Abstract
Susceptibility and protection against human autoimmune diseases, including type I diabetes, multiple sclerosis, and Goodpasture disease, is associated with particular human leukocyte antigen (HLA) alleles. However, the mechanisms underpinning such HLA-mediated effects on self-tolerance remain unclear. Here we investigate the molecular mechanism of Goodpasture disease, an HLA-linked autoimmune renal disorder characterized by an immunodominant CD4+ T-cell self-epitope derived from the α3 chain of type IV collagen (α3135–145)1,2,3,4. While HLA-DR15 confers a markedly increased disease risk, the protective HLA-DR1 allele is dominantly protective in trans with HLA-DR15 (ref. 2). We show that autoreactive α3135–145-specific T cells expand in patients with Goodpasture disease and, in α3135–145-immunized HLA-DR15 transgenic mice, α3135–145-specific T cells infiltrate the kidney and mice develop Goodpasture disease. HLA-DR15 and HLA-DR1 exhibit distinct peptide repertoires and binding preferences and present the α3135–145 epitope in different binding registers. HLA-DR15-α3135–145 tetramer+ T cells in HLA-DR15 transgenic mice exhibit a conventional T-cell phenotype (Tconv) that secretes pro-inflammatory cytokines. In contrast, HLA-DR1-α3135–145 tetramer+ T cells in HLA-DR1 and HLA-DR15/DR1 transgenic mice are predominantly CD4+Foxp3+ regulatory T cells (Treg cells) expressing tolerogenic cytokines. HLA-DR1-induced Treg cells confer resistance to disease in HLA-DR15/DR1 transgenic mice. HLA-DR15+ and HLA-DR1+ healthy human donors display altered α3135–145-specific T-cell antigen receptor usage, HLA-DR15-α3135–145 tetramer+ Foxp3− Tconv and HLA-DR1-α3135–145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes. Moreover, patients with Goodpasture disease display a clonally expanded α3135–145-specific CD4+ T-cell repertoire. Accordingly, we provide a mechanistic basis for the dominantly protective effect of HLA in autoimmune disease, whereby HLA polymorphism shapes the relative abundance of self-epitope specific Treg cells that leads to protection or causation of autoimmunity.
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Acknowledgements
We thank the staff of the Australian Synchrotron (beamline MX1 and MX2) for assistance with data collection and donors of the Australian Bone Marrow Donor Registry for blood samples. This study was supported by grants from the National Health and Medical Research Council of Australia (NHMRC), 1048575 and 1079648 to A.R.K., 334067 to A.R.K. and S.R.H., and 1071916 to N.L.L.G. N.L.L.G. is supported by a Sylvia and Charles Viertel Senior Medical Research Fellowship. A.W.P. is supported by an NHMRC Senior Research Fellowship. J.R. is supported by an Australian Research Council Laureate Fellowship.
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J.D.O., J.P., H.H.R, J.R. and A.R.K. initiated and designed the research and wrote the manuscript. J.D.O., J.P., Y.H.T., M.H., Z.J.W., N.L.D., P.J.E., K.L.L, K.A.W., P.Y.G., M.A.A., S.H.R. and H.H.R. performed experiments. D.A.P., S.G.H., P.T.C. and J.W.G. provided blood samples and clinicopathological information from patients with Goodpasture disease. A.H., B.G.H., L.F., A.W.P., S.R.H. and N.L.L.G. provided intellectual input and technical support. J.D.O. and J.P. are joint first authors. H.H.R., J.R. and A.R.K. are the co-corresponding and co-senior authors.
