Impaired IgM Memory B Cell Function Is Common in Coeliac Disease but Conjugate Pneumococcal Vaccination Induces Robust Protective Immunity.

Coeliac disease (CD) is associated with hyposplenism, an acquired impairment of spleen function associated with reduced IgM memory B cells and increased susceptibility to serious pneumococcal infection. Little is known about the immune implications of hyposplenism in CD or the optimal pneumococcal vaccination strategy. In this study, the immune effects of hyposplenism in CD, and the accuracy of screening approaches and protective responses induced by two different pneumococcal vaccines were examined. Active and treated CD cohorts, and healthy and surgically splenectomised controls underwent testing for the presence of Howell-Jolly bodies and pitted red cells, spleen ultrasound, and immune assessment of IgM memory B cell frequency and IgM memory B cell responses to T cell-dependent (TD) or T cell-independent (TI) stimulation. Responses following conjugate (TD) and polysaccharide (TI) pneumococcal vaccination were compared using ELISA and opsonophagocytic assays. Although hyposplenism is rare in treated CD (5.1%), functional B cell defects are common (28-61%) and are not detected by current clinical tests. Conjugate pneumococcal vaccination induced superior and sustained protection against clinically relevant serotypes. Clinical practice guidelines in CD should recommend routine pneumococcal vaccination, ideally with a conjugate vaccine, of all patients in lieu of hyposplenism screening.

Hydroxychloroquine inhibits the mitochondrial antioxidant system in activated T cells.

Although hydroxychloroquine (HCQ) has long been used to treat autoimmune diseases, its mechanism of action remains poorly understood. In CD4 T-cells, we found that a clinically relevant concentration of HCQ inhibited the mitochondrial antioxidant system triggered by TCR crosslinking, leading to increased mitochondrial superoxide, impaired activation-induced autophagic flux, and reduced proliferation of CD4 T-cells. In antigen-presenting cells, HCQ also reduced constitutive activation of the endo-lysosomal protease legumain and toll-like receptor 9, thereby reducing cytokine production, but it had little apparent impact on constitutive antigen processing and peptide presentation. HCQ's effects did not require endo-lysosomal pH change, nor impaired autophagosome-lysosome fusion. We explored the clinical relevance of these findings in patients with celiac disease-a prototypic CD4 T-cell-mediated disease-and found that HCQ limits ex vivo antigen-specific T cell responses. We report a T-cell-intrinsic immunomodulatory effect from HCQ and suggest potential re-purposing of HCQ for celiac disease.

A Sensitive Whole Blood Assay Detects Antigen-Stimulated Cytokine Release From CD4+ T Cells and Facilitates Immunomonitoring in a Phase 2 Clinical Trial of Nexvax2 in Coeliac Disease.

Improved blood tests assessing the functional status of rare gluten-specific CD4+ T cells are needed to effectively monitor experimental therapies for coeliac disease (CD). Our aim was to develop a simple, but highly sensitive cytokine release assay (CRA) for gluten-specific CD4+ T cells that did not require patients to undergo a prior gluten challenge, and would be practical in large, multi-centre clinical trials. We developed an enhanced CRA and used it in a phase 2 clinical trial ("RESET CeD") of Nexvax2, a peptide-based immunotherapy for CD. Two participants with treated CD were assessed in a pilot study prior to and six days after a 3-day gluten challenge. Dye-dilution proliferation in peripheral blood mononuclear cells (PBMC) was assessed, and IL-2, IFN-γ and IL-10 were measured by multiplex electrochemiluminescence immunoassay (ECL) after 24-hour gluten-peptide stimulation of whole blood or matched PBMC. Subsequently, gluten-specific CD4+ T cells in blood were assessed in a subgroup of the RESET CeD Study participants who received Nexvax2 (maintenance dose 900 µg, n = 12) or placebo (n = 9). The pilot study showed that gluten peptides induced IL-2, IFN-γ and IL-10 release from PBMCs attributable to CD4+ T cells, but the PBMC CRA was substantially less sensitive than whole blood CRA. Only modest gluten peptide-stimulated IL-2 release could be detected without prior gluten challenge using PBMC. In contrast, whole blood CRA enabled detection of IL-2 and IFN-γ before and after gluten challenge. IL-2 and IFN-γ release in whole blood required more than 6 hours incubation. Delay in whole blood incubation of more than three hours from collection substantially reduced antigen-stimulated IL-2 and IFN-γ secretion. Nexvax2, but not placebo treatment in the RESET CeD Study was associated with significant reductions in gluten peptide-stimulated whole blood IL-2 and IFN-γ release, and CD4+ T cell proliferation. We conclude that using fresh whole blood instead of PBMC substantially enhances cytokine secretion stimulated by gluten peptides, and enables assessment of rare gluten-specific CD4+ T cells without requiring CD patients to undertake a gluten challenge. Whole blood assessment coupled with ultra-sensitive cytokine detection shows promise in the monitoring of rare antigen-specific T cells in clinical studies.

