James A. Dromey

Comprehensive, Quantitative Mapping of T Cell Epitopes in Gluten in Celiac Disease

Three highly immunogenic peptides from gluten are primarily responsible for celiac disease, suggesting a rational immunotherapeutic approach that could replace the need for strict, lifelong dietary gluten avoidance. Taming of the Sprue Gluten, a complex protein in wheat, barley, and rye, forms the elastic network responsible for the airy texture of bread. But gluten can also trigger a prevalent inflammatory disorder—celiac disease (sprue)—which afflicts sufferers with problems such as gastrointestinal upset, fatigue, and anemia, and confers increased risks of osteoporosis, autoimmune disease, and cancer. The current therapy consists of strict lifelong avoidance of all foods containing gluten. The development of alternatives has been hampered by the inability to fully characterize the immune response to the toxic peptides within these grains. Several immunotoxic peptides from wheat have been implicated, but it has remained unclear how they contribute to the overall immune response in celiac disease, or whether other potentially toxic peptides from barley and rye exist. Tye-Din and colleagues have now comprehensively assessed the more than 16,000 potentially toxic peptides contained within wheat, barley, and rye, and identified which ones stimulate T cells from celiac disease patients. By feeding doses of wheat, barley, or rye to more than 200 people with celiac disease, the authors were able to examine the induced T cells appearing in the bloodstream several days afterward. These T cells were then tested for recognition of peptides from large libraries encompassing every possible toxic peptide from wheat, barley, and rye. Surprisingly, they found that just three highly active peptides were responsible for most of the immune response seen in patients with celiac disease after eating any of the toxic grains. Although the range of highly stimulatory or dominant peptides was very consistent between individuals, it was dependent on which grain was consumed. A previously described peptide from wheat α-gliadin was dominant only after wheat ingestion; another distinct peptide was dominant after wheat, barley, or rye ingestion. Of most interest was the fact that a combination of these peptides, plus another from barley, could elicit 90% of the response induced by the full complement of wheat, barley, and rye proteins. Because the authors assessed every possible toxic peptide from wheat, as well as barley and rye, they can be confident that their data paint a comprehensive picture of the immune response in celiac disease. This is important because alternative therapies to the complex, costly, and inconvenient gluten-free diet are likely to require a detailed molecular understanding of the peptides driving the immune response in celiac disease. Multiple doses of peptides corresponding to immunodominant T cell epitopes are effective in treating a mouse version of celiac disease, and the discovery that a small number of peptides can elicit the disease in patients suggests that a similar approach may be successful in humans as well. Celiac disease is a genetic condition that results in a debilitating immune reaction in the gut to antigens in grain. The antigenic peptides recognized by the T cells that cause this disease are incompletely defined. Our understanding of the epitopes of pathogenic CD4+ T cells is based primarily on responses shown by intestinal T-cells in vitro to hydrolysates or polypeptides of gluten, the causative antigen. A protease-resistant 33-amino acid peptide from wheat α-gliadin is the immunodominant antigen, but little is known about the spectrum of T cell epitopes in rye and barley or the hierarchy of immunodominance and consistency of recognition of T-cell epitopes in vivo. We induced polyclonal gluten-specific T cells in the peripheral blood of celiac patients by feeding them cereal and performed a comprehensive, unbiased analysis of responses to all celiac toxic prolamins, a class of plant storage protein. The peptides that stimulated T cells were the same among patients who ate the same cereal, but were different after wheat, barley and rye ingestion. Unexpectedly, a sequence from ω-gliadin (wheat) and C-hordein (barley) but not α-gliadin was immunodominant regardless of the grain consumed. Furthermore, T cells specific for just three peptides accounted for the majority of gluten-specific T cells, and their recognition of gluten peptides was highly redundant. Our findings show that pathogenic T cells in celiac disease show limited diversity, and therefore suggest that peptide-based therapeutics for this disease and potentially other strongly HLA-restricted immune diseases should be possible.

