Single-chain recombinant HLA-DQ2.5/peptide molecules block α2-gliadin-specific pathogenic CD4+ T-cell proliferation and attenuate production of inflammatory cytokines: a potential therapy for celiac disease

Abstract

Celiac disease (CD) is a disorder of the small intestine caused by intolerance to wheat gluten and related proteins in barley and rye. CD4(+) T cells have a central role in CD, recognizing and binding complexes of HLA-DQ2.5 bearing gluten peptides that have survived digestion and that are deamidated by tissue transglutaminase (TG2), propagating a cascade of inflammatory processes that damage and eventually destroy the villous tissue structures of the small intestine. In this study, we present data showing that recombinant DQ2.5-derived molecules bearing covalently tethered α2-gliadin-61-71 peptide have a remarkable ability to block antigen-specific T-cell proliferation and inhibited proinflammatory cytokine secretion in human DQ2.5-restricted α2-gliadin-specific T-cell clones obtained from patients with CD. The results from our in vitro studies suggest that HLA-DQ2.5-derived molecules could significantly inhibit and perhaps reverse the intestinal pathology caused by T-cell-mediated inflammation and the associated production of proinflammatory cytokines.

Figure 1. Primary amino acid sequence alignment of human DQ2-, DR2- and DP2-derived RTLs

“Empty” rDQ2.5 (RTL800) was derived from DQ2 (DQA1*0501/DQB1*0201), RTL302 from DR2 (DRA1*0101/DRB1*1501), and RTL600 from DP2 (DPA1*0103/DPB1*0201) primary sequences. Gaps in the sequences for optimal alignment (*) and the beta-1//alpha-1 junction (←|→) are shown. The conserved cysteines that form a disulfide bond are shaded yellow, and the cysteine residue present in DQ2-derived RTL800 that has been changed to Serine in RTL801 to prevent intermolecular disulfide bond formation and aggregation is underlined. Genes encoding RTL802 & RTL803, variants of 800 and 801, respectively, encode proteins that have the amino-terminal peptide sequence (MFPQPELPYPQPGSGSGSGSGSGSGSGS) instead of the starting methionine. These molecules bear antigenic α2-gliadin-61-71 (Q65E) “α-II” peptide and linker (the gliadin-derived sequence is underlined).

Figure 2. Characterization of human HLA-DQ2.5-derived RTL molecules

(A) Size exclusion chromatography (superdex-75) of purified and refolded “empty” RTL800 and RTL801 (αC44S mutation), & gliadin-61-71-covalently tethered RTL802 and 803. Inset, Samples of purified RTLs were boiled for 5 minutes in Laemmli sample buffer ± the reducing agent s-mercaptoethanol (s-ME), and then analyzed by SDS-PAGE (12%). Non-reduced RTLs (-s-ME) have a smaller apparent molecular weight than reduced RTLs (+s-ME), indicating the presence of a disulfide bond. First and last lanes show the molecular weight standards albumin (66 kD), ovalbumin (45 D), carbonic anhydrase (31 kD) and soybean trypsin inhibitor (21.5 kD). Note that the molecules with the αC44S mutation (RTL801 & 803) have dramatically reduced levels of intermolecular disulfide linked aggregates compared to RTLs 800 & 802 with the wild-type sequence. (B) DQ2.5-derived RTLs are recognized by DQ2.5-specific mAb 2.12.E11. 5ug DQ2-derived RTL800 and 802, DR4-derived RTL363 as a negative control (40), or full-length recombinant DQ2/CLIP as a positive control were blotted in duplicate onto nitrocellulose membranes. Membranes were blocked with 10% FCS in PBS overnight and then incubated with anti-DQ mAbs SPV-L3 (undetermined epitope) (41), 2.12.E11 (DQB1*0201, 0202 and 0203- specific) (42) and SFR-20α5 (undetermined epitope) (43) for 1hr. Blots were washed 2X and then secondary antibodies goat-anti-rat HRP (SFR-20α5) and rabbit-anti-mouse HRP antibodies were used for the detection. IgG were used as the control. All three antibodies recognize full-length DQ2/CLIP, only mAb 2.12.E11 recognized DQ2-derived RTL800 and RTL802, and none of these antibodies could detect DR4-derived RTL363. Our data is consistant with SPV-L3 and SFR-20α5 recognizing the α2 or β2 domains of full-length DQ2 and mAb 2.12.E11 recognizing a unique epitope present in the DQ2.5-derived RTLs that minimally consists of β1-domain residues G45, E46, and F47 (50). (C) Circular dichroism measurements were performed at 25°C on an Aviv Model 215 CD spectrometer using 0.1 mm cells, at 0.5 nm intervals from 260 to 180 nm. Concentration values for each protein solution were determined by amino acid analysis. Buffer, 20 mM Tris, pH 8.5. Deconvolution and analysis of the secondary structure presented in Table I was performed using the variable selection method (51). Data are expressed as Delta-epsilon per mole per cm.

