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Summary Background Refractory coeliac disease type 2 is a rare subtype of coeliac disease with high mortality rates; interleukin 15 (IL-15) is strongly implicated in its pathophysiology. This trial aimed to investigate the effects of AMG 714, an anti-IL-15 monoclonal antibody, on the activity and symptoms of refractory coeliac disease type 2. Methods This was a randomised, double-blind, placebo-controlled, phase 2a study of adults with a confirmed diagnosis of refractory coeliac disease type 2. Patients were randomly assigned at a 2:1 ratio to receive seven intravenous doses over 10 weeks of AMG 714 (8 mg/kg) or matching placebo. Biopsy samples were obtained at baseline and week 12 for cellular analysis and histology. The change in the proportion of aberrant intraepithelial lymphocytes from baseline to week 12 with respect to all intraepithelial lymphocytes was the primary endpoint and was quantified using flow cytometry. Secondary endpoints were the change in aberrant intraepithelial lymphocytes with respect to intestinal epithelial cells; intestinal histological scores (villous height-to-crypt depth ratio; VHCD); intraepithelial lymphocyte counts; Marsh score; and patient-reported symptom measures, including the Bristol stool form scale (BSFS) and gastrointestinal symptom rating scale (GSRS). Main analyses were done in the per-protocol population of patients who received their assigned treatment, provided evaluable biopsy samples, and did not have major protocol deviations; only patients with non-atypical disease were included in the analyses of aberrant intraepithelial lymphocytes, including the primary analysis. Safety was assessed in all patients who received at least one dose of study drug. This study is registered at ClinicalTrials.gov (NCT02633020) and EudraCT (2015-004063-36). Findings From April 13, 2016, to Jan 19, 2017, 28 patients were enrolled and randomly assigned to AMG 714 (n=19) and placebo (n=9). Six patients were not included in the primary analysis because of protocol deviation (one in the AMG 714 group), insufficient biopsy samples (one in the AMG 714 group), and atypical intraepithelial lymphocytes (three in the AMG 714 group and one in the placebo group). At 12 weeks, the least square mean difference between AMG 714 and placebo in the relative change from baseline in aberrant intraepithelial lymphocyte percentage was −4·85% (90% CI −30·26 to 20·56; p=0·75). The difference between the AMG 714 and placebo groups in aberrant intraepithelial lymphocytes with respect to epithelial cells at 12 weeks was −38·22% (90% CI −95·73 to 19·29; nominal p=0·18); the difference in change in Marsh score from baseline was 0·09% (95% CI −1·60–1·90; nominal p=0·92); the difference in VHCD ratio was 10·67% (95% CI −38·97 to 60·31; nominal p=0·66); and the difference in change in total intraepithelial lymphocyte count was −12·73% (95% CI −77·57–52·12); nominal p=0·69). Regarding symptoms, the proportion of patients with diarrhoea per the BSFS score decreased from ten (53%) of 19 at baseline to seven (37%) of 19 at week 12 in the AMG 714 group and increased from two (22%) of nine at baseline to four (44%) of nine at week 12 in the placebo group (nominal p=0·0008); and the difference between the groups in change in GSRS score was −0·14 (SE 0·19; nominal p=0·48). Eight (89%) patients in the placebo group and 17 (89%) in the AMG 714 group had treatment-emergent adverse events, including one (11%) patient in the placebo group and five (26%) in the AMG 714 group who had serious adverse events. The most common adverse event in the AMG 714 group was nasopharyngitis (eight [42%] patients vs one [11%] in the placebo group). Interpretation In patients with refractory coeliac disease type 2 who were treated with AMG 714 or placebo for 10 weeks, there was no difference between the groups in terms of the primary endpoint of aberrant intraepithelial lymphocyte reduction from baseline. Effects on symptoms and other endpoints suggest that further research of AMG 714 may be warranted in patients with refractory coeliac disease type 2. Funding Celimmune and Amgen.

Type 1 Innate lymphoid cells (ILC1) accumulate in the inflamed mucosa of patients with Crohn’s disease (CD) but their role in CD pathogenesis remains poorly known. In a recent issue of Nature materials, Jowett et al. (Nat. Mat. 2020) used a coculture model with intestinal organoids to show that ILC1 could promote intestinal epithelial growth and tissue remodeling through an unexpected mechanism that involves the transforming growth factor 1 (TGF-β1) and the metalloproteinase MMP9.

Objectives Assessment of the adaptive immune response against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is crucial for studying long‐term immunity and vaccine strategies. We quantified IFNγ‐secreting T cells reactive against the main viral SARS‐CoV‐2 antigens using a standardised enzyme‐linked immunospot assay (ELISpot). Methods Overlapping peptide pools built from the sequences of M, N and S viral proteins and a mix (MNS) were used as antigens. Using IFNγ T‐CoV‐Spot assay, we assessed T‐cell and antibody responses in mild, moderate and severe SARS‐CoV‐2 patients and in control samples collected before the outbreak. Results Specific T cells were assessed in 60 consecutive patients (mild, n = 26; moderate, n = 10; and severe patients, n = 24) during their follow‐up (median time from symptom onset [interquartile range]: 36 days [28;53]). T cells against M, N and S peptide pools were detected in n = 60 (100%), n = 56 (93.3%), n = 55 patients (91.7%), respectively. Using the MNS mix, IFNγ T‐CoV‐Spot assay showed a specificity of 96.7% (95% CI, 88.5–99.6%) and a specificity of 90.3% (75.2–98.0%). The frequency of reactive T cells observed with M, S and MNS mix pools correlated with severity and with levels of anti‐S1 and anti‐RBD serum antibodies. Conclusion IFNγ T‐CoV‐Spot assay is a reliable method to explore specific T cells in large cohorts of patients. This test may become a useful tool to assess the long‐lived memory T‐cell response after vaccination. Our study demonstrates that SARS‐CoV‐2 patients developing a severe disease achieve a higher adaptive immune response., We quantified IFNg‐secreting T cells reactive against the M, N and S viral SARS‐CoV‐2 proteins using a standardized enzyme‐linked immunospot assay in 60 patients. The frequency of reactive T cells correlated with severity, and with levels of anti‐S1 and anti‐RBD (receptor binding domain) serum antibodies, demonstrating a higher adaptive immune response after developing a more severe disease. IFNg T‐CoV‐Spot assay may also become a useful tool to assess the long‐lived memory T cell response after vaccination.