Naturally processed and presented epitopes of the islet cell autoantigen IA-2 eluted from HLA-DR4

During immune responses, antigen-presenting cells (APCs) process antigens and present peptide epitopes complexed with human leukocyte antigen (HLA) molecules. CD4 cells recognize these naturally processed and presented epitopes (NPPEs) bound to HLA class II molecules. Epitope identification is important for developing diagnostic and therapeutic tools for immune-mediated diseases and providing insight into their etiology, but current approaches overlook effects of natural processing on epitope selection. We have developed a technique to identify NPPEs using mass spectrometry (MS) after antigen is targeted onto APCs using a lectin-based antigen delivery system (ADS). We applied the technique to identify NPPEs of the intracellular domain of the type 1 diabetes mellitus-associated (type 1 DM-associated) autoantigen insulinoma-associated-2 (IA-2ic), presented by HLA-DR4 (0401). IA-2ic-derived NPPEs eluted from HLA-DR4 constitute 6 sets of peptides nested around distinct core regions. Synthetic peptides based on these regions bind HLA-DR4 and elicit primary T-cell proliferation frequently in HLA-DR4-positive type 1 DM patients, but rarely in non-HLA-DR4 patients, and in none of the HLA-DR4 nondiabetic controls we tested. This flexible, direct approach identifies an HLA allele-specific map of NPPEs for any antigen, presented by any HLA class II molecule. This method should enable a greater understanding of epitope selection and lead to the generation of sensitive and specific reagents for detecting autoreactive T cells.

T cell regulation mediated by interaction of soluble CD52 with the inhibitory receptor Siglec-10

Functionally diverse T cell populations interact to maintain homeostasis of the immune system. We found that human and mouse antigen-activated T cells with high expression of the lymphocyte surface marker CD52 suppressed other T cells. CD52hiCD4+ T cells were distinct from CD4+CD25+Foxp3+ regulatory T cells. Their suppression was mediated by soluble CD52 released by phospholipase C. Soluble CD52 bound to the inhibitory receptor Siglec-10 and impaired phosphorylation of the T cell receptor–associated kinases Lck and Zap70 and T cell activation. Humans with type 1 diabetes had a lower frequency and diminished function of CD52hiCD4+ T cells responsive to the autoantigen GAD65. In diabetes-prone mice of the nonobese diabetic (NOD) strain, transfer of lymphocyte populations depleted of CD52hi cells resulted in a substantially accelerated onset of diabetes. Our studies identify a ligand-receptor mechanism of T cell regulation that may protect humans and mice from autoimmune disease.

355 Immune Tolerance Induced By Peptide Immunotherapy in An HLA Dq2-Dependent Mouse Model of Gluten Immunity

Patients with Coeliac disease (CD) mount inflammatory T-cell responses to deamidated gluten peptides. Encounter of harmless protein via mucosal surfaces generally induces mucosal tolerance. Using animal models we have shown that oral tolerance to the harmless protein ovalbumin (OVA) depends on differentiation of regulatory T-cells (Treg) in the mucosa draining lymphoid tissue. How this translates to the uptake of gluten is difficult to predict as little is known with respect to localization and presentation of gluten after uptake via the gastrointestinal tract. The aim of this study was to identify the phenotype and localization of the gliadin specific T-cell response In Vivo. Mice transgenically expressing disease predisposing human HLA-DQ2 and human gliadin-specific TCR (CD-TCR) on an MHC-II-/background were used. CD-TCR+CD4+ T-cells from double transgenic mice were isolated and labelled with CFSE. After labelling, the cells were i.v. injected into HLA-DQ2tg x MHC-II-/mice. The acceptor mice received non-deamidated or deamidated chymotrypsin digested gliadin by gavage or deamidated gliadin in the thigh muscle (i.m.). After 72h the response of the gliadin-specific T-cells was analyzed in the gut-draining mesenteric lymph node (MLN), spleen and peripheral lymph nodes (PLN) and compared to the known tolerogenic T-cell response to OVA. At 72h after gavage T-cell response to non-deamidated gliadin was very low suggesting that during homeostasis gliadin is not deamidated by murine tissue transglutaminase. Conversely, a vigorous T-cell response was detected in MLN after gavage of deamidated gliadin. Unlike the response to harmless OVA, deamidated gliadin induced more proliferation in spleen than in MLN implying that the adaptive response to gliadin is not confined to the mucosa draining lymphoid tissue. Proliferating gliadin-specific T-cells in MLN contained minimal numbers of Foxp3+ Treg. By contrast, the cells phenotypically resembled activated T-cells and were comparable to differentiating effector T-cells in the PLN of mice that received deamidated gliadin i.m.. Culture of naive CD4+CD-TCR+ T-cells with HLADQ2+ APC in the presence of deamidated gliadin yielded phenotypically identical effector T-cells. Upon addition of factors that mediate mucosal Treg differentiation, Foxp3+CD4+CD-TCR+ T-cells did differentiate, indicating that the cells do have the intrinsic capacity to develop into adaptive mucosal Treg. Dissecting gliadin-specific T-cell responses in this novel murine system provides important opportunities to study the pathogenesis of CD, investigate efficacy of compounds and treatment strategies but also safety of novel dietary products.

