Methyldopa blocks MHC class II binding to disease-specific antigens in autoimmune diabetes

Abstract

Major histocompatibility (MHC) class II molecules are strongly associated with many autoimmune disorders. In type 1 diabetes (T1D), the DQ8 molecule is common, confers significant disease risk, and is involved in disease pathogenesis. We hypothesized that blocking DQ8 antigen presentation would provide therapeutic benefit by preventing recognition of self-peptides by pathogenic T cells. We used the crystal structure of DQ8 to select drug-like small molecules predicted to bind structural pockets in the MHC antigen-binding cleft. A limited number of the predicted compounds inhibited DQ8 antigen presentation in vitro, with 1 compound preventing insulin autoantibody production and delaying diabetes onset in an animal model of spontaneous autoimmune diabetes. An existing drug with a similar structure, methyldopa, specifically blocked DQ8 in patients with recent-onset T1D and reduced inflammatory T cell responses to insulin, highlighting the relevance of blocking disease-specific MHC class II antigen presentation to treat autoimmunity.

Keywords: Autoimmunity; Diabetes; Endocrinology; Immunotherapy; MHC class 2.

Conflict of interest statement

Conflict of interest: AWM and DAO are inventors on a patent titled “Compounds that modulate autoimmunity and methods of using the same,” licensed to ImmunoMolecular Therapeutics (US patent number 9,629,848). AWM and PAG are scientific cofounders of ImmunoMolecular Therapeutics and own shares in the company. KJS and BP are former employees of Novartis.

Figures

Figure 1. In silico screening of a small-molecule library by molecular docking to structural pockets within the HLA-DQ8 peptide–binding groove and in vitro screening of the top-scoring compounds.

(A) Molecular model of the DQ8 peptide binding cleft, with pockets 1, 4, 6, and 9 depicted by colored spheres. Using a supercomputer, 139,735 repository compounds were docked into each pocket in 1,000 different orientations and scored on the basis of the free energy of binding (ΔG). (B) The top-scoring compounds were screened with an in vitro bioassay, which used an immortalized T cell that responds to an insulin epitope presented by DQ8. The TCR α/β genes from an insulin B:9-23–restricted T cell clone were retrovirally transduced into a T cell line devoid of endogenous TCR to create a TCR transductant, named clone 5. Clone 5 was cultured with insulin peptide plus a small molecule in the presence of DQ8-Tg murine splenocytes. After overnight culture, secreted IL-2 was measured in the cell culture supernatant with a highly sensitive ELISA. In the heatmap, “No antigen” represents the response without in vitro–added peptide, and “Peptide” represents the response to peptide without an in vitro–added small molecule. Numbers 1–39 depict the tested compounds, and the compounds were different for each pocket. The insulin B:9-23 peptide was used to screen pockets 1 and 9 (p1 and p9), while insulin B:13-23 was used to screen pockets 4 and 6 (p4 and p6), which resulted in a greater baseline IL-2 response. Supplemental Tables 1–4 list the corresponding chemical structures, ΔG estimates from molecular docking, and rank based on the overall free energy score.

Figure 2. TATD blocks DQ8-restricted T cell responses and prevents diabetes in NOD mice.