ObjectiveEnteropathy-associated T-cell lymphoma (EATL) is a rare but severe complication of celiac disease (CeD), often preceded by low-grade clonal intraepithelial lymphoproliferation, referred to as type II refractory CeD (RCDII). Knowledge on underlying oncogenic mechanisms remains scarce. Here, we analysed and compared the mutational landscape of RCDII and EATL in order to identify genetic drivers of CeD-associated lymphomagenesis.DesignPure populations of RCDII-cells derived from intestinal biopsies (n=9) or sorted from blood (n=2) were analysed by whole exome sequencing, comparative genomic hybridization and RNA-sequencing. Biopsies from RCDII (n=50), EATL (n=19), type I refractory CeD (n=7) and uncomplicated CeD (n=7) were analysed by targeted next-generation sequencing. Moreover, functional in vitro studies and drug testing were performed in RCDII-derived cell lines.Results80% of RCDII and 90% of EATL displayed somatic gain-of-functions mutations in the JAK1-STAT3 pathway, including a remarkable p.G1097 hotspot mutation in the JAK1 kinase-domain in approximately 50% of cases. Other recurrent somatic events were deleterious mutations in NFκB-regulators TNFAIP3 and TNIP3 and potentially oncogenic mutations in TET2, KMT2D and DDX3X. JAK1 inhibitors and the proteasome inhibitor bortezomib could block survival and proliferation of malignant RCDII-cell lines.ConclusionMutations activating the JAK1-STAT3 pathway appear to be the main drivers of CeD-associated lymphomagenesis. In concert with mutations in negative regulators of NFκB, they may favour the clonal emergence of malignant lymphocytes in the cytokine-rich coeliac intestine. The identified mutations are attractive therapeutic targets to treat RCDII and block progression towards EATL.

Interleukin-15 (IL-15) is a critical regulator of immune responses, especially at mucosal interfaces within the gastro-intestinal tract. Here, we describe the discovery and characterization of a humanized antibody to IL-15. Data from its epitope and mode of action, cell biology and primate pharmacology, as well as translational studies in human samples and in vivo proof-of-concept experiments in mouse models demonstrate the therapeutic potential of this new antibody targeting IL-15 for refractory celiac disease and eosinophilic esophagitis.

ObjectivesPrimary GI T-cell lymphoproliferative diseases (T-LPD) are heterogeneous entities, which raise difficult diagnosis and therapeutic challenges. We have recently provided evidences that lymphomas complicating coeliac disease (CD) arise from innate-like lymphocytes, which may carry NK receptors (NKRs).DesignNKRs expression was compared by flow cytometry in intraepithelial lymphocytes (IEL) from CD, type I or type II refractory CD (RCD). NKp46 was next assessed by immunohistochemistry in paraffin-embedded biopsies from 204 patients with CD, RCDI, RCDII or GI T-cell lymphomas and from a validation cohort of 61 patients. The cytotoxic properties of an anti-NKp46 monoclonal antibody conjugated to pyrrolobenzodiazepine (PBD) was tested ex vivo in human primary tumour cells isolated from fresh duodenal biopsies.ResultsNKp46 (but not CD94, NKG2A, NKG2C, NKG2D) was significantly more expressed by malignant RCDII IEL than by normal IEL in CD and RCDI. In paraffin biopsies, detection of >25 NKp46+ IEL per 100 epithelial cells discriminated RCDII from CD and RCDI. NKp46 was also detected in enteropathy-associated T-cell lymphomas (EATL, 24/29) and in monomorphic epitheliotropic intestinal T-cell lymphomas (MEITL, 4/4) but not in indolent T-LPD (0/15). Treatment with anti-NKp46-PBD could efficiently and selectively kill human NKp46+ primary IEL ex vivo.ConclusionNKp46 is a novel biomarker useful for diagnosis and therapeutic stratification of GI T-LPD. Strong preclinical rationale identifies anti-NKp46-PBD as a promising therapy for RCDII, EATL and MEITL.

Cell sheet technology opens new perspectives in tissue regeneration therapy by providing readily implantable, scaffold-free 3D tissue constructs. Many studies have focused on the therapeutic effects of cell sheet implantation while relatively little attention has concerned the fate of the implanted cells in vivo. The aim of the present study was to track longitudinally the cells implanted in the cell sheets in vivo in target tissues. To this end we (i) endowed bone marrow-derived mesenchymal stem cells (BMMSCs) with imaging properties by double labeling with fluorescent and magnetic tracers, (ii) applied BMMSC cell sheets to a digestive fistula model in mice, (iii) tracked the BMMSC fate in vivo by MRI and probe-based confocal laser endomicroscopy (pCLE), and (iv) quantified healing of the fistula. We show that image-guided longitudinal follow-up can document both the fate of the cell sheet-derived BMMSCs and their healing capacity. Moreover, our theranostic approach informs on the mechanism of action, either directly by integration of cell sheet-derived BMMSCs into the host tissue or indirectly through the release of signaling molecules in the host tissue. Multimodal imaging and clinical evaluation converged to attest that cell sheet grafting resulted in minimal clinical inflammation, improved fistula healing, reduced tissue fibrosis and enhanced microvasculature density. At the molecular level, cell sheet transplantation induced an increase in the expression of anti-inflammatory cytokines (TGF-s2 and IL-10) and host intestinal growth factors involved in tissue repair (EGF and VEGF). Multimodal imaging is useful for tracking cell sheets and for noninvasive follow-up of their regenerative properties.

Celiac disease is a chronic inflammation of the small intestine with villous atrophy that can become refractory to a gluten-free diet. Two categories of refractory celiac disease can be distinguished by the phenotype of intraepithelial lymphocytes and the status of TRG genes. Their distinction is important because 30% to 50% of type II but only 0% to 14% of type I evolve to an aggressive enteropathy-associated T-cell lymphoma and therefore require intensive treatment. Currently, differential diagnosis integrates immunohistochemistry, immunophenotyping, and TRG clonality analyses, but each has limitations. A single-tube multiplex TRG PCR (ECN) was prospectively compared to an in-house two-tube TRG PCR (N2T) in 73 samples, including 67 cryopreserved intestine tissues. Thirteen formalin-fixed, paraffin-embedded (FFPE) samples were also analyzed retrospectively. The ECN PCR had comparable efficiency to detect major clonal rearrangements in highly infiltrated tissues from T-cell lymphoproliferative disorders and type II refractory celiac disease and to detect the persistence of minor clones in type II refractory celiac disease follow-up samples. The ECN PCR abolished the risk of amplification of false-positive weak clonal rearrangements in cryopreserved specimens and allowed improved detection of clonal rearrangements in DNA from FFPE samples. The ECN PCR allows robust assessment of cryopreserved and FFPE digestive tissues at diagnosis and follow-up of enteropathies with villous atrophy, thus guiding therapeutic management.