Generation and expansion of regulatory human CD4(+) T-cell clones specific for pancreatic islet autoantigens.

Autoantigen-specific regulatory T cells (Treg) are a potential cell therapy for human autoimmune disease, provided they could be generated in adequate numbers and with stable function. To this end, we determined the feasibility of cloning and expanding human CD4(+) Treg specific for the type 1 diabetes autoantigens, GAD65 and proinsulin. Blood CD4(+) cells stimulated to divide in response to GAD65 (in three healthy individuals) or proinsulin (in one type 1 diabetic) were flow sorted into single cells and cultured on feeder cells in the presence of anti-CD3 monoclonal antibody, IL-2 and IL-4. Clones were expanded over 4-6 weeks and screened for autoantigen-dependent suppression of tetanus toxoid-specific T-cell proliferation. Suppression by Treg clones was then confirmed against autoantigen-specific non-Treg clones. Of a total of 447 clones generated, 98 (21.9%) had autoantigen-dependent suppressor function. Treg clones were anergic but proliferated to autoantigen after addition of IL-2 or in co-culture with stimulated bulk T cells, without loss of suppressor function. Treg clones were stored over liquid N(2), thawed and further expanded over 12 days, whereupon they exhibited decreased suppressor function. Expansion of Treg clones overall was in the order 10⁷-10⁸-fold. Treg clones were not distinguished by markers of conventional CD4(+)CD25(+) Treg and suppressed independently of cell-cell contact but not via known soluble suppressor factors. This study demonstrates that autoantigen-specific CD4(+) Treg clones with potential application as a cell therapy for autoimmune disease can be generated and expanded from human blood.

Proinsulin is encoded by an RNA splice variant in human blood myeloid cells

Genes for peripheral tissue-restricted self-antigens are expressed in thymic and hematopoietic cells. In thymic medullary epithelial cells, self-antigen expression imposes selection on developing autoreactive T cells and regulates susceptibility to autoimmune disease in mouse models. Less is known about the role of self-antigen expression by hematopoietic cells. Here we demonstrate that one of the endocrine self-antigens expressed by human blood myeloid cells, proinsulin, is encoded by an RNA splice variant. The surface expression of immunoreactive proinsulin was significantly decreased after transfection of monocytes with small interfering RNA to proinsulin. Furthermore, analogous to proinsulin transcripts in the thymus, the abundance of the proinsulin RNA splice variant in blood cells corresponded with the length of the variable number of tandem repeats 5′ of the proinsulin gene, known to be associated with type 1 diabetes susceptibility. Self-antigen expression by peripheral myeloid cells extends the umbrella of “immunological self” and, by analogy with the thymus, may be implicated in peripheral immune tolerance.

A novel population of regulatory CD4 t cells is deficient after stimulation by autoantigen in type 1 diabetes

Regulatory T cells (Tregs) suppress pro-inflammatory immune responses and prevent autoimmune disease. Many types of Tregs have been described, the prototypic in mice defined as CD4 +CD25 +with high expression of the Foxp3 transcription factor. However, Fox P3 is not a reliable marker of human CD4 +CD25 +Tregs and Tregs activated by disease-associated antigens have not been well characterised. By comparing autoantigen-activated CD4 +T-cell clones, we identified CD52 as a marker of suppressor clones. We then showed that high expression of CD52 on antigen-activated T cells identified a unique Treg population in human blood. CD52hi Tregs were not distinguished by markers of CD4 +CD25 +Tregs and did not require cell contact for suppressor function. Following activation by glutamic acid decarboxylase 65 or proinsulin, pancreatic islet autoantigens in type 1 diabetes, generation of CD4 + CD52hi Tregs was reduced in individuals at high risk for type 1 diabetes. Autoantigen-specific Tregs should allow monitoring of autoimmune disease susceptibility and response to immunotherapy, in addition to being directly applicable as a cell therapy for autoimmune disease.