Stuart I. Mannering

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.

Human Dendritic Cell Subsets from Spleen and Blood Are Similar in Phenotype and Function but Modified by Donor Health Status

Mouse dendritic cells (DC) have been extensively studied in various tissues, especially spleen, and they comprise subsets with distinct developmental origins, surface phenotypes, and functions. Considerably less is known about human DC due to their rarity in blood and inaccessibility of other human tissues. The study of DC in human blood has revealed four subsets distinct in phenotype and function. In this study, we describe four equivalent DC subsets in human spleen obtained from deceased organ donors. We identify three conventional DC subsets characterized by surface expression of CD1b/c, CD141, and CD16, and one plasmacytoid DC subset characterized by CD304 expression. Human DC subsets in spleen were very similar to those in human blood with respect to surface phenotype, TLR and transcription factor expression, capacity to stimulate T cells, cytokine secretion, and cross-presentation of exogenous Ag. However, organ donor health status, in particular treatment with corticosteroid methylprednisolone and brain death, may affect DC phenotype and function. DC T cell stimulatory capacity was reduced but DC were qualitatively unchanged in methylprednisolone-treated deceased organ donor spleen compared with healthy donor blood. Overall, our findings indicate that human blood DC closely resemble human spleen DC. Furthermore, we confirm parallels between human and mouse DC subsets in phenotype and function, but also identify differences in transcription factor and TLR expression as well as functional properties. In particular, the hallmark functions of mouse CD8α+ DC subsets, that is, IL-12p70 secretion and cross-presentation, are not confined to the equivalent human CD141+ DC but are shared by CD1b/c+ and CD16+ DC subsets.

The insulin A-chain epitope recognized by human T cells is posttranslationally modified

The autoimmune process that destroys the insulin-producing pancreatic β cells in type 1 diabetes (T1D) is targeted at insulin and its precursor, proinsulin. T cells that recognize the proximal A-chain of human insulin were identified recently in the pancreatic lymph nodes of subjects who had T1D. To investigate the specificity of proinsulin-specific T cells in T1D, we isolated human CD4+ T cell clones to proinsulin from the blood of a donor who had T1D. The clones recognized a naturally processed, HLA DR4–restricted epitope within the first 13 amino acids of the A-chain (A1–13) of human insulin. T cell recognition was dependent on the formation of a vicinal disulfide bond between adjacent cysteine residues at A6 and A7, which did not alter binding of the peptide to HLA DR4. CD4+ T cell clones that recognized this epitope were isolated from an HLA DR4+ child with autoantibodies to insulin, and therefore, at risk for T1D, but not from two healthy HLA DR4+ donors. We define for the first time a novel posttranslational modification that is required for T cell recognition of the insulin A-chain in T1D.

Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion

T cells target peptide combos One of the enduring mysteries of autoimmunity is the identity of the specific proteins targeted by autoimmune T cells. Delong et al. used mass spectrometry to elucidate the peptide targets of autoimmune T cells isolated from a mouse model of type 1 diabetes. T cells targeted hybrid peptides formed by the covalent linking of a peptide derived from pro-insulin to other peptides derived from proteins found in pancreatic beta cells. T cells isolated from the pancreatic islets of two individuals with type 1 diabetes also recognized such hybrid peptides, suggesting that they may play an important role in driving disease. Science, this issue p. 711 Autoimmune T cells recognize covalently linked peptides derived from two distinct proteins. T cell–mediated destruction of insulin-producing β cells in the pancreas causes type 1 diabetes (T1D). CD4 T cell responses play a central role in β cell destruction, but the identity of the epitopes recognized by pathogenic CD4 T cells remains unknown. We found that diabetes-inducing CD4 T cell clones isolated from nonobese diabetic mice recognize epitopes formed by covalent cross-linking of proinsulin peptides to other peptides present in β cell secretory granules. These hybrid insulin peptides (HIPs) are antigenic for CD4 T cells and can be detected by mass spectrometry in β cells. CD4 T cells from the residual pancreatic islets of two organ donors who had T1D also recognize HIPs. Autoreactive T cells targeting hybrid peptides may explain how immune tolerance is broken in T1D.

Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society.

Autoimmune T cell responses directed against insulin‐producing β cells are central to the pathogenesis of type 1 diabetes (T1D). Detection of such responses is therefore critical to provide novel biomarkers for T1D ‘immune staging’ and to understand the mechanisms underlying the disease. While different T cell assays are being developed for these purposes, it is important to optimize and standardize methods for processing human blood samples for these assays. To this end, we review data relevant to critical parameters in peripheral blood mononuclear cell (PBMC) isolation, (cryo)preservation, distribution and usage for detecting antigen‐specific T cell responses. Based on these data, we propose recommendations on processing blood samples for T cell assays and identify gaps in knowledge that need to be addressed. These recommendations may be relevant not only for the analysis of T cell responses in autoimmune disease, but also in cancer and infectious disease, particularly in the context of clinical trials.