Background: The immune response causing celiac disease (CD) depends on the activation of intestinal CD4+ T cells by gluten-derived peptides presented by HLA-DQ2 or HLA-DQ8 molecules, the main genetic risk factor. However, additional factors are necessary to impair immune tolerance to dietary gluten, to stimulate intraepithelial lymphocytes (IEL) and to induce intestinal damage. Key Messages: Current data point to a central role of interleukin-15 (IL-15). In situ and ex vivo studies indicate that IL-15 stimulates the accumulation and cytotoxic activation of CD8+ T IEL in active CD, and that of the malignant innate-like IEL in type II refractory CD (RCDII). Other studies show that IL-15 impairs the immunoregulatory control of effector T cells, notably CD8+. Recently, animal models have been designed to investigate the respective role of CD4+ T cells and IL-15 in CD. We discuss more particularly our results in such a model, which shows that IL-15 produced in excess in the intestine can cooperate with CD4+ T cells specific for a dietary antigen to trigger a celiac-like enteropathy. In this mouse model, CD4+ T cells activated by dietary ovalbumin secreted IL-2 which, along with IL-15, stimulated the expansion of noncognate intestinal cytotoxic CD8+ T cells containing large amounts of granzyme B. In the presence of IL-15, the latter cells did not respond to regulatory T cells, and accumulated in the intestine close to epithelial damage. Conclusion: On the basis of these data, we propose that, in CD, gluten-specific CD4+ T cells synthesize cytokines that synergize with IL-15 to license the expansion and activation of cytotoxic IEL, which drive tissue damage. We suggest that IL-15 is a meaningful therapeutic target, notably in patients with RCDII in which malignant IEL can respond to IL-15 independently of signals provided by CD4+ T cells.

The NKG2 family of NK receptors includes activating and inhibitory members. With the exception of the homodimer-forming NKG2D, NKG2 receptors recognize the nonclassical MHC class I molecule HLA-E, and they can be subdivided into two groups: those that associate with and signal through DAP12 to activate cells, and those that contain an ITIM motif to promote inhibition. The function of NKG2 family member NKG2E is unclear in humans, and its surface expression has never been conclusively established, largely because there is no Ab that binds specifically to NKG2E. Seeking to determine a role for this molecule, we chose to investigate its expression and ability to form complexes with intracellular signaling molecules. We found that NKG2E was capable of associating with CD94 and DAP12 but that the complex was retained intracellularly at the endoplasmic reticulum instead of being expressed on cell surfaces, and that this localization was dependent on a sequence of hydrophobic amino acids in the extracellular domain of NKG2E. Because this particular sequence has emerged and been conserved selectively among higher order primates evolutionarily, this observation raises the intriguing possibility that NKG2E may function as an intracellular protein. Copyright © 2014 by The American Association of Immunologists, Inc.

Background & Aims CD4 + T cells specific for dietary gluten and interleukin 15 (IL15) contribute to the pathogenesis of celiac disease. We investigated whether and how they interact to damage the intestine using mice that overexpress human IL15 in the intestinal epithelium and have CD4 + T cells specific for ovalbumin, a dietary antigen. Methods We crossed mice with CD4 + T cells specific for ovalbumin (OTII) with mice that overexpress human IL15 under an intestine-specific promoter (B6 × IL15Tge). The offspring (OTII × IL15Tge mice) received control or ovalbumin-containing diets until 3 months of age. Enteropathy was monitored by weight, ratio of villous:crypt length, and the number of intestinal lymphocytes. Phenotype, cytokine production, and degranulation of mucosal and spleen lymphocytes were analyzed by multicolor flow cytometry or enzyme-linked immunosorbent assay. Regulatory T-cell function and CD8 + T-cell activation were analyzed in co-culture assays. Results Exposure to ovalbumin reduced growth and led to enteropathy in OTII × IL15Tge mice but not in control OTII × B6 littermates. Enteropathy was associated with expansion of mucosal granzyme B + CD8 + T cells, and developed despite increased frequency of functional ovalbumin-specific regulatory T cells. Ovalbumin-activated CD4 + T cells secreted IL2, which along with IL15 stimulated expansion of noncognate intestinal cytotoxic CD8 + T cells, which did not respond to regulatory T cells and induced epithelial damage. Conclusions We observed that in mice given food antigen, cooperation between IL15 and CD4 + T cells is necessary and sufficient to activate CD8 + T cells and damage the small intestine. We propose that this process is involved in the development of celiac disease.

Refractory celiac disease is defined by the persistence of symptoms of malnutrition and intestinal villous atrophy for more than 6-12 months despite strict gluten-free diet in celiac patients. Diagnosis of this rare condition is made after excluding other causes of chronic small intestinal inflammation and villous atrophy and inadvertent intake of gluten. Over the past 15 years, multidisciplinary approaches have been developed to assess the mechanism of resistance to the diet, and two distinct entities have been delineated. Type II refractory celiac disease (RCD) can be defined as a low-grade intraepithelial lymphoma. RCD II is characterised by a massive accumulation of abnormal IEL that display an aberrant hybrid NK/T cell phenotype, contain clonal T cell rearrangement(s) and can mediate a cytolytic attack of the gut epithelium. This condition has a severe prognosis, largely due to the frequent transformation of RCDII IEL into overt aggressive enteropathy-type-associated T cell lymphoma. In contrast, in type I RCD, intestinal lymphocytes have a normal phenotype, and this generally milder condition remains often difficult to differentiate from uncomplicated CD except for the resistance to gluten-free diet (GFD). Several mechanisms may underlie resistance to gluten. Herein, we review the distinctive characteristics of RCD I and RCD II, the mechanisms underlying the onset of resistance to GFD, the risk of developing high grade lymphoma and possible clues to improve their treatment.

International audience; With the goal in mind to define how interleukin-15 (IL-15) contributes to acute intestinal inflammation, we have used a mouse model of ileitis induced by oral infection with Toxoplasma gondii. We observed that a crosstalk between IL-15 and interleukin-18 (IL-18) promoted intestinal recruitment of inflammatory monocytes, where these cells participated in parasite control but also in tissue damage. A stromal source of IL-15 controlled the development of lamina propria NKp46(+)NK1.1(+) cells, whereas IL-18 produced during T. gondii infection stimulated their production of the chemokine CCL3. In turn, CCL3 attracted inflammatory monocytes via their chemokine receptor CCR1, which was indispensable for their recruitment into the inflamed gut. Collectively, these results identify the IL-15-dependent subset of intestinal NKp46(+) cells as an important source of CCL3, which can amplify intestinal inflammation via the recruitment of CCR1(+) inflammatory monocytes. Preliminary evidence suggests that this pathway might operate in Crohn's disease.

Summary Introduction Enteropathy-associated T-cell lymphoma (EATL) is a rare complication of celiac disease ( Methods International literature review with PubMed search (up to January 2009) of pathophysiological, clinical and therapeutic data. Results EATL is found in patients with a mean age of 59 years, often with a complication that signals its diagnosis. Refractory celiac disease (RCD), equivalent to low-grade intraepithelial T-cell lymphoma, could be an intermediary between celiac disease and high-grade invasive T-cell lymphoma. The median survival is 7 months, with no significant difference between stages; the cumulative 5-year survival is less than 20%. The poor prognosis is determined by disease that has often spread before it is diagnosed (50%), multifocal involvement of the small bowel (50%), poor general health status and undernutrition, and recurrence of complications (infections, perforations, gastrointestinal haemorrhages, occlusions), thus delaying the chemotherapy and contributing to frequent chemotherapy resistance. There is currently no effective and consensual treatment: preventive surgery for complications is controversial, and the results of chemotherapy are disappointing. The classic CHOP protocol (combination of doxorubicin–cyclophosphamide–vincristine–prednisone) does not have satisfactory results and survival remains poor, especially in patients with underlying RCD. High-dose chemotherapy with autotransplantion seems to only improve the prognosis in localised forms. Allogeneic bone marrow transplantation was not evaluated. In all, 1/3 of patients, being unfit for treatment, die before 3 months and half of treated patients stop chemotherapy prematurely due to inefficacy, intolerance and/or complications. Conclusion Improvement of the prognosis requires collaboration in order to compose a national cohort, to evaluate new diagnostic and therapeutic strategies and to define prognostic factors.

