutive therapy, as performed nowadays.

Introduction Les anticorps anti-cellules d'ilot (ICA) sont un marqueur diagnostique du DT1. Ils sont presents chez 85–90 % des patients a la decouverte du DT1 mais il est admis qu'ils disparaissent dans les annees suivantes. La recherche d'anticorps anti-GAD et anti-IA-2 remplace desormais celle des ICA. Nous avons evalue la persistance a long terme de ces deux anticorps chez 227 patients DT1 suivis dans le service. Patients et Methodes Les anticorps anti-GAD et anti-IA-2 ont ete recherches chez des patients (122 femmes, 105 hommes) ayant un DT1 evoluant depuis plus de 10 ans. Le diagnostic de DT1 a ete etabli sur les criteres suivants : âge au diagnostic inferieur a 20 ans et/ou existence d'une cetose et/ou presence d'autoanticorps au diagnostic, insulinodependance d'emblee. Les parametres associes a la persistance des anticorps ont ete etudies en analyse univariee. Resultats L'âge median au diagnostic de DT1 etait de 13 ans (1–70), l'anciennete du DT1 au moment de l'etude de 19 ans (10–65). La prevalence des anticorps etait de 61 % (un anticorps dans 38 %, les deux dans 23 % des cas), plus elevee chez les femmes (68 % vs 52 %, p=0,02), ou en cas d'autoimmunite extra-pancreatique associee au DT1 (70 % vs 56 %, p=0,05). La persistance des anticorps etait inversement associee a la duree d'evolution du DT1 (10–14 ans : 82 % ; 15–19 ans : 68 % ; 20–24 ans : 58 % ; 25 ans et plus : 35 %, p=0,0001). La positivite des anticorps n'etait pas associee aux alleles HLA DR3 et/ou DR4. La persistance des anti-GAD etait plus frequente (46 %) que celle des anti-IA-2 (37 %), significativement chez les femmes (p = 0,002). Chez une patiente, des anticorps anti-GAD etaient presents 65 ans apres le debut du DT1. Il n'y avait pas de difference concernant les doses d'insuline (0,73 vs 0,70 UI/kg. j) et l'HbA1c (en HPLC, 7,8 vs 7,8 %) au moment de l'analyse chez les patients ayant ou non des anticorps. Conclusion Contrairement a une idee recue, la persistance a long terme d'autoanticorps est un phenomene frequent au cours du DT1. Cette observation a des consequences pratiques pour le diagnostic etiologique d'un diabete, meme tres a distance de sa survenue. Elle suggere egalement la persistance a long terme d'une reponse immune anti-cellules d'ilot au cours du DT1.

The association of insulin-dependent diabetes mellitus (IDDM) with certain HLA alleles is well documented in pediatric patients. Whether a similar association is found in adult-on-set IDDM is not clear, although the disease occurs after the age of 20 in 50% of cases. HLA class II DRB1, DQA1, and DQB1 alleles were studied in 402 type I diabetics and 405 healthy controls (all Caucasian) using oligonucleotide typing after gene amplification. Alleles DRB1*03, DRB1*04, DQB1*0201, DQB1*0302, DQA1*0301, and DQA1*0501 were indeed enriched in diabetics and the highest relative risk was observed in patients carrying both the DRB1*03-DQB1*0201 and the DRB1*0402 or DRB1*0405-DQB1*0302 haplotypes. However none of these alleles, or specific residues, could alone account for the susceptibility to IDDM. Furthermore, there were major differences in HLA class II gene profiles according to the age of onset. Patients with onset after 15 yr (n = 290) showed a significantly higher percentage of non-DR3/non-DR4 genotypes than those with childhood onset (n = 112) and a lower percentage of DR3/4 genotypes. These non-DR3/non-DR4 patients, although presenting clinically as IDDM type 1 patients, showed a lower frequency of islet cell antibodies at diagnosis and a significantly milder initial insulin deficiency. These subjects probably represent a particular subset of IDDM patients in whom frequency increases with age. The data confirm the genetic heterogeneity of IDDM and call for caution in extrapolating to adult patients the genetic concepts derived from childhood IDDM.