(A) Chemical structure of TATD. (B) TATD blocked an in vitro DQ8–restricted T cell response to insulin B:13-23 (Clone 5) and deamidated α-gliadin228-240 (489 TCR). Data represent the mean ± SEM and are representative of 3 independent experiments. No antigen addition to the culture resulted in IL-2 levels below 3 pg/ml. (C) TATD was cultured with recombinant α-gliadin/DQ8 protein and free peptide in conditions to allow peptide exchange. After washing, the recombinant protein was used to stimulate 489, and IL-2 secretion was measured. Data are from triplicate wells and are representative of 3 independent experiments. 489 in culture without protein resulted in IL-2 levels below 3 pg/ml. (D) Female NOD mice were treated from 4 to 12 weeks of age with 20 mg/kg TATD (n = 10) or PBS (n = 13) by intraperitoneal injection daily for 5 days each week. P = 0.006, by log-rank test. (E) Peak serum insulin autoantibody (IAA) levels were measured using a fluid-phase RIA during the 40-week prevention study. **P = 0.003, by Mann-Whitney U test. The dotted line at 0.01 indicates a positive value. (F) Blood glucose levels during the late prevention study, in which female NOD mice were treated with 30 mg/kg TATD orally each day (n = 9), 50 μg anti-CD3 monoclonal antibody intraperitoneally for 5 consecutive days (n = 10), or PBS (n = 10) beginning at 12 weeks of age and ending at 25 weeks. Data represent the mean ± SEM. *P < 0.05, by ANOVA for comparison of TATD versus PBS. (G) Intraperitoneal GTT following cessation of the study treatments; each dot represents an individual mouse. ***P < 0.01, by 2-tailed, unpaired t test. (H) Representative H&E-stained pancreatic sections from PBS- and TATD-treated mice; insulin staining is shown in brown. Original magnification, ×15. (I) Insulitis scoring from at least 100 separate islets from TATD-treated (n = 3) and PBS-treated (n = 5) mice.

Figure 3. Methyldopa blocks DQ8 antigen presentation in vitro.

(A) Methyldopa chemical structure. Methyldopa blocked an in vitro DQ8–restricted T cell response by (B) clone 5 to insulin B:13-23 and (C) TCR 489 to deamidated α-gliadin presented by DQ8. The negative controls (no antigen added to culture) resulted in IL-2 levels below 3 pg/ml. (D) Methyldopa specifically inhibited 489, a DQ8-restricted T cell response, while not altering C7CH17, a DR4-restricted T cell response to the HA peptide HA306-318. Cells from an EBV-transformed B cell line homozygous for DQ8 and DR4 were used as APCs in this experiment, such that each APC had DQ8 and DR4 on the cell surface. (E) Methyldopa did not inhibit a DQ8-restricted T cell response to the influenza peptide HA102-118. (B–E) Data represent the mean ± SEM and are representative of 3 independent experiments. (F) Representative isothermal titration calorimetric data for titration of methyldopa into DQ8 protein. Top: Heat released as a function of time from 2-μl injections of 4 mM methyldopa titrated into the sample cell containing 4 μM DQ8 protein at 25oC. Bottom: Fitted binding curve along with the measured thermodynamic parameters and the calculated KD from 3 independent experiments, reported as the mean ± SEM. (G) Molecular docking model of methyldopa in the DQ8 antigen–binding cleft shows potential H bonds between a methyldopa hydroxyl group and DQα62 asparagine (DQα N62) on the DQα-helix and between the methyldopa carboxylic acid and DQβ30 tyrosine (DQβ Y30) on the floor of the cleft.

Figure 4. Methyldopa blocks DQ8 antigen presentation in vivo.

(A) Diagram of the assay used to monitor the potency of methyldopa to block DQ8 in Tg mice. (B) Adult DQ8-Tg mice were gavaged with vehicle or 200 mg/kg methyldopa 3 times per day for 4 days (n = 4/group). Ex vivo splenocytes were used as APCs to present different concentrations of the insulin B:13-23 peptide or a deamidated α-gliadin peptide to clone 5 or 489, respectively. No methyldopa was added to the in vitro culture. A dose of 200 mg/kg is equivalent to 1,000 mg 3 times per day in a human weighing 60 kg. Data represent the mean ± SEM and are representative of 3 independent experiments. (C) Percentages and (D) numbers of CD11b+ and CD11c+ cells in the spleens of treated mice. (E) MFI of DQ8 cell-surface staining on each cell population. Each dot represents an individual mouse.

Figure 5. Methyldopa treatment specifically blocks DQ8 antigen presentation in recent-onset T1D patients with the DQ8 allele.