Celiac disease is an enteropathy related to autoimmune diseases induced by gluten in genetically predisposed individuals. Its prevalence is of 1% in Europe and United States. Its clinical presentation is extremely various and diagnosis relies on the detection of specific serum antibodies and on the demonstration of intestinal villous atrophy. Treatment relies on a life-long gluten free diet which prevents bone, autoimmune and malignant complications. The keystone of its pathogenesis is the interaction of gliadin peptides with HLA DQ2/8 molecules, the main genetic risk factor, which induces the activation of CD4+ T-cells in the lamina propria. Yet, complementary mechanisms are necessary to provoke the loss of tolerance to gluten which involves the cytokine IL-15 responsible of the activation/expansion of intraepithelial lymphocytes, a hallmark of the origin of the severe lymphomatous complications. The burden of the gluten-free diet leads to a strong demand for alternative treatments. Numerous strategies have been identified to prevent the recognition of gliadin peptides by the immune system. Their efficiency and safety remained to be evaluated, the most attainable strategy today being oral therapy by enzymes able to eliminate gluten immunogenicity.

Refractory celiac disease (RCD) was recently subdivided into 2 subtypes (RCD I and II) based on a normal or abnormal phenotype of intraepithelial lymphocytes (IELs), respectively. It is not clear, however, if these 2 entities differ in their presentation at diagnosis or long-term outcome. We compared the clinical and biological characteristics of RCD I and RCD II at diagnosis, the risk of developing an overt lymphoma, and the predictive factors of survival.Medical files of 14 patients with RCD I and 43 with RCD II were analyzed retrospectively. Predictive factors of overt lymphoma and survival were studied in univariate and multivariate analyses.At diagnosis, malnutrition, ulcerative jejunitis, and lymphocytic gastritis were more common in patients with RCD II than RCD I (P.05). Overt lymphomas occurred in 2 patients with RCD I and 16 with RCD II. In the univariate analysis, abnormal IEL phenotype and increased age at diagnosis of RCD were predictive factors for overt lymphoma. Abnormal IEL phenotype (P.01), clonality (P= .01), and overt lymphoma (P= .001) predicted short survival time. Only abnormal IEL phenotype (P= .03) and overt lymphoma (P= .04) were predictive in the multivariate analysis. The 5-year survival rate was 93% in patients with RCD I and 44% with RCD II.RCD II has a much more severe presentation and prognosis than patients with RCD I;44% of patients with RCD II survive 5 years after diagnosis. Abnormal IEL phenotype is a predictive factor but not a necessary condition for the development of overt lymphoma.

Celiac disease is a multifactorial disorder and provides a privileged model to decipher how the interplay between environmental and genetic factors can alter mucosal tolerance to a food antigen, lead to chronic intestinal inflammation, and ultimately promote T-cell lymphomagenesis. Here we summarize how HLA-DQ2/8 molecules, the main genetic risk factor for this disease can orchestrate a CD4+ T-cell adaptive immune response against gluten, and discuss recent data which shed light on the innate and adaptive immune stimuli that collaborate to induce a proinflammatory TH1 response, a massive expansion of intraepithelial lymphocytes, and a cytolytic attack of the epithelium. The intestinal immune response driven in genetically predisposed patients by chronic exposure to gluten emerges as the pathological counterpart of normal acute intestinal responses to intracellular pathogens.

International audience; Background and Objectives : Anti-hypertensive treatment with the angiotensin II receptor antagonist olmesartan is a rare cause of severe Sprue-like enteropathy. To substantiate the hypothesis that olmesartan interferes with gut immune homeostasis, clinical, histopathological and immune features were compared in olmesartan-induced-enteropathy (OIE) and in autoimmune enteropathy (AIE).Methods : Medical files of seven patients with OIE and 4 patients with AIE enrolled during the same period were retrospectively reviewed. Intestinal biopsies were collected for central histopathological review, T cell Receptor clonality and flow cytometric analysis of isolated intestinal lymphocytes. Results : Among seven olmesartan-treated patients who developed villous atrophy refractory to a gluten free diet, three had extra-intestinal autoimmune diseases, two had antibodies reacting with the 75 kilodalton antigen characteristic of AIE and one had serum anti-goblet cell antibodies. Small intestinal lesions and signs of intestinal lymphocyte activation were thus reminiscent of the four cases of AIE diagnosed during the same period. Before olmesartan discontinuation, remission was induced in all patients (7/7) by immunosuppressive drugs. After interruption of both olmesartan and immunosuppressive drugs in six patients, remission was maintained in 4 but anti-TNF-α therapy was needed in two.Conclusion : This case-series shows that olmesartan can induce intestinal damage mimicking AIE. OIE usually resolved after olmesartan interruption but immunosuppressive drugs may be necessary to achieve remission. Our data sustain the hypothesis that olmesartan interferes with intestinal immuno regulation in predisposed individuals.

A major function of NKG2D linking innate and adaptive immunity is to upregulate antigen-specific CTL-mediated cytotoxicity in tissues expressing stress-induced NKG2D ligands, such as MIC, by coactivating TCR signaling. Here, we show that, under conditions of dysregulated IL15 expression in vivo in patients with celiac disease and in vitro in healthy individuals, multiple steps of the NKG2D/DAP10 signaling pathway leading to ERK and JNK activation are coordinately primed to activate direct cytolytic function independent of TCR specificity in effector CD8 T cells. These findings may not only explain previous reports of transformation of CTL into NK-like “lymphokine-activated killers” (LAK cells) under high doses of IL2 (a substitute for IL15) but may also have significant implications for understanding and treating immunopathological diseases.

Coeliac disease is a complex small intestinal enteropathy that develops consequently to a breach of tolerance to gliadin, a storage protein abundantly found in cereals such as wheat, rye and barley. The understanding of the mechanisms underlying the development of coeliac disease in HLA-DQ2 and HLA-DQ8 genetically susceptible individuals has greatly improved during the last decades but so far did not allow to develop curative therapeutics, leaving a long-life gluten free diet as the only treatment option for the patients. In order to bring new therapeutic targets to light and to test the safety and efficacy of putative drugs, animal models recapitulating features of the disease are needed. Here, we will review the existing animal models and the clinical features of coeliac disease they reflect and discuss their relevance for modelling immune pathways that may lead to potential therapeutic approaches.