Conventional detection of islet cell antibodies is based on indirect immunofluorescence performed on frozen human pancreas sections. The number and nature of epitopes recognized by antibodies detected by such techniques are unknown. To determine the existence of heterogeneous fluorescence patterns of islet cell antibodies on pancreatic sections, we selected two sera showing a distinctive granular fluorescence. We then tested random sera from patients with Type 1 (insulin-dependent) diabetes mellitus for their ability to block ultimate binding of fluorescein isothiocyanate-labelled immunoglobulins purified from these two sera with a characteristic granular pattern. Among 102 subjects with recent-onset Type 1 diabetes, 79 had detectable anti-islet cell antibodies; 21 showed complete blockade of the binding to islets of granular fluorescein isothiocyanate-labelled immunoglobulins. The majority of these 21 patients were women carrying a DR3 non-DR4 DQB1*0201 allele, with under-representation of DRB1*0402 and 0405. Discrimination between islet cell antigenic specificities may help in identifying islet cell autoantibodies in autoimmune Type 1 diabetes.

Abstract 346 Introduction: Invariant Natural Killer T (iNKT) cells express a highly restricted T cell receptor (TCR) repertoire composed of a single invariant Va14Ja18 chain in mice and a Va24Ja18 chain in humans. In contrast to their conventional counterpart, iNKT cells recognize CD1d-bound glycolipids rather than peptides. iNKT cells have elicited a lively interest in the last years because of their implication in several immune responses including experimental graft-versus-host-disease (GVHD). In this study, we addressed the question whether human iNKT cells could be associated with the outcome of allogeneic haematopoietic stem cell transplantation (HSCT). Materials and methods: Forty-seven patients allografted for diverse hematological malignancies in our institution entered prospectively the study. We sequentially analyzed the reconstitution of peripheral blood CD1d tetramer + iNKT, CD56+ NK, and CD4+, CD8+, CD4+CD25high T cells for 90 days after allogeneic HSCT by flow cytometry. Results were correlated to the clinical evolution of allogeneic HSCT. Results: We observed two groups of patients according to their iNKT/106 T cell ratios on days 30, 60 and 90 after HSCT. Patients with at least one point above the normal ratio of 1000 iNKT/106 T cells were considered in the iNKT high group (n=17), while those with a poor reconstitution, defined by all points below that threshold, were analyzed in the iNKT low group (n=30). iNKT reconstitution was the only significant difference in terms of immune reconstitution between the two groups since reconstitution of T CD4+, CD8+, or CD4+CD25high and NK cells was similar at all time points in both groups. Chimerism, analyzed by PCR amplification of short tandem repeat markers, showed that in all cases iNKT displayed a 100% donor origin. Pre-transplant characteristics of patients were similar between the two groups except for the conditioning regimen and the source of stem cells. Patients in the iNKT high group had more often received a reduced-intensity conditioning (80.5% versus 46.7%, p=0.004) and peripheral blood stem cells (88.5% versus 53.6%, p=0.015). Occurrence and severity of acute GVHD was significantly reduced in the iNKT high group (23.5 % grade I-II and 0% grade III-IV) compared to the iNKT low group (66.7 % grade I-II and 26.6% grade III-IV), (p Conclusion: Our data suggest that donor-derived iNKT cell reconstitution after allogeneic HSCT may represent a predictive factor of acute GVHD and a prognostic factor of transplant outcome. Disclosures: No relevant conflicts of interest to declare.

Microchimerism after allogeneic organ transplantation may be a mechanism for induction of donor-specific graft acceptance. However, the frequency of chimerism and its relevance in long-term tolerance are uncertain. We studied 15 long-surviving (more than 20 years) cadaveric-kidney transplant recipients for the systemic presence of donor alleles with allele-specific genomic amplification of DRB1 and H-Y loci. Microchimerism was observed in 1 case in peripheral blood and in 4 cases in skin. Chimerism and number of HLA alleles shared by donor and recipient were not correlated. This low frequency of microchimerism in long-term kidney allograft recipients raises doubts about a major participation of chimerism in donor-specific tolerance.

