Pathogenic Autoreactive T and B Cells Cross-React with Mimotopes Expressed by a Common Human Gut Commensal to Trigger Autoimmunity

Abstract

Given the immense antigenic load present in the microbiome, we hypothesized that microbiota mimotopes can be a persistent trigger in human autoimmunity via cross-reactivity. Using antiphospholipid syndrome (APS) as a model, we demonstrate cross-reactivity between non-orthologous mimotopes expressed by a common human gut commensal, Roseburia intestinalis (R. int), and T and B cell autoepitopes in the APS autoantigen β2-glycoprotein I (β2GPI). Autoantigen-reactive CD4+ memory T cell clones and an APS-derived, pathogenic monoclonal antibody cross-reacted with R. int mimotopes. Core-sequence-dependent anti-R. int mimotope IgG titers were significantly elevated in APS patients and correlated with anti-β2GPI IgG autoantibodies. R. int immunization of mice induced β2GPI-specific lymphocytes and autoantibodies. Oral gavage of susceptible mice with R. int induced anti-human β2GPI autoantibodies and autoimmune pathologies. Together, these data support a role for non-orthologous commensal-host cross-reactivity in the development and persistence of autoimmunity in APS, which may apply more broadly to human autoimmune disease.

Keywords: Bacteroides thetaiotaomicron; DNA methyltransferase; IgA-coated bacteria; Th1 cell clones; apolipoprotein H; calprotectin; microbiotme; molecular mimicry; systemic autoimmunity; thrombosis.

Conflict of interest statement

DECLARATION OF INTERESTS

M.A.K. received salary, consulting fees, honoraria, and research funds from Roche, Bristol-Meyers Squibb, AbbVie, and Cell Applications and is an employee of Roche. M.A.K. and S.M.V. hold an international patent on the use of antibiotics and commensal vaccination to treat autoimmunity and received royalties. The remaining authors declare no conflict of interests.

Copyright © 2019 Elsevier Inc. All rights reserved.

Figures

Figure 1.. APS Patients Exhibit Signs of Gut Inflammation with Systemic Adaptive Immune Responses to β2-Glycoprotein I-Mimotope-Expressing Roseburia intestinalis

(A) Clustal Omega alignment of β2GPI B cell domain I epitope (left panel) aligned to Roseburia intestinalis L1-82 (R. int) mimotope (WP_118597735.1). Clustal Omega alignment of HLA-DRB4*0103-restricted immunodominant T cell domain V epitope within β2GPI (p276-290, KVSFFCKNKEKKCSY) (right panel) aligned to R. int mimotope (EEU99424.1). (B) In silico modeling of the core domain I epitope R39-R43 exposed on human β2GPI (PDB_ID: 1qubA) and R. int mimotope (SWISS-MODEL, WP_118597735.1). (C–F) 16S rRNA sequencing (C and D) and IgA-seq (E and F) was performed as described in STAR Methods. (C) Alpha diversity between APS (n = 35) and NHD (n = 37) by Shannon-Weiner diversity index for all time points and the average of visits from each patient. (D) Principal-coordinate analysis of weighted UniFrac distances shows no difference (PERMANOVA 999 permutations, p = 0.164). (E) IgA-coated fraction of fecal bacteria have decreased Shannon-Weiner diversity in APS (n = 9) compared to NHD (n = 9), p = 0.0003. (F) IgA-coated fraction of fecal bacteria have significantly different weighted UniFrac diversity (PERMANOVA 999 permutations, p = 0.001). (G) Elevated fecal calprotectin in APS (all time points n = 38, p = 0.0002; average n = 13, p = 0.002) compared to NHD (all time points n = 40, average n = 18). (H) Elevated plasma lipocalin-2 in APS (all time points n = 42, p = R. int IgG to anti-R. int IgM is increased in APS (all time points n = 42, p < 0.0001; average n = 15, p = 0.024) compared to NHD (all time points n = 43; average n = 17, p = 0.04). (J) Ratio of plasma anti-R. int IgG to anti-R. int IgA is increased in APS (all time points n = 42, p = 0.0008; average n = 15, p = 0.022) compared to NHD (all time points n = 43; average n = 17). (K) Peripheral blood mononuclear cells from HLA-DRB4*01 (DR53)+ APS (n = 5) proliferate more in response to R. int lysate compared to HLA-DRB4*01 (DR53)− APS (n = 3, p = 0.036), NHD HLA-DRB4*01 (DR53)+ (n = 3, p = 0.036), and NHD HLA-DRB4*01 (DR53)− (n = 4, p = 0.0016) donors, respectively. Treatment of lysates with proteinase K abrogated the response. Counts per minute (CPM) as measured by 3[H]-thymidine incorporation was used to calculate proliferation with averages of triplicates shown as single points. Stimulation index was calculated as CPM of stimulated PBMCs divided by CPM of unstimulated PMBCs. All points, all time points; avg, average; APS, antiphospholipid syndrome; NHD, normal healthy donors. Two-tailed Mann-Whitney U test was performed unless noted. Error bars represent ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2.. β2GPI-Reactive Memory CD4+ T Cells from APS Patients Cross-React with the Corresponding R. int Mimotope