International audience; BACKGROUND: Recently, a new enteropathy has been described: olmesartan-associated enteropathy. However, the association has been questioned: a phase 3 trial and a cohort study found no association between gastrointestinal events and olmesartan. AIM: We collected French cases of sartan-associated enteropathy to describe further this entity, confirm or refute causality, and determine if the association exists with other sartans. METHODS: French gastroenterologists were invited to report cases of sartan-associated enteropathy and collect clinical, biological and histological data. Patients with diarrhoea and histological duodenal abnormalities were included. RESULTS: Thirty-six patients with olmesartan-associated enteropathy were reported, including 32 with villous atrophy and four without. There was only one patient with irbesartan-associated enteropathy. None of the patients died. Patients with villous atrophy had diarrhoea, vomiting, renal failure, hypokalaemia, body weight loss and hypoalbuminaemia. Thirty-one patients were hospitalised; four required intensive care. Anti-transglutaminase and anti-enterocyte antibodies were negative; anti-nuclear antibodies were positive (9/11). Endoscopic duodenal biopsies showed villous atrophy (32/32) and polyclonal intra-epithelial CD3+CD8+ lymphocytosis (11/11). Exactly, 14/15 patients responded to steroids and/or immunosuppressants, prescribed because of suspected autoimmune enteropathy. Ten olmesartan interruptions were followed by reintroductions before steroids or immunosuppressants. Interruptions were followed by remissions (9/10), but reintroductions were followed by relapses (9/9). Twenty-nine patients were in remission since olmesartan interruption, including 26 without immunosuppressants. Patients with normal villi had similar clinical characteristics, but mild histological abnormalities (intra-epithelial lymphocytosis and lamina propria lymphocytic infiltration). CONCLUSIONS: Olmesartan causes a severe and immune-mediated enteropathy, with or without villous atrophy. Enteropathy associated with other sartans seems to be very rare.

Over the past 25 years, studies led in humans have considerably improved our understanding of celiac disease, a complex disease that is generally defined as an autoimmune-like enteropathy induced by dietary gluten in genetically predisposed individuals. Recently, large efforts were also invested in the development of mouse models in order to explore pathogenic hypotheses, and also with the goal to design pretherapeutic models that could be used to test innovative therapies. Yet, modeling this complex multifactorial disease has been a very challenging task. Herein, we review how approaches in rodents have provided insight into celiac disease pathophysiology and also highlight the difficulties met to fully recapitulate the human disease.

Coeliac disease is an immune-mediated enteropathy induced by gluten. Generation of gluten-specific CD4+ T cells is necessary but not sufficient to induce overt disease. Now, epithelial stress has been proposed as a distinct factor that synergizes with the anti-gluten adaptive immune response to induce intraepithelial lymphocyte activation and tissue damage.

International audience; : In celiac disease, enhanced permeability to gliadin peptides can result from their apico-basal transport by secretory immunoglobulin A1 (SIgA1) binding to the CD71 receptor ectopically expressed at the gut epithelial surface. Herein, we have established a mouse model in which there is apico-basal transport of the model antigen ovalbumin (OVA) by specific SIgA1 and have analyzed local T-cell activation. Transgenic DO11.10 mice were grafted with a hybridoma-secreting OVA-specific humanized IgA1, which could bind mouse CD71 and which were released in the intestinal lumen as SIgA. CD71 expression was induced at the gut apical surface by treating the mice with tyrphostin A8. Following gavage of the mice with OVA, OVA-specific CD4(+) T cells isolated from the mesenteric lymph nodes displayed higher expression of the activation marker CD69 and produced more interferon gamma in mice bearing the hybridoma-secreting OVA-specific IgA1, than in ungrafted mice or in mice grafted with an irrelevant hybridoma. These results indicate that the protective role of SIgA1 might be jeopardized in human pathological conditions associated with ectopic expression of CD71 at the gut surface.Mucosal Immunology advance online publication 10 July 2013; doi:10.1038/mi.2013.49.

Introduction Prognosis of enteropathy-associated T cell lymphoma is poor but predictors of survival remain ill-defined. How clinical presentation, pathological features and therapies influence outcome was evaluated in 37 thoroughly characterized patients with celiac disease and T-cell lymphoma. Patients and methods Medical files were studied retrospectively. Lymphoma and intestinal mucosa were analysed by histopathology, multiplex PCR and intestinal intraepithelial lymphocytes phenotyping. Survival and prognostic factors were analysed using Kaplan–Meier curves with Logrank test and Cox Model. Results Lymphoma complicated non clonal enteropathy, celiac disease (n = 15) and type I refractory celiac disease (n = 2) in 17 patients and clonal type II refractory celiac disease in 20 patients. Twenty-five patients underwent surgery with resection of the main tumour mass in 22 cases. In univariate analysis, non clonal celiac disease, serum albumin level > 21.6 g/L at diagnosis, chemotherapy and surgical resection predicted good survival (p = 0.0007, p 21.6 g/L, chemotherapy and reductive surgery were all significantly associated with increased survival (p

International audience; OBJECTIVES:Regulatory T cells (Tregs) are instrumental for tolerance to self-antigens and dietary proteins. We have previously shown that interleukin (IL)-15, a cytokine overexpressed in the intestine of patients with celiac disease (CD), does not impair the generation of functional Tregs but renders human T cells resistant to Treg suppression. Treg numbers and responses of intestinal and peripheral T lymphocytes to suppression by Tregs were therefore compared in CD patients and controls.METHODS:Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) were isolated from duodenal biopsy specimens of CD patients and controls. Concomitantly, CD4+CD25+ T lymphocytes (Tregs) were purified from blood. Responses of IELs and of LPLs, and peripheral lymphocytes (PBLs) to suppression by Tregs were tested by analyzing anti-CD3-induced proliferation and interferon (IFN)-γ production in the presence or absence of peripheral Tregs. Lamina propria and peripheral CD4+CD25+FOXP3+ T cells were assessed by flow cytometry.RESULTS:Although percentages of CD4+CD25+FOXP3+ LPLs were significantly increased in patients with active CD, proliferation and IFN-γ production of intestinal T lymphocytes were significantly less inhibited by autologous or heterologous Tregs in CD patients than in controls (P

After initial acceptance and on-line publication it was decided that this article warranted translation into English. This English translation has been published in Gastroenterologie Clinique et Biologique, 34/11, pp. 590–605 ( http://dx.doi.org/10.1016/j.gcb.2010.09.008 ) and to avoid unnecessary duplication this version of the article has been withdrawn.