(A) Diagram of the assay used to monitor specific MHC class II antigen presentation. PBMCs from participants were isolated and frozen after each study visit. These primary PBMCs were then thawed and used as APCs to stimulate engineered T cells (TCR transductants) that respond to a specific peptide presented by a given MHC class II molecule (DQ8, DR4, or DQ2). Secreted IL-2 from each TCR transductant was measured using a highly sensitive ELISA. (B) Individual study subject response for 489 (DQ8-restricted) and (C) C7CH17 (DR4-restricted). Data represent the mean ± SEM from triplicate wells at each time point during the study. Colors represent the durations on and off the drug. Participants underwent dose titration, with a low dose (500 mg 2 times/day), moderate dose (500 mg 3 times/day), and high dose (2–3 grams over the course of the day), and then went off the drug. “No antigen” indicates that there was no antigen added in vitro in the assay. (D) Summative data on study participants (n = 20) showing their responses to 489 and (E) clone 5, both of which were DQ8 restricted. *P = 0.001, **P < 0.001, ***P = 0.02, and #P < 0.01, using a longitudinal mixed-effects model that compared the least-squares mean at each time point with baseline. (F) Data from study participants with DR4 subtypes (n = 18) able to present and stimulate the HA306-318–restricted TCR transductant C7CH17. (G) Response to a DQ2 TCR transductant (233 responding to α-gliadin62-73) for the study duration in subjects with a DQ2 allele (n = 7). The negative control (no antigen added to culture) resulted in IL-2 levels below 2 pg/ml for each subject and for individual TCR transductants. Data in D–G are depicted as the least-squares mean ± SEM for the study cohort; individual responses are shown in Supplemental Figure 10.

Figure 6. Methyldopa treatment reduces primary antigen–specific T cells restricted to HLA-DQ8 but not those presented by DR4.

Cryopreserved PBMCs were thawed, cultured in the presence or absence of protein/peptide for 48 hours, washed, and then transferred to an IFN-γ monoclonal antibody–coated plate for overnight culture, followed by development and enumeration of ELISPOTs. IFN-γ ELISPOT results from study subjects for (A) an insulin B chain mimotope, B:9-23 (B22E), known to be presented by DQ8, (B) whole tetanus toxin (TT) protein, (C) an epitope of TT consisting of amino acids 506-525 known to be presented by DR4, and (D) another DR4-restricted TT epitope, amino acids 922-941. Seven study subjects responded to the insulin B chain mimotope at baseline and were further evaluated for responses to TT protein and epitopes. Each data point represents the total number of spots for a given condition from triplicate wells, minus the total number of spots without antigen (background) for an individual. Symbols represent the same individual tested for each condition. *P = 0.016, by Wilcoxon matched-pairs, signed-rank test; P = NS for TT, TT506-525, and TT922-941. Supplemental Table 8 provides ELISPOT counts at baseline and 3 months for each individual and condition, including no antigen as a negative control and whole TT as a positive control.

Figure 7. Measures of glycemic control and β cell function among study participants.

Individual responses showed (A) an improvement in hemoglobin A1c, a measure of average blood glucose over the preceding 3 months, from baseline to study completion. *P = 0.008, by Wilcoxon matched-pairs, signed-rank test. (B) The amounts of exogenous insulin used per kilogram of body weight were similar from the beginning to the end of the study. (C) Maintenance of β cell function, as measured by the C-peptide 2-hour AUC following a MMTT compared with baseline levels. C-peptide is a measure of endogenous insulin secretion, as both are secreted at a 1:1 molar ratio. Individuals ingested a liquid meal (Boost) with a fixed amount of protein, fat, and carbohydrate in the fasting state, followed by the timed measurements of serum C-peptide at 0, 15, 30, 60, 90, and 120 minutes to compute the AUC. Each data point represents a single individual, with a line connecting the same study participant; n = 20 subjects in A–C, with the mean ± SD reported for the entire cohort at the start of study and upon study completion.