Biased T Cell Receptor Usage Directed against Human Leukocyte Antigen DQ8-Restricted Gliadin Peptides Is Associated with Celiac Disease

Celiac disease is a human leukocyte antigen (HLA)-DQ2- and/or DQ8-associated T cell-mediated disorder that is induced by dietary gluten. Although it is established how gluten peptides bind HLA-DQ8 and HLA-DQ2, it is unclear how such peptide-HLA complexes are engaged by the T cell receptor (TCR), a recognition event that triggers disease pathology. We show that biased TCR usage (TRBV9(∗)01) underpins the recognition of HLA-DQ8-α-I-gliadin. The structure of a prototypical TRBV9(∗)01-TCR-HLA-DQ8-α-I-gliadin complex shows that the TCR docks centrally above HLA-DQ8-α-I-gliadin, in which all complementarity-determining region-β (CDRβ) loops interact with the gliadin peptide. Mutagenesis at the TRBV9(∗)01-TCR-HLA-DQ8-α-I-gliadin interface provides an energetic basis for the Vβ bias. Moreover, CDR3 diversity accounts for TRBV9(∗)01(+) TCRs exhibiting differing reactivities toward the gliadin epitopes at various deamidation states. Accordingly, biased TCR usage is an important factor in the pathogenesis of DQ8-mediated celiac disease.

Proinsulin-Specific, HLA-DQ8, and HLA-DQ8-Transdimer–Restricted CD4+ T Cells Infiltrate Islets in Type 1 Diabetes

Type 1 diabetes (T1D) develops when insulin-secreting β-cells, found in the pancreatic islets of Langerhans, are destroyed by infiltrating T cells. How human T cells recognize β-cell-derived antigens remains unclear. Genetic studies have shown that HLA and insulin alleles are the most strongly associated with risk of T1D. These long-standing observations implicate CD4+ T-cell responses against (pro)insulin in the pathogenesis of T1D. To dissect the autoimmune T-cell response against human β-cells, we isolated and characterized 53 CD4+ T-cell clones from within the residual pancreatic islets of a deceased organ donor who had T1D. These 53 clones expressed 47 unique clonotypes, 8 of which encoded proinsulin-specific T-cell receptors. On an individual clone basis, 14 of 53 CD4+ T-cell clones (26%) recognized 6 distinct but overlapping epitopes in the C-peptide of proinsulin. These clones recognized C-peptide epitopes presented by HLA-DQ8 and, notably, HLA-DQ8 transdimers that form in HLA-DQ2/-DQ8 heterozygous individuals. Responses to these epitopes were detected in the peripheral blood mononuclear cells of some people with recent-onset T1D but not in HLA-matched control subjects. Hence, proinsulin-specific, HLA-DQ8, and HLA-DQ8-transdimer–restricted CD4+ T cells are strongly implicated in the autoimmune pathogenesis of human T1D.

Failed CTL/NK cell killing and cytokine hypersecretion are directly linked through prolonged synapse time

Jenkins et al. discover that failure of perforin and granzyme cytotoxicity by human and mouse CTLs/NK cells prolongs the immunological synapse, leading to repetitive calcium signaling and hypersecretion of inflammatory mediators that subsequently activate macrophages. Disengagement from target cells is dependent on apoptotic caspase signaling. The findings may provide mechanistic understanding for immunopathology in familial hemophagocytic lymphohistiocytosis.

Intranasal Vaccination with Proinsulin DNA Induces Regulatory CD4+ T Cells That Prevent Experimental Autoimmune Diabetes

Insulin, an autoantigen in type 1 diabetes, when administered mucosally to diabetes-prone NOD mice induces regulatory T cells (Treg) that protect against diabetes. Compared with protein, Ag encoded as DNA has potential advantages as a therapeutic agent. We found that intranasal vaccination of NOD mice with plasmid DNA encoding mouse proinsulin II-induced CD4+ Treg that suppressed diabetes development, both after adoptive cotransfer with “diabetogenic” spleen cells and after transfer into NOD mice given cyclophosphamide to accelerate diabetes onset. In contrast to prototypic CD4+CD25+ Treg, CD4+ Treg induced by proinsulin DNA were both CD25+ and CD25− and not defined by markers such as glucocorticoid-induced TNFR-related protein (GITR), CD103, or Foxp3. Intriguingly, despite induction of Treg and reduced islet inflammation, diabetes incidence in proinsulin DNA-treated mice was unchanged. However, diabetes was prevented when DNA vaccination was performed under the cover of CD40 ligand blockade, known to prevent priming of CTL by mucosal Ag. Thus, intranasal vaccination with proinsulin DNA has therapeutic potential to prevent diabetes, as demonstrated by induction of protective Treg, but further modifications are required to improve its efficacy, which could be compromised by concomitant induction of pathogenic immunity.

Proinsulin-a pathogenic autoantigen in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterised by immunity to pancreatic beta-cell autoantigens, associated with beta-cell destruction leading to insulin deficiency and hyperglycaemia. The rigorous definition of an autoimmune disease requires evidence that an autoantigen elicits pathological immune responses. Using criteria for the pathogenicity of an autoantigen, we examine the evidence for proinsulin as an autoantigen in T1D. We conclude that proinsulin satisfies these criteria. As a corollary, proinsulin is a potential immunotherapeutic tool for the prevention of T1D.