Circulating gluten-specific, but not CMV-specific, CD39+ regulatory T cells have an oligoclonal TCR repertoire.

OBJECTIVES: Understanding the T cell receptor (TCR) repertoire of regulatory CD4+ T-cell (Treg) populations is important for strategies aiming to re-establish tolerance in autoimmune diseases. We studied circulating deamidated gluten-epitope-specific CD39+ Tregs in patients with coeliac disease following an oral gluten challenge, and we used cytomegalovirus (CMV)-specific CD39+ Tregs from healthy controls as a comparator population. METHODS: We used the OX40 assay to isolate antigen-specific Tregs by induced surface co-expression of CD25, OX40 and CD39. RACE PCR amplification and Sanger sequencing of the TCR ß chain were used to analyse repertoire diversity. RESULTS: We found that, following oral gluten challenge, circulating gluten-specific CD39+ Tregs had an oligoclonal TCR repertoire that contained public clonotypes. Conversely, the TCR repertoire of CMV-epitope-specific CD39+ Tregs from healthy controls was polyclonal. DISCUSSION: These data indicate that a biased TCR repertoire is not inherent to CD39+ Tregs, and, in this case, is apparently driven by the HLA-DQ2.5-restricted deamidated gluten peptide in coeliac disease patients. CONCLUSION: This is the first assessment of the TCR repertoire within circulating human Tregs specific for foreign antigen. These data enhance our understanding of antigen-specific CD4+ responses in the settings of chronic inflammation and infection and may help guide immunomonitoring strategies for CD4+ T cell-based therapies, particularly for coeliac disease.

Characterisation of clinical and immune reactivity to barley and rye ingestion in children with coeliac disease.

OBJECTIVE: Barley and rye are major components of the Western diet, and historic feeding studies indicate that they cause clinical effects in patients with coeliac disease (CD). This toxicity has been attributed to sequence homology with immunogenic wheat sequences, but in adults with CD, these cereals stimulate unique T cells, indicating a critical contribution to gluten immunity independent of wheat. Clinical and immune feeding studies with these grains in children with CD are sparse. We undertook a barley and rye feeding study to characterise the clinical and T-cell responses in children with CD. DESIGN: 42 children with human leucocyte antigen (HLA)-DQ2.5+ (aged 3-17 years) consumed barley or rye for 3 days. Blood-derived gluten-specific T cells were tested for reactivity against a panel of barley (hordein) and rye (secalin) peptides. Hordein and secalin-specific T-cell clones were generated and tested for grain cross-reactivity. T-cell receptor sequencing was performed on sorted single cells. T-cell responses were compared with those observed in adults with CD. RESULTS: 90% of the children experienced adverse symptoms, mostly GI, and 61% had detectable gluten-specific T-cell responses targeting peptides homologous to those immunogenic in adults. Deamidation was important for peptide reactivity. Homozygosity for HLA-DQ2.5 predicted a stronger T-cell response. Gluten-specific T cells showed striking similarities in their cross-reactivity between children and adults. CONCLUSIONS: Barley and rye induce a consistent range of clinical and T-cell responses in children with CD. The findings highlight the importance of a series of dominant hordein and secalin peptides pathogenic in children with CD, some independent of wheat, which closely correspond to those seen in adults.

