A bispecific antibody targeting HLA-DQ2.5-gluten peptides potently blocks gluten-specific T cells induced by gluten ingestion in patients with celiac disease.

The gluten-free diet for celiac disease (CeD) is restrictive and often fails to induce complete symptom and/or mucosal disease remission. Central to CeD pathogenesis is the gluten-specific CD4+ T cell that is restricted by HLA-DQ2.5 in over 85% of CeD patients, making HLA-DQ2.5 an attractive target for suppressing gluten-dependent immunity. Recently, a novel anti-HLA-DQ2.5 antibody that specifically recognizes the complexes of HLA-DQ2.5 and multiple gluten epitopes was developed (DONQ52). OBJECTIVE: To assess the ability of DONQ52 to inhibit CeD patient-derived T-cell responses to the most immunogenic gluten peptides that encompass immunodominant T cell epitopes. METHODS: We employed an in vivo gluten challenge model in patients with CeD that affords a quantitative readout of disease-relevant gluten-specific T-cell responses. HLA-DQ2.5+ CeD patients consumed food containing wheat, barley, or rye for 3 days with collection of blood before (D1) and 6 days after (D6) commencing the challenge. Peripheral blood mononuclear cells were isolated and assessed in an interferon (IFN)-γ enzyme-linked immunosorbent spot assay (ELISpot) testing responses to gluten peptides encompassing a series of immunodominant T cell epitopes. The inhibitory effect of DONQ52 (4 or 40 µg/mL) was assessed and compared to pan-HLA-DQ blockade (SPVL3 antibody). RESULTS: In HLA-DQ2.5+ CeD patients, DONQ52 reduced T cell responses to all wheat gluten peptides to an equivalent or more effective degree than pan-HLA-DQ antibody blockade. It reduced T cell responses to a cocktail of the most immunodominant wheat epitopes by a median of 87% (IQR 72-92). Notably, DONQ52 also substantially reduced T-cell responses to dominant barley hordein and rye secalin derived peptides. DONQ52 had no effect on T-cell responses to non-gluten antigens. CONCLUSION: DONQ52 can significantly block HLA-DQ2.5-restricted T cell responses to the most highly immunogenic gluten peptides in CeD. Our findings support in vitro data that DONQ52 displays selectivity and broad cross-reactivity against multiple gluten peptide:HLA-DQ2.5 complexes. This work provides proof-of-concept multi-specific antibody blockade has the potential to meaningfully inhibit pathogenic gluten-specific T-cell responses in CeD and supports ongoing therapeutic development.

Whole blood interleukin-2 release test to detect and characterize rare circulating gluten-specific T cell responses in coeliac disease.

Whole blood cytokine release assays (CRA) assessing cellular immunity to gluten could simplify the diagnosis and monitoring of coeliac disease (CD). We aimed to determine the effectiveness of electrochemiluminescence CRA to detect responses to immunodominant gliadin peptides. HLA-DQ2·5+ CD adults (cohort 1, n = 6; cohort 2, n = 12) and unaffected controls (cohort 3, n = 9) were enrolled. Cohort 1 had 3-day gluten challenge (GC). Blood was collected at baseline, and for cohort 1 also at 3 h, 6 h and 6 days after commencing 3-day GC. Gliadin peptide-stimulated proliferation, interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and 14- and 3-plex electrochemiluminescence CRA were performed. Poisson distribution analysis was used to estimate responding cell frequencies. In cohort 1, interleukin (IL)-2 dominated the gliadin peptide-stimulated cytokine release profile in whole blood. GC caused systemic IL-2 release acutely and increased gliadin peptide-stimulated IFN-γ ELISPOT and whole blood CRA responses. Whole blood CRA after GC was dominated by IL-2, but also included IFN-γ, C-X-C motif chemokine ligand 10/IFN-γ-induced protein 10 (CXCL10/IP-10), CXCL9/monokine induced by IFN-γ (MIG), IL-10, chemokine (C-C motif) ligand 3/macrophage inflammatory protein 1-alpha (CCL3/MIP-1α), TNF-α and IL-8/CXCL8. In cohorts 2 and 3, gliadin peptide-stimulated whole blood IL-2 release was 100% specific and 92% sensitive for CD patients on a gluten-free diet; the estimated frequency of cells in CD blood secreting IL-2 to α-gliadin peptide was 0·5 to 11 per ml. Whole blood IL-2 release successfully mapped human leucocyte antigen (HLA)-DQ2·5-restricted epitopes in an α-gliadin peptide library using CD blood before and after GC. Whole blood IL-2 release assay using electrochemiluminescence is a sensitive test for rare gliadin-specific T cells in CD, and could aid in monitoring and diagnosis. Larger studies and validation with tetramer-based assays are warranted.

Increased chondrocyte sclerostin may protect against cartilage degradation in osteoarthritis.

OBJECTIVES: To investigate the regulation of sclerostin (SOST) in osteoarthritis (OA) and its potential effects on articular cartilage degradation. METHODS: SOST and other Wnt-ß-catenin components were immuno-localised in osteochondral sections of surgically-induced OA in knees of sheep and mice, and human OA samples obtained at arthroplasty. Regulation of SOST mRNA and protein expression by ovine chondrocytes in response to interleukin-1α (IL-1α) or tumour necrosis factor-α (TNFα) was examined in explant cultures. The effect of 25 or 250 ng/ml recombinant SOST alone or in combination with IL-1α, on ovine articular cartilage explant aggrecan degradation, and chondrocyte gene expression of Wnt-ß-catenin pathway proteins, metalloproteinases and their inhibitors, and cartilage matrix proteins was quantified. RESULTS: Contrary to being an osteocyte-specific protein, SOST was expressed by articular chondrocytes, and mRNA levels were upregulated in vitro by IL-1α but not TNFα. Chondrocyte SOST staining was significantly increased only in the focal area of cartilage damage in surgically-induced OA in sheep and mice, as well as end-stage human OA. In contrast, osteocyte SOST was focally decreased in the subchondral bone in sheep OA in association with bone sclerosis. SOST was biologically active in chondrocytes, inhibiting Wnt-ß-catenin signalling and catabolic metalloproteinase [matrix metalloproteinases (MMP) and distintegrin and metalloproteinase with thrombospndin repeats (ADAMTS)] expression, but also decreasing mRNA levels of aggrecan, collagen II and tissue inhibitors of metalloproteinaes (TIMPs). Despite this mixed effect, SOST dose-dependently inhibited IL-1α-stimulated cartilage aggrecanolysis in vitro. CONCLUSIONS: These results implicate SOST in regulating the OA disease processes, but suggest opposing effects by promoting disease-associated subchondral bone sclerosis while inhibiting degradation of cartilage.