Figure 3. Deamidated gliadin peptide binds specifically to HLA-DQ2.5-derived RTL800

RTLs (20 pMol; ~500 ng) were mixed for 60h at 37 C with 4 nMol of rhodamine-labeled peptides in 100 mM Phosphate, pH 6.0, 0.01% sodium azide and 1 mM EDTA. The reactions were stopped by adding one volume (60 ul) of 1.5 M Tris, 0.1% SDS and 20% glycerol pH 8.8. Forty ul were analyzed by 18% SDS-PAGE and scanned at 580 nm to monitor binding of amino-terminal rhodamine labeled peptides (top panel). Equivalent amounts of RTLs were loaded in each lane, as indicated by staining with Coomassie Blue (middle panel). (Lower panel), the amino-terminal rhodamine-labeled peptides used in this binding study, aligned relative to their position within the immunodominant α2-gliadin-57-89 33-mer. Differences in the sequences of each peptide at key positions are indicated. Boxed regions of the α2-gliadin 33-mer correspond to the α-I (red), α-II (green), and α-III (blue) gliadin minimal epitopes, of which one, three and two copies of each, respectively, are found within the 33-mer peptide. Data are representative of three independent experiments.

Figure 4. HLA-DQ2.5-derived RTL802 blocks antigen specific T cell proliferation

Proliferation assays were performed using CD patient biopsy-derived antigen-specific T clones. TCC were pre-incubated with 4 or 8 uM of the study drug DQ2.5-derived RTL802, negative controls “empty” RTL800, DR4-derived RTL361, or buffer alone for 48 hours. (A) TCC364.1.0.14 proliferated in response to TG2-treated gluten (10 ug/ml) or the α-II p1274 (PQPELPYPQPQLPY) peptide (2 uM and 10 uM). (B) TCC387.19 proliferates in response to TG2-treated gluten (10 ug/ml) or the γ-VII p1729 (PQTQQPEQPFPQPQ) peptide (10 ug/ml). EBV-transformed human B cells expressing HLA-DQ2.5 loaded with antigens were used as APCs. The T:APC ratio was 1:1, with 60,000 T cells/well. Antigens used included 10 μg/ml of gluten, TG2-treated gluten, p1274 (PQPELPYPQPQLPY) or p1729 (PQTQQPEQPFPQPQ) gluten-derived peptides. Data from triplicate wells are presented as average CPM +/- SD. A representative example of three individual experiments is shown.

Figure 5. HLA-DQ2.5-derived RTL802 significantly attenuates antigen-specific cytokine production

Aliquots of supernatants harvested from the RTL proliferation assay at 48 hr after culture with APCs was used for cytokine analysis. A customized human Bio-Plex cytokine kit was used to detect IL-2, IL-5, IL-6, IL-10, IL-17, IFN-γ, and TNF-α. Each data point represented triplicate samples from each treatment group. Significance between the control and treatment groups was determined by Student’s t test. A p value of < 0.05 was considered statistically significant.