(A–C) β2GPI p276-290 (DV epitope)-specific CD4+, CCR6−, β7+ memory T cells (n = 2, p < 0.001) cross-react with R. int mimotope peptide (DV mimotope) in (A) proliferation assays (n = 2, p < 0.0087). These clones secrete (B) GM-CSF (n = 10, p < 0.023) and (C) IFN-γ (n = 8, p < 0.001) in the culture supernatant 72 h post-antigen stimulation. (D–F) Representative single cell-sorted β2GPI p276-290, DRB4*0103 MHC class II tetramer-positive CD4+, CD45RA−, CD45RO+, and CCR6− T cell clones that proliferate (D) significantly (n = 3, p < 0.05) in response to whole, heat-killed R. int, DV tetramer epitope (n = 2, p < 0.005), and β2GPI (n = 2, p < 0.0042). Phytohemagglutinin (PHA) plus rhIL-2 serve as positive control. Clones secrete (E) GM-CSF (n = 4, p < 0.002) and (F) IFN-γ (n = 3, p < 0.0001). CPM measured by 3[H]-thymidine incorporation with averages of triplicates shown. Tetramer proliferation was determined using ATP luminescence. Fold increase determined by dividing the average of triplicates from individual clones by the average of the unstimulated (“no antigen”) clones in relative light units (RLU). Unpaired, two-tailed Student’s t test. Error bars represent ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p< 0.0001.

Figure 3.. A Pathogenic, APS-Derived β2GPI R39-R43-Specific Autoantibody Cross-Reacts with R. int DNA Methyltransferase

APS-derived monoclonal antibodies P1-117 and P2-6 were cloned as full-length human IgG1 and purified. Full-length mature rhβ2GPI, a mutant version containing alanines in positions R39-R43 (rhβ2GPIΔ39-43), R. int DNA methyltransferase (R. int DNMT, WP_118597735.1), and a mutant version containing alanines in positions R122-R126 (R. int DNMTΔ122-126) were expressed and purified. (A) Representative ELISA titration curve showing P1-117 binds to R39-R43 within domain I (DI) of β2GPI. (B) Representative R. int ELISA showing P1-117, P2-6 negative control (binding domain I outside R39-R43), R. int-immunized, and sham-immunized mouse sera, respectively. X-axis shows serum dilutions (1:100 serum = 10 μg/mL antibody) in 2-fold dilutions. (C) Representative ELISA titration curve of P1-117 and P2-6 binding to R. int DNMT or R. int DNMTΔ122-126, respectively. (D) Human trophoblast cells were treated with media, P1-117 (50 μg/mL) or IgG isotype control (50 μg/mL). After 48 h, cell migration was measured (n = 4, *p

Figure 4.. APS Patients Have Significantly Elevated Levels of Anti-R. int DNMT IgG that Positively Correlate with Anti-β2GPI IgG Autoantibodies

ELISAs of R. int DNMT and R. int DNMTΔ122-126 plasma diluted 1:1000 and probed for IgG. (A) Averages from 2-3 time points are shown as individual points. APS patients have significantly higher anti-R. int DNMT IgG (n = 15, p = 0.0112) compared to NHD (n = 20). APS patient plasma contains significantly less anti-R. int DNMTΔ122-126 IgG compared to wild-type DNMT (n = 15, p = 0.0103). APS plasma anti-R. int DNMTΔ122-126 IgG values were not significantly different from NHD plasma anti-R. int DNMT IgG or anti-R. int DNMTΔ122-126 IgG, respectively (n = 20 each). (B) Average APS anti-R. int DNMT IgG levels (x axis) significantly correlate with increasing anti-β2GPI IgG (y axis). Pearson r = 0.569, R2 = 0.323, p = 0.0271 two-tailed. (C) Average NHD R. int DNMT IgG levels (x axis) do not correlate with average anti-β2GPI IgG (y axis). Pearson r = 0.216, R2 = 0.047, p = 0.361 two-tailed. Two-tailed Mann-Whitney U test performed for inter-group comparison and two-tailed Wilcoxon signed-rank test performed for intra-group comparison. APS, antiphospholipid syndrome; NHD, normal healthy donor. Error bars represent ± SEM. *p < 0.05, ns = not significant.