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Reviewer Information Nature thanks H.-G. Rammensee, D. Wraith and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Figure 1 HLA-DR15-α3135–145 tetramer+ CD4+ T cells from humans and mice respond to α3135–145 and to whole α3(IV)NC1.
a, HLA-DR15-α3135–145 tetramer+ CD4+ T cells isolated from patients with Goodpasture disease (n = 8) respond to α3135–145 and recombinant human (rh)α3(IV)NC1. HLA-DR15-α3135–145 tetramer+ CD4+ T cells were isolated by magnetic bead separation from the blood of patients with Goodpasture disease, then cultured at a frequency of 400 HLA-DR15-α3135–145 tetramer+ CD4+ T cells per well in the presence of mitomycin C-treated HLA-DR15-α3135–145 tetramer+-cell-depleted white blood cells and either hα3135–145 or rhα3(IV)NC1. Antigen-specific responses were assessed by ELISPOTs for IFN-γ and IL-17A and expressed as numbers of spots per well. b, To induce experimental autoimmune anti-GBM disease, DR15+Fcgr2b−/− mice (n = 5) were immunized with hα3135–145 on days 0, 7 and 14. Disease was measured at day 42, by which time DR15+Fcgr2b−/− mice had developed kidney disease similar to human Goodpasture disease including glomerular segmental necrosis, crescents (PAS stain), linear IgG deposition (direct immunofluorescence) and pathological albuminuria. Each dot represents one mouse. Photomicrographs taken at 400× depict a crescentic glomerulus with segmental necrosis (bottom left) and linear glomerular IgG deposition (bottom right) seen in DR15+Fcgr2b−/− mice immunized with hα3135–145. Scale bars, 30 μm. c, HLA-DR15-α3135–145 tetramer+ CD4+ T cells isolated from DR15+Fcgr2b+/+ mice respond to mouse (m)α3136–146, hα3135–145, rmα3(IV)NC1 and rhα3(IV)NC1. HLA-DR15-α3135–145 tetramer+ CD4+ T cells were isolated by magnetic bead separation from pooled spleen and lymph node cells of two DR15+ transgenic mice 10 days after immunization with mα3136–146 (DWVSLWKGFSF, hα3135–145 GWISLWKGFSF), then cultured at a frequency of 400 HLA-DR15-α3135–145 tetramer+ CD4+ T cells per well in the presence of mitomycin C-treated HLA-DR15-α3135–145 tetramer+-cell-depleted spleen and lymph node cells. Antigen-specific responses were assessed by ELISPOTs for IFN-γ and IL-17A and expressed as numbers of spots per well. *P < 0.05; **P < 0.01; ***P < 0.001 by Kruskal–Wallis test (a, c) or Mann–Whitney U-test (b).
Extended Data Figure 2 Immune responses in DR15+DR1+Fcgr2b+/+ mice.
a, Naive FcγRIIb intact HLA transgenic mice have similar immune properties. Shown are the proportion of splenocytes expressing HLA-DR; HLA-DR15 and HLA-DR1 intensities (MFI) in naive DR15+ (n = 4), DR1+ (n = 4) and DR15+DR1+ transgenic mice (n = 5) showing half the amount of expression in DR15+DR1+ relative to the single transgenic counterparts; total number of splenocytes; proportion of CD4+ and CD4+Foxp3+ splenocytes; total number of lymph node cells retrieved from the brachial, axillary and inguinal lymph nodes; proportion of CD4+ and CD4+Foxp3+ lymph node cells and the overall Vβ repertoire of HLA transgenic mice showing no skewing of any one Vβ chain. Data expressed as mean ± s.e.m., analysed by analysis of variance. b, Co-expression of HLA-DR1 abrogates pro-inflammatory high autoreactivity to peptide mα3129–148 but not to other parts of α3(IV)NC1. FcγRIIb intact DR15+, DR1+ and DR15+DR1+ mice were immunized with murine α3 peptide pools (see Methods for peptide pools) and responses to individual 20-mer peptides measured by re-stimulating the draining lymph node cells ex vivo (x axis numbering represents the N terminus amino-acid number of the individual 20-mer peptide) using [3H]thymidine proliferation assays, and IFN-γ and IL-17A ELISPOTs. Each dot represents the mean response from triplicate determinations in an individual mouse and each bar represents the mean response in each group (DR15+ mice: pool 1, n = 6; pool 2, n = 6; pool 3, n = 5; DR1+ mice: pool 1, n = 5; pool 2, n = 6; pool 3, n = 5; DR15+DR1+ mice: pool 1, n = 6; pool 2, n = 6; pool 3, n = 5). Blue bars indicate no reactivity (stimulation index (SI) < 2; spots < 5), yellow bars indicate low reactivity (2 < SI < 5; 5 < spots < 25) and red bars indicate high reactivity (SI > 5, spots > 25).