T cell receptor cross-reactivity between gliadin and bacterial peptides in celiac disease.

The human leukocyte antigen (HLA) locus is strongly associated with T cell-mediated autoimmune disorders. HLA-DQ2.5-mediated celiac disease (CeD) is triggered by the ingestion of gluten, although the relative roles of genetic and environmental risk factors in CeD is unclear. Here we identify microbially derived mimics of gliadin epitopes and a parental bacterial protein that is naturally processed by antigen-presenting cells and activated gliadin reactive HLA-DQ2.5-restricted T cells derived from CeD patients. Crystal structures of T cell receptors in complex with HLA-DQ2.5 bound to two distinct bacterial peptides demonstrate that molecular mimicry underpins cross-reactivity toward the gliadin epitopes. Accordingly, gliadin reactive T cells involved in CeD pathogenesis cross-react with ubiquitous bacterial peptides, thereby suggesting microbial exposure as a potential environmental factor in CeD.

Preparation and Characterization of Avenin-Enriched Oat Protein by Chill Precipitation for Feeding Trials in Celiac Disease.

The safety of oats for people with celiac disease remains unresolved. While oats have attractive nutritional properties that can improve the quality and palatability of the restrictive, low fiber gluten-free diet, rigorous feeding studies to address their safety in celiac disease are needed. Assessing the oat prolamin proteins (avenins) in isolation and controlling for gluten contamination and other oat components such as fiber that can cause non-specific effects and symptoms is crucial. Further, the avenin should contain all reported immunogenic T cell epitopes, and be deliverable at a dose that enables biological responses to be correlated with clinical effects. To date, isolation of a purified food-grade avenin in sufficient quantities for feeding studies has not been feasible. Here, we report a new gluten isolation technique that enabled 2 kg of avenin to be extracted from 400 kg of wheat-free oats under rigorous gluten-free and food grade conditions. The extract consisted of 85% protein of which 96% of the protein was avenin. The concentration of starch (1.8% dry weight), ß-glucan (0.2% dry weight), and free sugars (1.8% dry weight) were all low in the final avenin preparation. Other sugars including oligosaccharides, small fructans, and other complex sugars were also low at 2.8% dry weight. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the proteins in these preparations showed they consisted only of oat proteins and were uncontaminated by gluten containing cereals including wheat, barley or rye. Proteomic analysis of the avenin enriched samples detected more avenin subtypes and fewer other proteins compared to samples obtained using other extraction procedures. The identified proteins represented five main groups, four containing known immune-stimulatory avenin peptides. All five groups were identified in the 50% (v/v) ethanol extract however the group harboring the epitope DQ2.5-ave-1b was less represented. The avenin-enriched protein fractions were quantitatively collected by reversed phase HPLC and analyzed by MALDI-TOF mass spectrometry. Three reverse phase HPLC peaks, representing ~40% of the protein content, were enriched in proteins containing DQ2.5-ave-1a epitope. The resultant high quality avenin will facilitate controlled and definitive feeding studies to establish the safety of oat consumption by people with celiac disease.

Circulating gluten-specific FOXP3+CD39+ regulatory T cells have impaired suppressive function in patients with celiac disease.