Enteropathy-associated T cell lymphoma is a severe complication of celiac disease (CD). One mechanism suggested to underlie its development is chronic exposure of intraepithelial lymphocytes (IELs) to potent antiapoptotic signals initiated by IL-15, a cytokine overexpressed in the enterocytes of individuals with CD. However, the signaling pathway by which IL-15 transmits these antiapoptotic signals has not been firmly established. Here we show that the survival signals delivered by IL-15 to freshly isolated human IELs and to human IEL cell lines derived from CD patients with type II refractory CD (RCDII) - a clinicopathological entity considered an intermediary step between CD and enteropathy-associated T cell lymphoma - depend on the antiapoptotic factors Bcl-2 and/or Bcl-xL. The signals also required IL-15Rbeta, Jak3, and STAT5, but were independent of PI3K, ERK, and STAT3. Consistent with these data, IELs from patients with active CD and RCDII contained increased amounts of Bcl-xL, phospho-Jak3, and phospho-STAT5. Furthermore, incubation of patient duodenal biopsies with a fully humanized human IL-15-specific Ab effectively blocked Jak3 and STAT5 phosphorylation. In addition, treatment with this Ab induced IEL apoptosis and wiped out the massive IEL accumulation in mice overexpressing human IL-15 in their gut epithelium. Together, our results delineate the IL-15-driven survival pathway in human IELs and demonstrate that IL-15 and its downstream effectors are meaningful therapeutic targets in RCDII.

One arm of the gut-associated immune system is represented by a vast collection of T lymphocytes which participate in the subtle interplay between innate and adaptive immune mechanisms and maintain homeostasis at the main body external surface. Mounting data are providing exciting new insight into the innate-like mechanisms which enable intestinal T cells to rapidly sense local conditions and which broaden the spectrum of their functions and regulation at this strategic location. Herein we discuss how innate-like T cell recognition by unconventional T cell subsets and expression of innate NK receptors might modulate immune T cell responses in the human normal or diseased intestine.

In this issue of Gut ,1 Fina et al demonstrate that interleukin 21 (IL21) expression is increased in the intestinal mucosa of patients with active coeliac disease (CD) but not that of treated patients ( see page 887 ). The authors show that IL21 is produced by CD4+ lamina propria T cells, a finding consistent with previous work indicating that this cytokine is exclusively produced by CD4+ T cells. Using a neutralising anti-IL21 antibody, they observed that blocking IL21 activity reduced the increase in interferon γ mRNA induced by gliadin digests in intestinal organ cultures from treated CD patients. Since the anti-IL21 antibody also reduced the induction of mRNA for T-bet, the transcription factor that controls the differentiation of interferon γ-producing CD4+ T lymphocytes (so-called T helper cell type 1 or Th1 lymphocytes), the authors conclude that IL21 is an important contributor to the mucosal Th1 proinflammatory response.1 The mucosal Th1 response in CD is generally ascribed to lamina propria CD4+ T cells that recognise gluten peptides presented by the human leucocyte antigen (HLA)-DQ2/8 molecules at the surface of mucosal dendritic cells. This response accounts for the interplay between the major genetic risk factor, the MHC (major histocompatibility complex) class II genes encoding the HLA-DQ2/8 molecules, and the triggering environmental factor gluten, and is considered instrumental in the pathogenesis of CD enteropathy.2 These results are of particular interest following the first genome-wide association study recently carried out in CD by van Heel et al .3 The latter study has indeed provided convincing evidence that the chromosomal 4q27 region harbouring the genes encoding the IL2 and IL21 cytokines might be involved in CD. In this study based on the use of single-nucleotide polymorphisms (SNPs), several variants were highly significant when analysed in three distinct populations of UK, Dutch and …

Celiac disease (CD) is an inflammatory enteropathy induced by cereal-derived prolamines (gluten) in genetically predisposed individuals. Prolamines, due to their high proline content, are incomplet

International audience; Toxoplasma gondii is an intracellular parasite that frequently infects a large spectrum of warm-blooded animals. This parasite induces abortion and establishes both chronic and silent infections' particularly in the brain, Parasite penetration into the host activates a strong arti-parosite immune response. In the present paper, we will discuss the interplay between innate end adaptive immunity that occurs within the infected intestine to clear the parasite and to maintain intestinal homeostasis despite the exacerbation of an inflammatory immune response.

It is well recognized that the nature of the immune response is different in the intestinal tract than in peripheral lymphoid organs. The immunologic tone of the gut-associated lymphoid tissue is one of suppression rather than active immunity, distinguishing pathogens from normal flora. Failure to control mucosal immune responses may lead to inflammatory diseases such as Crohn's disease (CD) and ulcerative colitis (UC) and celiac disease. It has been suggested that this normally immunosuppressed state may relate to unique antigen-presenting cells and unique T-cell populations. The intestinal epithelial cell (IEC) has been proposed to act as a nonprofessional antigen-presenting cell (APC). Previous studies have suggested that antigens presented by IECs result in the activation a CD8+ regulatory T-cell subset in a nonclassical MHC I molecule restricted manner. We therefore analyzed the expression of nonclassical MHC I molecules by normal IECs and compared this to those expressed by inflammatory bowel disease (IBD) IECs. Normal surface IEC from the colon and, to a much lesser extent, the small bowel express nonclassical MHC I molecules on their surface. In contrast, mRNA is expressed in all intestinal epithelial cells. Surface IEC express CD1d, MICA/B, and HLA-E protein. In contrast, crypt IECs express less or no nonclassical MHC I molecules but do express mRNA for these molecules. Furthermore, the regulation of expression of distinct nonclassical class I molecules is different depending on the molecule analyzed. Interestingly, IECs derived from patients with UC fail to express any nonclassical MHC I molecules (protein and HLA-E mRNA). IECs from CD patients express HLA-E and MICA/B comparable to that seen in normal controls but fail to express CD1d. Thus, in UC there may be a failure to activate any nonclassical MHC I molecule restricted regulatory T cells that may result in unopposed active inflammatory responses. In CD only the CD1d-regulated T cells would be affected. (Inflamm Bowel Dis 2007)

Celiac disease is an intestinal inflammatory disorder induced by dietary gluten in genetically susceptible individuals. The mechanisms underlying the massive expansion of interferon γ–producing intraepithelial cytotoxic T lymphocytes (CTLs) and the destruction of the epithelial cells lining the small intestine of celiac patients have remained elusive. We report massive oligoclonal expansions of intraepithelial CTLs that exhibit a profound genetic reprogramming of natural killer (NK) functions. These CTLs aberrantly expressed cytolytic NK lineage receptors, such as NKG2C, NKp44, and NKp46, which associate with adaptor molecules bearing immunoreceptor tyrosine-based activation motifs and induce ZAP-70 phosphorylation, cytokine secretion, and proliferation independently of T cell receptor signaling. This NK transformation of CTLs may underlie both the self-perpetuating, gluten-independent tissue damage and the uncontrolled CTL expansion leading to malignant lymphomas in severe forms of celiac disease. Because similar changes were detected in a subset of CTLs from cytomegalovirus-seropositive patients, we suggest that a stepwise transformation of CTLs into NK-like cells may underlie immunopathology in various chronic infectious and inflammatory diseases.

NKG2 receptors and their ligands play an essential role in the control of CTL activation in the tissue microenvironment. We discuss the regulation of NKG2 receptor expression by CTL and how uncontrolled activation of NKG2 receptors can lead to organ-specific autoimmune and inflammatory disorders.