Extended Data Figure 3 Self-peptide repertoires of HLA-DR15/DR51 and HLA-DR1 using human cell lines, with electron density maps for the α3135–145 peptide.
Peptide repertoire analysis of 9-mer core sequences of HLA-DR-presented self-peptides in the human B lymphoblastoid cell lines IHW09013 (DR15+/DR51+) and IHW09004 (HLA-DR1+). Amino-acid frequencies in each peptide position p1–p9 were plotted using IceLogo32, with the human proteome as the frequency reference. Relatively enriched amino acids are plotted above the horizontal bar, and depleted amino acids below. The scale of each letter is proportional to the frequency difference to the reference. Peptides were eluted from (a) IHW09013 HLA-DR15+/DR51+ cells and (b) IHW09004 HLA-DR1+ cells. For HLA-DR1, preferred amino acids at p1, p4, p6, p7 and p9 are similar between the human and mouse DR1+ cells. For DR15+/DR51+ human cells compared with DR15+ mouse antigen-presenting cells, the difference at p9, with Lys and Arg being the most frequently bound residues, reflects the known DR51-related motif. c, d, Simulated annealing 2Fobs − Fcalc omit maps for the peptide of (c) HLA-DR15 and (d) HLA-DR1. e, f, Final 2Fobs − Fcalc maps for the peptide in (e) HLA-DR15 and (f) HLA-DR1. Electron density maps contoured at 1σ are shown as blue mesh.
Extended Data Figure 4 HLA-DR1-α3135–145 tetramer+ CD4+ T cells have differential TRAJ usage and the CD4+CD25+ Treg cell gene transcription profile is similar to that of tTreg cells.
a, The TRAJ and TRBJ usage of α3135–145-specific CD4+ T cells was compared using the HLA-DR15-α3135–145 tetramer (in naive DR15+ Fcgr2b+/+ mice) and the HLA-DR1-α3135–145 tetramer (in naive DR1+Fcgr2b+/+ mice) by single-cell sequencing. The α3135–145-specific CD4+ tetramer+ T cells from naive DR15+ (n = 3) and DR1+ (n = 3) transgenic mice were pooled then single-cell sorted, TCR genes amplified by multiplex PCR, then sequenced to determine TRAJ (DR15, n = 81; DR1 n = 84) and TRBJ (DR15, n = 100; DR1 n = 87) usage. For each TCR type/region (TRAV, TRBV, TRAJ, TRBJ; TRAV and TRBV shown in Fig. 2g), we compared the TCR distribution (frequencies of different TCRs) between DR15 and DR1, ***P < 0.001 by Fisher’s exact test. b, HLA-DR1-α3135–145 tetramer− CD4+CD25− Tconv cells, HLA-DR1-α3135–145 tetramer−CD4+CD25+ Treg cells and HLA-DR1-α3135–145 tetramer+ CD4+CD25+ Treg cells were isolated from naive FcγRIIb intact DR1+ or DR15+DR1+ transgenic mice by flow cytometry, RNA extracted and the relative expression of tTreg genes expressed relative to 18S. The HLA-DR1-α3135–145 tetramer+ CD4+CD25+ Treg cells express genes consistent with a tTreg origin, similar to other Treg cells that are not α3135–145-specific from the same mice.