BACKGROUND: Celiac disease is a chronic immune-mediated inflammatory disorder of the gut triggered by dietary gluten. Although the effector T-cell response in patients with celiac disease has been well characterized, the role of regulatory T (Treg) cells in the loss of tolerance to gluten remains poorly understood. OBJECTIVE: We sought to define whether patients with celiac disease have a dysfunction or lack of gluten-specific forkhead box protein 3 (FOXP3)+ Treg cells. METHODS: Treated patients with celiac disease underwent oral wheat challenge to stimulate recirculation of gluten-specific T cells. Peripheral blood was collected before and after challenge. To comprehensively measure the gluten-specific CD4+ T-cell response, we paired traditional IFN-γ ELISpot with an assay to detect antigen-specific CD4+ T cells that does not rely on tetramers, antigen-stimulated cytokine production, or proliferation but rather on antigen-induced coexpression of CD25 and OX40 (CD134). RESULTS: Numbers of circulating gluten-specific Treg cells and effector T cells both increased significantly after oral wheat challenge, peaking at day 6. Surprisingly, we found that approximately 80% of the ex vivo circulating gluten-specific CD4+ T cells were FOXP3+CD39+ Treg cells, which reside within the pool of memory CD4+CD25+CD127lowCD45RO+ Treg cells. Although we observed normal suppressive function in peripheral polyclonal Treg cells from patients with celiac disease, after a short in vitro expansion, the gluten-specific FOXP3+CD39+ Treg cells exhibited significantly reduced suppressive function compared with polyclonal Treg cells. CONCLUSION: This study provides the first estimation of FOXP3+CD39+ Treg cell frequency within circulating gluten-specific CD4+ T cells after oral gluten challenge of patients with celiac disease. FOXP3+CD39+ Treg cells comprised a major proportion of all circulating gluten-specific CD4+ T cells but had impaired suppressive function, indicating that Treg cell dysfunction might be a key contributor to disease pathogenesis.

Coeliac disease: a unique model for investigating broken tolerance in autoimmunity.

Coeliac disease, a prevalent immune-mediated enteropathy driven by dietary gluten, provides an exceptional human model to dissect the genetic, environmental and immunologic factors operating in autoimmunity. Despite the causative antigen being an exogenous food protein, coeliac disease has many features in common with autoimmune disease including a strong HLA class II association and the presence of pathogenic CD4+ T cells and autoantibodies. CD8+ intraepithelial lymphocytes specifically target and destroy intestinal epithelium in response to stress signals and not a specific antigen. A unique feature of coeliac disease is the ability to remove gluten to induce disease remission and reintroduce it to trigger a memory response. This provides an unparalleled opportunity to study disease-relevant CD4+ T cells that have been expanded in vivo. As a result, the causative peptides have been characterised at a level unprecedented for any autoimmune disease. Despite the complexity of the gluten proteome, resistance to gastrointestinal proteolysis and susceptibility to post-translational modification by transglutaminase help shape a restricted repertoire of immunogenic gluten peptides that have high affinity for disease-associated HLA. The critical steps in coeliac disease pathogenesis have been broadly elucidated and provide the basis for experimental therapies in pre-clinical or clinical development. However, little is known about how and why tolerance to gluten sometimes breaks or fails to develop. Understanding the interactions between genes, the environment, gluten immunity and the microbiome may provide novel approaches for the prevention and treatment of disease.

Consistency in polyclonal T-cell responses to gluten between children and adults with celiac disease.

BACKGROUND & AIMS: Developing antigen-specific approaches for diagnosis and treatment of celiac disease requires a detailed understanding of the specificity of T cells for gluten. The existing paradigm is that T-cell lines and clones from children differ from those of adults in the hierarchy and diversity of peptide recognition. We aimed to characterize the T-cell response to gluten in children vs adults with celiac disease. METHODS: Forty-one children with biopsy-proven celiac disease (median age, 9 years old; 17 male), who had been on strict gluten-free diets for at least 3 months, were given a 3-day challenge with wheat; blood samples were collected and gluten-specific T cells were measured. We analyzed responses of T cells from these children and from 4 adults with celiac disease to a peptide library and measured T-cell receptor bias. We isolated T-cell clones that recognized dominant peptides and assessed whether gluten peptide recognition was similar between T-cell clones from children and adults. RESULTS: We detected gluten-specific responses by T cells from 30 of the children with celiac disease (73%). T cells from the children recognized the same peptides that were immunogenic to adults with celiac disease; deamidation of peptides increased these responses. Age and time since diagnosis did not affect the magnitude of T-cell responses to dominant peptides. T-cell clones specific for dominant α- or ω-gliadin peptides from children with celiac disease had comparable levels of reactivity to wheat, rye, and barley peptides as T-cell clones from adults with celiac disease. The α-gliadin-specific T cells from children had biases in T-cell receptor usage similar to those in adults. CONCLUSIONS: T cells from children with celiac disease recognize similar gluten peptides as T cells from adults with celiac disease. The findings indicate that peptide-based diagnostics and therapeutics for adults may also be used for children.