Background & Aims: Interleukin (IL)-15 delivers signals that drive chronic inflammation in several diseases, including celiac disease. Smad3–transforming growth factor-beta (TGF-β) signaling is instrumental to counteract proinflammatory signals and maintain immune homeostasis. Our goal has been to investigate why the proinflammatory effects of IL-15 cannot be efficiently controlled by TGF-β in celiac disease. Methods: The impact of IL-15 on TGF-β signaling in T cells and in the intestinal mucosa of celiac disease patients was analyzed by combining cell and organ cultures, immunohistochemistry, flow cytometry, real-time polymerase chain reaction, electromobility gel shift, and Western blot. Results: IL-15 impaired Smad3-dependent TGF-β signaling in human T lymphocytes downstream from Smad3 nuclear translocation. IL-15-mediated inhibition was associated with a long-lasting activation of c-jun-N-terminal kinase and reversed by c-jun antisense oligonucleotides, consistent with the demonstrated inhibitory effect of phospho-c-jun on the formation of Smad3–DNA complexes. In active celiac disease, intestinal lymphocytes showed impaired TGF-β–Smad3-dependent transcriptional responses and up-regulation of phospho-c-jun. Anti-IL-15 antibody and c-jun antisense both downmodulated phospho-c-jun expression and restored TGF-β–Smad-dependent transcription in biopsies of active celiac disease. c-jun antisense decreased interferon gamma transcription. Conclusions: Impairment of TGF-β-mediated signaling by IL-15 might promote and sustain intestinal inflammation in celiac disease. More generally, our data provide a new rationale for the potent proinflammatory effects of IL-15, and further support the concept that IL-15 is a meaningful therapeutic target in inflammatory diseases associated with irreducible elevation of IL-15.

The protective effects of different mucin gene profiles on gut protection were assessed by the evaluation of TNFalpha production by intestinal epithelial cells infected by Shigella flexneri. Three HT-29 cell lines were used: HT29-G(-) (enterocyte-like cells, secreting no mucins), HT29-FU (highly expressing MUC2 and MUC4) and HT29-MTX (highly expressing MUC3 and MUC5AC). These cells were infected either by an invasive (M90T) or the control isogenic (BS176) strains of S. flexneri, and TNFalpha mRNA production was quantified by competitive PCR. In the HT29-G(-) cells, M90T induced an increased production of TNFalpha mRNA compared to BS176, giving a TNFalpha ratio of 5.6 +/- 3.3. In contrast, similar levels of TNFalpha mRNA were detected in HT29-FU and HT29-MTX cells stimulated with either M90T or BS176, giving ratios of 1.4 +/- 1.3 and 1.0 +/- 0.1, respectively. The results suggest that mucin genes have abilities to protect epithelial cells against S. flexneri. Furthermore, the difference in the TNFalpha ratio between the HT29-FU and HT29-MTX cells suggests distinct protective effects for these two mucin-secreting epithelial cells.

Interleukin-10 (IL-10) is a potent anti-inflammatory and immunosuppressive cytokine that modulates disease expression in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). In previous studies, two dinucleotide repeat elements (microsatellites G and R) were identified, respectively located about 1.1 and 4.0 kb upstream of the IL-10 gene transcription initiation site. Several of their alleles were found to be associated with the level of IL-10 production. The aim of our study was to determine whether sequence variations in the IL-10 gene were associated with MS susceptibility and progression. To do so, we analyzed the distribution of IL-10.R and IL-10.G alleles and genotype polymorphisms in MS patients and healthy controls. We then correlated our findings with disease severity in MS patients using the progression index (PI). Patients were classified as experiencing mild (PI0.5) or severe (PI0.5) disease progression. Our results show no association between the IL-10.R microsatellite and MS, regardless of disease severity. However, IL-10.G microsatellite genotyping showed that IL-10.G9/9, G10/13, G11/13 and G13/14 were more frequently found in patients with mild disease progression (p = 0.005). We also found that in patients with severe disease progression, IL-10.G9/10, G9/11, G9/13 and G12/13 were over-represented (p = 0.002). Our study indicates that neither the IL-10.R or the IL-10.G alleles are associated with predisposition to MS. However, several IL-10.G genotypes might emerge as markers of disease progression.

Background: Endoscopic recurrence after surgery in Crohn's disease is frequent and unpredictable. Abnormal intestinal production of pro- (interleukin (IL)-1β, tumour necrosis factor α (TNF-α)) and anti- (IL-10) inflammatory cytokines has been associated with severe outcome in experimental models of colitis. Patients and methods: We evaluated if ileal TNF-α, IL-1β, or IL-10 mRNA levels measured at the time of surgery predict endoscopic recurrence, and if ileal IL-10 levels are associated with particular IL-10 promoter alleles. Ileal biopsies were obtained peroperatively from the healthy neoileum of patients undergoing a right ileocolectomy for Crohn's disease. Mucosal TNF-α, IL-1β, and IL-10 mRNA levels were quantified by competitive polymerase chain reaction. A cut off value was determined using a receiver operating curve. IL-10.G promoter haplotypes were analysed using a polymorphic dinucleotide repeat in the IL-10 promoter region. Results: Three months after surgery, 53% of patients had endoscopic recurrence while 47% remained free of disease. The risk of endoscopic recurrence correlated with ileal IL-10 mRNA concentrations (r2=0.81). Endoscopic recurrence occurred more frequently in patients classified as low IL-10 producers than in those that were high producers (80% v 40%) (p=0.02). Patients with at least one of the two alleles G7–8 or G10–13 produced, respectively, higher (p=0.006) and lower (p=0.029) ileal IL-10 mRNA. The distribution of IL-10.G microsatellite genotypes was similar in patients with or without endoscopic recurrence. Conclusion: Low ileal IL-10 mRNA concentration is a good marker of endoscopic recurrence in Crohn's disease but the distribution of IL-10.G haplotypes cannot predict the postoperative evolution of the disease.

Background & Aims Little is known about intestinal CD4+ T-cell lymphoma; this rare malignancy is misdiagnosed frequently. We evaluated diagnostic criteria and factors that might affect its development and outcome. Methods In a retrospective analysis, we analyzed medical records and intestinal specimens from 10 patients diagnosed with intestinal CD4+ T-cell lymphoma among 115 consecutive patients examined for severe enteropathy with villous atrophy. Samples were analyzed by histology, flow cytometry, and comparative genomic hybridization. Results Small-intestine epithelial and lamina propria tissues from patients who presented with chronic diarrhea and malnutrition had variable levels of infiltration of CD3+ CD4+ T cells. Flow cytometry showed a high frequency of CD4+ intraepithelial cells, which frequently expressed a specific Vβ chain. T-cell receptor β clonality was confirmed by DNA sequencing. Two patients had HLA and serology results compatible with celiac disease and autoimmune enteropathy, respectively. Two patients were found to have antibodies against human T-cell leukemia virus and 2 patients had signs of a recent infection with the herpes viruses. Comparative genomic hybridization analyses showed heterogeneous chromosomal abnormalities. Symptoms were reduced in patients treated with steroids (n = 5), but not in patients given purine analogues or chemotherapy. Antibodies against CD52 produced clinical and histologic responses in 2 of 2 patients, whereas severe adverse effects developed in 1 patient. At the latest follow-up evaluation, all patients were alive. Conclusions There is much heterogeneity in the onset and genetic features of intestinal CD4+ T-cell lymphomas, despite their common presentation as indolent lymphoproliferations of the intestinal mucosa. Patients should be treated with steroids, and possibly antibodies against CD52 (for the most aggressive forms of this disorder).