Extended Data Figure 5 Responses and effects of antigen-specific DR1-associated Treg cells.
a, Measurement of pro- and anti-inflammatory mouse (m)α3136–146-specific CD4+ T-cell responses by ex vivo stimulation. CD4+ T cells, isolated and pooled from naive DR15+Fcgr2b+/+ (n = 2), DR1+Fcgr2b+/+ (n = 2) and DR15+DR1+Fcgr2b+/+ (n = 2) transgenic mice, were cultured for 8 days in the presence of mα3136–146- and mitomycin C-treated syngeneic CD4+ cell-depleted splenocytes. Treg cells were depleted by removing CD4+CD25+ T cells by cell sorting. IFN-γ, IL-17A, IL-6 and IL-10 were measured in the cultured supernatant by cytometric bead array and TGF-β by ELISA. The experiment was performed in triplicate and the data presented as mean ± s.e.m. To measure α3136–146-specific CD4+ Treg proliferation, CD4+ T cells were labelled with CTV then stained for Foxp3 on day 8. Results were similar to those performed using human (h)α3135–145 presented in Fig. 3b. b, HLA-DR1-α3135–145-specific Treg cells are potent suppressors of HLA-DR15-α3135–145-induced pro-inflammatory responses. CD4+CD25− T cells were isolated and pooled from naive DR15+DR1+Fcgr2b+/+ mice (n = 2) and co-cultured with either CD4+CD25+ Treg cells (which included HLA-DR1-α3135–145 tetramer+ Treg cells) or CD4+CD25+ HLA-DR1-α3135–145 tetramer− Treg cells from naive DR1+Fcgr2b+/+ mice (n = 4) in the presence of mα3136–146 and CD4+ cell-depleted spleen and lymph node cells from DR15+DR1+ mice. Cells were cultured for 8 days. Proliferation of α3135–145-specific CD4+CD25− cells was measured by labelling only the DR15+DR1+-derived naive CD4+CD25− cells with CTV; IFN-γ, IL-17A, IL-6 and IL-10 were measured in the cultured supernatant by cytometric bead array. In the absence of CD4+CD25+ HLA-DR1-α3135–145 tetramer+ cells, the capacity of Treg cells to prevent the induction of autoreactivity to α3135–145 was impaired. These experiments were performed in triplicate and the data presented as mean ± s.e.m. c, Treg depletion expands HLA-DR15-α3135–145-specific TFH cells. Anti-CD25 monoclonal antibodies (or control rat IgG) were administered 2 days before hα3135–145 immunization and boost (n = 4 per group), then the number of α3135–145-specific PD-1+CXCR5+ TFH cells was enumerated in the draining lymph nodes. Fluorescence-activated cell sorting plots show the expansion of PD1+CXCR5+ TFH cells after Treg depletion and the detection of HLA-DR15-α3135–145-specific cells within that population. *P < 0.05; **P < 0.01; ***P < 0.001 by Mann–Whitney U-test (a, b) or Kruskal–Wallis test (c).
Extended Data Figure 6 In vivo Treg depletion in HLA transgenic mice.