A phase I clinical trial of CD1c (BDCA-1)+ dendritic cells pulsed with HLA-A*0201 peptides for immunotherapy of metastatic hormone refractory prostate cancer.

Preclinical studies have suggested that purified populations of CD1c (BDCA-1) blood-derived dendritic cells (BDC) loaded with tumor-specific peptides may be a feasible option for prostate cancer immunotherapy. We performed an open-label dose-finding Phase I study to evaluate the safe use of CD1c BDC in patients with advanced metastatic hormone refractory prostate cancer. HLA-A*0201-positive patients with advanced metastatic prostate cancer were recruited and consented. The vaccine was manufactured by pulsing autologous CD1c BDC, prepared by magnetic bead immunoselection from apheresed peripheral blood mononuclear cells, with a cocktail of HLA-A*0201-restricted peptides (prostate-specific antigen, prostate acid phosphatase, prostate specific membrane antigen, and control influenza peptide) and keyhole limpet hemocyanin. The vaccine was administered intradermally or intravenously and peripheral blood was taken at predetermined intervals for clinical and immunologic monitoring. The vaccine was manufactured with a median purity of 82% CD1c BDC and administered successfully to 12 patients. Each patient received between 1 and 5 × 10 fresh CD1c BDC on day 0, followed by cryopreserved product in the same dose on days 28 and 56. The vaccine was well tolerated in all patients, with the most frequent adverse events being grade 1-2 fever, pain, or injection-site reactions. Vaccination with CD1c BDC is therefore feasible, safe, and well tolerated in patients with advanced-stage metastatic prostate cancer.

Ingestion of oats and barley in patients with celiac disease mobilizes cross-reactive T cells activated by avenin peptides and immuno-dominant hordein peptides.

Celiac disease (CD) is a common CD4(+) T cell mediated enteropathy driven by gluten in wheat, rye, and barley. Whilst clinical feeding studies generally support the safety of oats ingestion in CD, the avenin protein from oats can stimulate intestinal gluten-reactive T cells isolated from some CD patients in vitro. Our objective was to establish whether ingestion of oats or other grains toxic in CD stimulate an avenin-specific T cell response in vivo. We fed participants a meal of oats (100 g/day over 3 days) to measure the in vivo polyclonal avenin-specific T cell responses to peptides contained within comprehensive avenin peptide libraries in 73 HLA-DQ2.5(+) CD patients. Grain cross-reactivity was investigated using oral challenge with wheat, barley, and rye. Avenin-specific responses were observed in 6/73 HLA-DQ2.5(+) CD patients (8%), against four closely related peptides. Oral barley challenge efficiently induced cross-reactive avenin/hordein-specific T cells in most CD patients, whereas wheat or rye challenge did not. In vitro, immunogenic avenin peptides were susceptible to digestive endopeptidases and showed weak HLA-DQ2.5 binding stability. Our findings indicate that CD patients possess T cells capable of responding to immuno-dominant hordein epitopes and homologous avenin peptides ex vivo, but the frequency and consistency of these T cells in blood is substantially higher after oral challenge with barley compared to oats. The low rates of T cell activation after a substantial oats challenge (100 g/d) suggests that doses of oats commonly consumed are insufficient to cause clinical relapse, and supports the safety of oats demonstrated in long-term feeding studies.

A flow cytometry based assay for the enumeration of regulatory T cells in whole blood.