Figure 5.. Immunization of BALB/c Mice with R. int Induces Human β2GPI Cross-Reactivity

(A) Clustal Omega alignment of RGGMR DI epitope in β2GPI, Roseburia intestinalis L1-82 (R. int) and Bacteroides thetaiotaomicron VPI-5482 (B. theta). B. theta lacks T cell mimotope homology but contains a partial B cell mimotope (GGMR) homology. (B and C) Proliferative response to immunization was determined using ATP luminescence in triplicates. Fold increase was determined by averaging triplicates from stimulated populations and dividing by the average of the unstimulated (“no antigen”) cells in RLU. Splenocytes (B) and peripheral lymphocytes isolated from inguinal and axillary lymph nodes (C) were restimulated ex vivo with rhβ2GPI. (B) Splenocytes from R. int-immunized mice (n = 7) respond significantly more to rhβ2GPI than sham-immunized mice (n = 7, p = 0.0017) and B. Theta-immunized mice (n = 6, p = 0.0047). (C) Peripheral lymphocytes from R. int-immunized mice (n = 7) respond significantly more to rhβ2GPI than sham-immunized mice (n = 7, p = 0.0006) and B. theta-immunized mice (n = 6, p = 0.0012). (D and E) Mice were immunized s.c. with lysates from R. int, B. theta, or no lysates (sham) in IFA followed by three s.c. IFA boosts. Representative ELISA using 1:100 diluted serum. Values represent the average OD450 of individual mice in triplicates. (D) R. int-immunized mice have significantly more serum anti-β2GPI IgG (n = 8) compared to B. theta-immunized mice (n = 7, p = 0.0003) and sham-immunized mice (n = 7, p = 0.0003). R. int-immunized and B. theta-immunized sera lose significantly binding to rhβ2GPI39-43 compared to rhβ2GPI. (E) R. int-immunized mice have significantly more serum anti-R. int DNMT IgG (n = 8) compared to B. theta-immunized mice (n = 7, p = 0.0205) and sham-immunized mice (n = 7, p = 0.0003). R. int-immunized and B. theta-immunized sera lose significantly binding to R. int DNMTΔ122-126 compared to R. int DNMT. Two-tailed Mann-Whitney U test performed for inter-group comparison and two-tailed Wilcoxon signed-rank test performed for intra-group comparison. Error bars represent ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.

Figure 6.. Gavage of (NZW × BXSB)F1 Male Mice with R. intestinalis Induces Anti-Human β2GPI IgG Autoantibodies and Thromboses

(NZW × BXSB)F1 mice were gavaged weekly with R. intestinalis L1-82 (R. int) or media after two weeks of vancomycin treatment. (A and B) Sera from 16-week-old mice were tested for anti-human β2GPI IgG or anti-R. int DNMT IgG by ELISA at 1:100 dilution. (A) R. int gavage of male mice (n = 17) induces significantly elevated anti-human β2GPI IgG compared to males gavaged with media (n = 16, p = 0.006) and females gavaged with R. int (n = 6, p = 0.0002). R. int gavage of male mice induces significantly elevated anti-R. int DNMT IgG compared to males gavaged with media (p < 0.0001) and females gavaged with R. int (p < 0.0001). (B) Anti-human β2GPI IgG positively correlates with anti-R. int DNMT in male R. int-gavaged mice at 16 weeks of age. Pearson r = 0.626, R2 = 0.392, p = 0.007 two-tailed. (C) Shown are representative micrographs of H&E-stained myocardium from two R. int-gavaged males that died spontaneously because of autoimmunity at 17 weeks (upper panel) and 16 weeks (lower panel) of age, respectively. R. int-gavaged males show widespread myocardial and subendocardial lymphocytic infiltrates. (D) Shown are representative micrographs of H&E-stained myocardium from two male mice gavaged with media that survived beyond 34 weeks at which point they were euthanized (representative images of two separate mice, upper and lower). Surviving mice show unremarkable myocardium without any evidence of inflammation or myocardial infarction. Scale bar, 500 μm. Two-tailed Mann-Whitney U test performed for inter-group comparison and two-tailed Wilcoxon signed-rank test performed for intra-group comparison. Error bars represent ± SEM. **p