a, Efficiency and duration of Treg depletion using anti-CD25 monoclonal antibodies (clone PC61) in DR15+DR1+Fcgr2b−/− mice. Timeline showing the administration of anti-CD25 monoclonal antibodies 2 days before immunizing mice with human (h)α3135–145. Detection of CD4+Foxp3+ Treg cells in the blood showing depletion of Treg cells at days 7 and 14 in mice that received the anti-CD25 monoclonal antibody (n = 5 each group). White bars represent mice that received control antibodies and solid bars represent mice that received anti-CD25 monoclonal antibodies. b, DR15+Fcgr2b−/− (n = 6 each group), DR1+Fcgr2b−/− (n = 4 each group) and DR15+DR1+Fcgr2b−/− (n = 6 each group) mice immunized with a control peptide, OVA323–339, do not develop renal injury. Functional injury measured by albuminuria and blood urea nitrogen, and histological injury assessed by scoring of PAS-stained histological sections for segmental necrosis and glomerular crescents. Representative PAS-stained histological sections. White bars represent mice that received control antibodies and solid bars represent mice that received anti-CD25 monoclonal antibodies. c, Treg depletion leads to cell-mediated injury in DR15+DR1+Fcgr2b−/− mice (further data collected in the experiment presented in Fig. 4a). Anti-CD25 monoclonal antibodies (or control rat IgG) were administered 2 days before the induction of experimental autoimmune anti-GBM GN by hα3135–145 immunization in DR15+Fcgr2b−/− (n = 6 per group), DR1+Fcgr2b−/− (n = 4 per group) and DR15+DR1+Fcgr2b−/− (n = 8 (Treg intact), 9 (Treg depleted)) mice. Cell-mediated injury was assessed by quantifying glomerular fibrin deposition and enumerating and inflammatory cell infiltrates (CD4+ T cells, macrophages and neutrophils). Renal inflammation was measured by RT–PCR of inflammatory cytokines (TNF, IL-6 and IL-1α) on kidney digests. Scale bars, 30 μm. d, Repeating the experiment presented in Fig. 4a using mouse (m)α3136–146 instead of hα3135–145 as the immunogen showed similar results (DR15+Fcgr2b−/−, n = 6 (Treg intact), 7 (Treg depleted); DR1+Fcgr2b−/−, n = 4 (Treg intact), 5 (Treg depleted); and DR15+DR1+Fcgr2b−/−, n = 6 per group), with the emergence of autoimmune anti-GBM glomerulonephritis in DR15+DR1+Fcgr2b−/− mice only after Treg depletion. Values are mean ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001 by Mann–Whitney U-test (a–d).
Extended Data Figure 7 Single-cell sequencing data from healthy humans and patients with Goodpasture disease validate and corroborate findings in HLA transgenic mice.
a, Comparing the TCR usage of α3135–145-specific CD4+ T cells using the HLA-DR15-α3135–145 tetramer and the HLA-DR1-α3135–145 tetramer by TCR single-cell sequencing, TRAV (DR15, n = 20; DR1 n = 28), TRBV (DR15, n = 20; DR1 n = 24), TRAJ (DR15, n = 20; DR1 n = 28) and TRBJ (DR15, n = 20; DR1 n = 24). For each TCR type/region (TRAV, TRBV, TRAJ, TRBJ), we compared the TCR distribution (frequencies of different TCRs) between DR15 and DR1. *P < 0.05 by Fisher’s exact test. The full HLA-types of the DR15 homozygous donor (HD1) and the DR1 homozygous donor (HD7) are listed in Extended Data Table 3. b, In anti-GBM patients, α3135–145-specific T cells clonally expand. The α3135–145-specific CD4+ T cells from the blood of two anti-GBM patients were single-cell sorted using HLA-DR15-α3135–145 tetramer by flow cytometry. TCR genes were amplified by multiplex PCR, then sequenced to determine TRAV (n = 12 and 45) and TRBV (n = 16 and 44) usage and CDR3 amino-acid sequence. Differently coloured slices within the pie chart highlight the TRAV or TRBV in which repeated CDR3 sequences were found. All CDR3 sequences within the highlighted TRAV or TRBV are shown in coloured letters (one row is one CDR3 sequence).
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This file contains peptide repertoires eluted from human BLCLs IHW09013 (DR15+/DR51+) and IHW09004 (HLA-DR1+). (XLSX 303 kb)
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Ooi, J., Petersen, J., Tan, Y. et al. Dominant protection from HLA-linked autoimmunity by antigen-specific regulatory T cells. Nature 545, 243–247 (2017). https://doi.org/10.1038/nature22329
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DOI: https://doi.org/10.1038/nature22329
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