MICA molecules interact with the NKG2D-activating receptor on human NK and CD8 T cells. We investigated the participation of the MICA/NKG2D pathway in the destruction of intestinal epithelium by intraepithelial T lymphocytes (IEL) in Celiac disease and its premalignant complication, refractory sprue. We show that MICA is strongly expressed at epithelial cell surface in patients with active disease and is induced by gliadin or its p31-49 derived peptide upon in vitro challenge, an effect relayed by IL-15. This triggers direct activation and costimulation of IEL through engagement of NKG2D, leading to an innate-like cytotoxicity toward epithelial targets and enhanced TCR-dependent CD8 T cell-mediated adaptive response. Villous atrophy in Celiac disease might thus be ascribed to an IEL-mediated damage to enterocytes involving NKG2D/MICA interaction after gliadin-induced expression of MICA on gut epithelium. This supports a key role for MIC/NKG2D in the activation of intraepithelial immunity in response to danger.

OKT3 exerts its in vivo immunosuppressive effects by inducing major peripheral T cell depletion as well as antigenic modulation of the T3/Ti T cell receptor complex. Modulated cells, which reversibly lose the expression of the CD3 T cell receptor molecular complex but still share the CD4 and CD8 antigens, have been shown to be functionally immunoincompetent. Antigenic modulation is maintained as long as significant OKT3 serum levels are present. Cells infiltrating renal allografts from seven OKT3 treated patients were studied by double immunofluorescence to assess whether antigenic modulation could affect cells located in profound organs such as renal allografts. Needle biopsies were obtained in patients given OKT3 (5 mg/day) for at least 10 consecutive days in association with conventional immunosuppressive drugs for treatment of a rejection episode (5 cases) or prophylactically (2 cases). In all patients at the time of biopsy, CD3 positive cells were absent from the circulation, significant OKT3 serum levels were present, and neither IgG nor IgM anti-OKT3 antibodies were detected. Infiltrating cells were double-labeled using a combination of either anti-CD3 and anti-CD4 or anti-CD3 and anti-CD8 monoclonal antibodies. Following 7-14 consecutive days of treatment, all patients given OKT3 for a rejection episode showed a significant decrease in the number of graft-infiltrating lymphocytes. Importantly, all T cells still infiltrating the allograft were CD3-CD4+ or CD3-CD8+ cells, which is exactly the same phenotypical pattern of CD3 circulating modulated T cells. In 6 out of the 7 patients, this phenotypical pattern was associated with clinically normal graft function. These results further underline the fact that antigenic modulation is an important mechanism mediating the immunosuppressive effect of OKT3 both in peripheral blood and in renal allografts.

The genetics of resistance to infection by HIV-1 cohort consists of 200 slow and 75 rapid progressors to AIDS corresponding to the extremes of HIV disease outcome of 20,000 Caucasians of European descent. A comprehensive analysis of HLA class I and class II genes in this highly informative cohort has identified HLA alleles associated with fast or slow progression, including several not described previously. A quantitative analysis shows an overall HLA influence independent of and equal in magnitude (for the protective effect) to the effect of the CCR5-Δ32 mutation. Among HLA class I genes, A29 (p = 0.001) and B22 (p < 0.0001) are significantly associated with rapid progression, whereas B14 (p = 0.001) and C8 (p = 0.004) are significantly associated with nonprogression. The class I alleles B27, B57, C14 (protective), and C16, as well as B35 (susceptible), are also influential, but their effects are less robust. Influence of class II alleles was only observed for DR11. These results confirm the influence of the immune system on disease progression and may have implications on peptide-based vaccine development.

International audience; Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease characterized by a lymphocytic exocrinopathy. However, patients often have evidence of systemic autoimmunity, and they are at markedly increased risk for the development of non- Hodgkin's lymphoma. Similar to other autoimmune disorders, a strong interferon (IFN) signature is present among subsets of pSS patients, although the precise etiology remains uncertain. NCR3/NKp30 is a natural killer (NK)-specific activating receptor regulating the cross talk between NK and dendritic cells and type II IFN secretion. We performed a case-control study of genetic polymorphisms of the NCR3/NKp30 gene and found that rs11575837 (G>A) residing in the promoter was associated with reduced gene transcription and function as well as protection to pSS. We also demonstrated that circulating levels of NCR3/NKp30 were significantly increased among pSS patients compared with controls and correlated with higher NCR3/NKp30 but not CD16-dependent IFN-γ secretion by NK cells. Excess accumulation of NK cells in minor salivary glands correlated with the severity of the exocrinopathy. B7H6, the ligand of NKp30, was expressed by salivary epithelial cells. These findings suggest that NK cells may promote an NKp30-dependent inflammatory state in salivary glands and that blockade of the B7H6/NKp30 axis could be clinically relevant in pSS.

Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (Ad) cause significant morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation. We have established a procedure to generate polyclonal cytotoxic T lymphocyte (CTL) populations with specificity against Ad and CMV or against Ad and EBV. Healthy donor-derived dendritic cells (DCs) were transduced with recombinant adenovirus encoding either CMV pp65 or EBV EBNA3C and used to stimulate autologous T cells. Stimulated T lymphocytes displayed specific simultaneous cytotoxicity against CMV and adenovirus and to a lesser extent against adenovirus and EBV. Recombinant vaccinia virus encoding individual adenovirus proteins showed that the T cell response to the adenovirus was directed mainly against the capsid protein hexon. The frequency of IFN-gamma-secreting T cells was 0.02% for adenovirus alone, and 0.05 and 0.14% for adenoviruses encoding EBNA3C and pp65, respectively. pp65-specific CTLs killed autologous fibroblasts infected with the laboratory strain CMV AD169. The culture conditions were specific as alloreactive T cells were not expanded. Therefore, this approach could be considered in order to generate efficient virus cytolytic T cells to be used as adoptive immunotherapy in transplanted patients.

The nonclassical MHC class I molecule MHC class I-related chain A (MICA) interacts with the NKG2D receptor expressed at the surface of most peripheral CD8 T cells, γδ T cells, and NK cells. We investigated the role of MICA-NKG2D interactions in the selection or maturation of the T cell repertoire within the thymus using MICA tetramers and anti-MICA mAbs. MICA tetramers identified a small population of late stage CD8 single-positive, CD45RA+ CD62L+ CCR7+ CD69− thymocytes, a phenotype compatible with that of fully mature CD8+ cells ready to emigrate to the periphery as naive cells. MICA molecules were expressed in the outer layer of Hassal’s corpuscles within the medulla of normal thymus. In thymomas, an overexpression of MICA in cortical and medullar epithelial cells was observed. This was associated with a decreased percentage of NKG2D-positive thymocytes, which expressed a less mature phenotype than in normal thymus. These results indicate that CD8+ thymocytes up-regulate NKG2D as they complete their developmental program before leaving the thymic medulla to seed the periphery, and identify NKG2D as a potential regulator of the developmental processes in T cells that are essential for immune homeostasis.

Maternally inherited diabetes and deafness (MIDD), which is seen in 0.5% to 2.8% of patients with type 2 diabetes mellitus, is related to a point mutation at position 3243 of mitochondrial (mt) DNA. Its clinical description is incomplete.To study the clinical presentation and complications of diabetes in patients with MIDD and to identify clinical characteristics that may help select diabetic patients for mtDNA mutation screening.Multicenter prospective descriptive study.16 French departments of internal medicine, diabetes and metabolic diseases, or both.54 patients with type 2 diabetes mellitus and the mtDNA 3243 mutation.Characteristics of diabetes, metabolic control (glycosylated hemoglobin level), complications of diabetes, and involvement of other organs.On average, patients with MIDD were young at diabetes onset and presented with a normal or low body mass index. None were obese. Seventy-three percent of probands had a maternal family history of diabetes. Diabetes was non-insulin-dependent at onset in 87% of patients; however, 46% of patients had non-insulin-dependent disease at onset but progressed to insulin therapy after a mean duration of approximately 10 years. Neurosensory hearing loss was present in almost all patients. Eighty-six percent of patients who received an ophthalmologic examination had macular pattern dystrophy (a specific retinal lesion). Forty-three percent of patients had myopathy, 15% had cardiomyopathy, and 18% (9 of 51) had neuropsychiatric symptoms. Although the prevalence of diabetic retinopathy was 8% among patients who received an ophthalmologic examination, lower than expected after a mean 12-year duration of diabetes, prevalence of kidney disease was 28%. This suggests that a specific renal involvement was the result of mitochondrial disease.Maternally inherited diabetes and deafness has a specific clinical profile that may help identify diabetic patients for mtDNA testing.