The analysis of regulatory T cells (T-reg(s)) is becoming an increasingly important consideration in the development of novel immunotherapeutic strategies. Accurate quantification of T-regs during treatment protocols is crucial, particularly where the therapeutic strategy is targeting T-regs. The TruCOUNT™ method has utility for enumerating different immune cells but has not been used to detect T-regs. We have utilized this technology to develop an assay to enumerate human T-regs in whole blood, based on CD127 expression. The mean number of CD4(+)CD25(+)CD127(lo) T-regs per µl of whole blood was 48±16.9 with a range of 18 - 79 (n=22) and the average percentage was 6.1±1.9% (range 2.2-10.4%). The percentages of CD4(+)CD25(+)CD127(lo) T-regs were similar when detected in whole blood or density-gradient separated PBMC, and were comparable to those distinguished using the T-reg marker FoxP3. The assay was robust and reliable for enumeration of the lower frequency T-regs, with CV's for intra-assay repeatability and inter-assay precision of <9% and <35%, respectively. The CV's for the detection of total CD4(+) T lymphocytes using this assay were <2% for intra-assay repeatability and <18% for inter-assay precision, providing further evidence for reproducibility. This assay has a number of advantages over current methods, including small sample volume, the ability to determine absolute cell counts, and no need for hematology cell analyzers. This assay will simplify clinical trial immune monitoring and can be used to provide crucial data on patient T-reg numbers before, during, and after therapeutic interventions.

Human CD1c (BDCA-1)+ myeloid dendritic cells secrete IL-10 and display an immuno-regulatory phenotype and function in response to Escherichia coli.

Human blood myeloid DCs can be subdivided into CD1c (BDCA-1)(+) and CD141 (BDCA-3)(+) subsets that display unique gene expression profiles, suggesting specialized functions. CD1c(+) DCs express TLR4 while CD141(+) DCs do not, thus predicting that these two subsets have differential capacities to respond to Escherichia coli. We isolated highly purified CD1c(+) and CD141(+) DCs and compared them to in vitro generated monocyte-derived DCs (MoDCs) following stimulation with whole E. coli. As expected, MoDCs produced high levels of the proinflammatory cytokines TNF, IL-6, and IL-12, were potent inducers of Th1 responses, and processed E. coli-derived Ag. In contrast, CD1c(+) DCs produced only low levels of TNF, IL-6, and IL-12 and instead produced high levels of the anti-inflammatory cytokine IL-10 and regulatory molecules IDO and soluble CD25. Moreover, E. coli-activated CD1c(+) DCs suppressed T-cell proliferation in an IL-10-dependent manner. Contrary to their mouse CD8(+) DC counterparts, human CD141(+) DCs did not phagocytose or process E. coli-derived Ag and failed to secrete cytokines in response to E. coli. These data demonstrate substantial differences in the nature of the response of human blood DC subsets to E. coli.

Human kallikrein 4 signal peptide induces cytotoxic T cell responses in healthy donors and prostate cancer patients.

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201+ KLK4 + cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.

NK cells enhance the induction of CTL responses by IL-15 monocyte-derived dendritic cells.

Dendritic cells differentiated from monocytes (MoDC) in the presence of GM-CSF and IL-15 (IL-15 MoDC) exhibit superior migration and cytotoxic T-lymphocyte (CTL) induction compared with MoDC differentiated in IL-4 and GM-CSF (IL-4 MoDC) and are promising candidates for DC immunotherapy. We explored the mechanisms by which IL-15 MoDC induce CTL. IL-15 MoDC expressed higher levels of CD40 and secreted high levels of TNF-alpha, but little or no IL-12p70 compared with IL-4 MoDC. Despite immuno-selecting monocytes to >97% purity before MoDC generation, a tiny population (0.2%) of natural killer (NK) cells was identified that was increased sevenfold during IL-15 MoDC, but not IL-4 MoDC differentiation. These NK cells produced high levels of IFN-gamma and were responsible for the enhanced CTL-inducing capacity of the IL-15 MoDC, but not for their increased expression of CD40 or secretion of TNF-alpha. Interestingly, a proportion of IL-15 MoDC were found to express the NK cell marker, CD56, but these did not secrete IFN-gamma. These data implicate a role for small percentages of NK cells in the enhanced capacity of IL-15 MoDC to induce tumour-specific CTL independent of IL-12p70.