Crohn disease is a chronic inflammatory bowel disease that involves all the intestine but predominantly alters the ileum. The disease largely depends on T cells, but the biologic role of intestinal intraepithelial lymphocytes (IEL) in transmural inflammation remains poorly characterized. To address this issue, a comparison of IEL and lamina propria lymphocytes (LPL) isolated from the uninvolved and the inflamed ileal mucosa of Crohn disease patients was performed. More CD8+ IEL (26% versus 8%) from the inflamed ileal mucosa expressed the CD28 receptor and the CD11a integrin than IEL from the uninvolved ileal mucosa, which were mostly CD28-. IEL had longer telomeres in the inflamed than in the uninvolved areas and a TCR Vbeta repertoire more similar to circulating T cells, suggesting that the increased proportion of CD28+ TCRalphabeta+ IEL within the inflamed mucosa is more likely due to recruited lymphocytes from the periphery that populate the epithelial layer than to the acquisition of the CD28 molecule by activated resident lymphocytes. In the uninvolved ileal mucosa, IEL from Crohn disease patients had shorter telomeric lengths than IEL from control patients, suggesting that they have been chronically stimulated. Such perturbation of the IEL population within the ileal mucosa could contribute to the inflammation in Crohn disease.

Introduction Enteropathy-associated T-cell lymphoma (EATL) is an intestinal tumor of intraepithelial T lymphocytes. The 2008 WHO classification distinguishes two types of EATL: the first type of EATL (type I), the most frequent (80-90%) is strongly associated with celiac disease (CD) and the HLA-DQ2/DQ8 haplotypes. The tumor cells are CD3+CD8-/+CD4-CD56- and contain cytotoxic granules. In almost all cases, a varying proportion of the tumor cells express CD30. The second type of EATL (type II), the monomorphic form, has a distinct immunophenotype (CD3+CD4-CD8+CD56+). In two types, TCRgamma genes are often clonally rearranged. EATL may be preceded by refractory celiac disease (RCD), corresponding to CD refractory to gluten free diet (GFD). RCD is divided in two types based on the absence (type I) or presence (type II) of abnormal intraepithelial lymphocytes (IEL) showing down-regulation of CD8 and often TCRgamma genes rearrangement. RCD II is now considered as a small cell intraepithelial T-cell lymphoma that could be an intermediate stage between CD and EATL. The aim of the present study was to establish the pattern of CD30 expression in EATL. This could have therapeutic implications with the use of anti-CD30 monoclonal antibody like brentuximab vedotin (BV). Methods Consecutive adult patients (pts) diagnosed with EATL between 2007 and 2013 in two university hospitals in Paris (Necker University Hospital and Georges Pompidou European Hospital) were eligible for this study. Diagnosis was confirmed after histopathologic and immunohistochemical review. For the purpose of the present study, two expert hematopathologists (V.V. and N.B.) reviewed all EATL and extended the phenotypic analysis to reclassify them according to the 2008 WHO classification. A panel of antibodies directed against CD20, CD3, CD4, CD5, CD8, CD56, granzyme B and ALK1 was used. CD30 staining was performed with Monoclonal Mouse Anti-Human CD30, Clone Ber-H2 (Dako). Consecutive RCDI and RCDII cases with complete phenotype and clonality analyses diagnosed in the same period were used as control. Diagnosis of CD was based on HLA-DQ2/8 typing, detection of celiac specific antibodies and of villous atrophy with increased counts of IEL on normal diet. Pts were further classified in RCDI or II depending on their clinical and histological response to a GFD and the presence of abnormal IEL. Results Twenty five adults were diagnosed with EATL on consensus review (median age 53 years [range 34-76], M/F ratio 12/13). Twenty five RCDI pts (median age 51 years [range 16-75], M/F ratio 6/19) and 20 RCDII pts (median age 62 years [range 29-81], M/F ratio 7/13) were used as control. A clinical history of CD was found in 17/20 (85%) evaluable EATL pts. Histological features of CD/RCD were seen in all cases (20/20) of EATL in which the mucosa adjacent to the tumor could be investigated (half of these were RCDII). Primary sites of EATL were small intestine (20/25), mesenteric lymph nodes (3/25), peritoneal nodules (1/25) and spleen (1/25). Phenotypic analysis showed that EATL cases were all WHO type I (25/25). ALK1 was constantly negative. IEL were CD3+ in all 70 cases. CD8 was normally expressed in all RCDI IEL and downregulated in all RCDII IEL and 35% of EATL. In all cases of EATL (25/25), CD30 was strongly expressed by all large tumor cells. In RCDI and II, CD30 was negative in most cases (∼90%), and was rarely expressed by dispersed atypical lymphocytes (IEL or in lamina propria) in some cases. TCRgamma genes were clonally rearranged in 11/14 (79%) EATL, 3/25 (12%) RCDI and 18/20 (90%) RCDII. Based on these results, we initiated in 2012 a pilot study combining BV with chemotherapy followed by autologous stem-cell transplantation (ASCT) as frontline treatment of EATL. Five pts have currently been treated. The associated chemotherapy regimen was IVE/MTX (Sieniawski M, Blood 2010) for the first two pts. After presentation at ASH 2012 Annual Meeting of preliminary results of a phase 1 study combining BV with CHP regimen as frontline treatment of systemic ALCL and other CD30-positive mature T–cell and NK–cell lymphomas (Fanale MA, Abstract #60), we replaced IVE/MTX by CHP regimen, and treated 3 other pts. The treatment was well tolerated, and the 5 pts reached CR and underwent ASCT. Conclusion CD30 is strongly expressed in EATL type I. Promising results of the combination of BV with CHP led us to plan a phase 2 study of BV and CHP followed by ASCT as frontline treatment of EATL. Disclosures: Off Label Use: Brentuximab vedotin was used in enteropathy-associated T-cell lymphoma (EATL).

Large granular lymphocyte leukemia (LGL) is characterized by clonal expansion of CD3+ T cells or CD3(-) natural killer cells and frequently is associated with autoimmune diseases. We describe 2 patients with celiac disease who no longer responded to gluten-free diets after they developed T-cell LGL, with intestinal localization of malignant lymphocytes. Flow cytometry phenotyping of isolated intestinal intraepithelial and lamina propria cells eliminated type II refractory celiac disease, identifying large-sized CD8(+)CD57(+) T cells. Treatment with a combination of cyclosporine and methotrexate restored the patients' sensitivity to gluten-free diets. LGL therefore might be a cause of refractory celiac disease that is sensitive to immunosuppressive therapy.