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Aim: To use Goodpasture's disease (GPD), a classical form of HLA-linked autoimmunity, to define the mechanism of HLA-mediated risk, and dominant protection from risk of disease. Background(s): The mechanisms underpinning HLA-mediated susceptibility to, and protection from autoimmune diseases are unknown. GPD is HLAlinked and characterized by an immunodominant CD4+ self-epitope derived from the alpha3 chain of Type IV collagen (alpha3135-145). Method(s): HLA-DR transgenic mice, cells from HLA-typed healthy humans and from patients with GPD were used, with HLA-DR-alpha3135-145 tetramers, Xray crystallography, and in vivo/in vitro models of autoimmunity. Result(s): Autoreactive alpha3135-145-specific T cells clonally expanded in GPD patients. In alpha3135-145-immunized HLA-DR15 transgenic mice with GPD, alpha3135-145-specific T cells infiltrated the kidney. Structurally, HLA-DR15 and HLA-DR1 presented alpha3135-145 in different binding registers, resulting in differential T cell receptor (TCR) usage. HLA-DR15-alpha3135-145 tetramer+ CD4+ T cells in disease susceptible HLA-DR15 transgenic mice exhibited a conventional phenotype (Tconv), secreting pro-inflammatory cytokines. In contrast, HLA-DR1-alpha3135-145 tetramer+ cells in disease resistant HLA-DR1 and HLADR15/ DR1 transgenic mice were largely CD4+Foxp3+ regulatory T cells (Tregs) expressing tolerogenic cytokines. In HLA-DR15/DR1 transgenic mice, HLA-DR1-alpha3135-145 specific Tregs conferred protection to alpha3135-145-specific autoimmunity and protected from GPD (alpha3135-145-immunized, Treg intact vs Treg depleted: segmental necrosis 0+/-0 vs 50+/-13% P<0.01, serum urea 9+/-1 vs 31+/-10 P<0.001). HLA-DR15+ and HLA-DR1+ healthy human donors displayed altered alpha3135-145-specific TCRs, with HLA-DR15-alpha3135-145 tetramer+ Foxp3- Tconv and HLA-DR1-alpha3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes (alpha3135-145 tetramer+ Treg:Tconv ratios: DR15 0.04+/-0.00, DR1 9.61+/-1.79, P<0.001). Conclusio

Objectives: Anti-glomerular basement membrane (GBM) disease is a small vessel vasculitis caused by anti-a3(IV)NC1 T and B cell autoreactivity. An immunodominant T cell epitope, a3135-145, has been defined. HLA-DR15 confers an increased disease risk (OR 8.5) while HLA-DR1 is dominantly protective from this risk. These studies aimed to define the mechanisms by which different HLA-DR alleles lead to susceptibility or protection from disease. Method(s): a3135-145-specific CD4+ T cells were enumerated in anti-GBM patients, HLA-typed healthy humans and HLA-DR transgenic mice using HLA-DR-a3135-145 tetramers. The crystal structures of HLADR15-a3135-145 and HLA-DR1-a3135-145 were solved. a3135-145-specific T cell receptor (TCR) usage was determined by single cell sequencing. T cell phenotype was determined by flow cytometry. T cell responses were determined in vivo, ex vivo and by culturing naive T cells with a3135-145 in vitro. Experimental autoimmune anti-GBM disease was induced by a3135-145 immunization of HLA-DR transgenic mice (Treg intact or Treg depleted). Result(s): a3135-145-specific CD4+ T cells were expanded in anti-GBM patients and infiltrated kidneys of a3135-145-immunized HLA-DR15 transgenic mice. Mice transgenic for both HLA-DR15 and HLA-DR1 showed similar reactivity to most a3 peptides, but lacked proinflammatory responses to a3135-145. Structurally, HLA-DR15 and HLA-DR1 presented a3135-145 in different binding registers. Compared to the conformation of a3135-145 in HLA-DR15, in HLA-DR1 a3135-145 adopted a more pronounced conformation with a flipped backbone. These different HLA-peptide conformations altered a3135-145-specific TCR usage and T cell phenotypes. HLA-DR15-a3135-145-specific T cells exhibited a Foxp3-conventional T cell phenotype resulting in proinflammatory T cell autoreactivity, anti-GBM antibody production and disease. In contrast, HLA-DR1-a3135-145-specific T cells were predominantly anti-inflammatory Foxp3+ Tregs that proliferated and respon

Aim: To use Goodpasture's disease (GPD), a classical form of HLA-linked autoimmunity, to define the mechanism of HLA-mediated risk, and dominant protection from risk of disease. Background(s): The mechanisms underpinning HLA-mediated susceptibility to, and protection from autoimmune diseases are unknown. GPD is HLAlinked and characterized by an immunodominant CD4+ self-epitope derived from the alpha3 chain of Type IV collagen (alpha3135-145). Method(s): HLA-DR transgenic mice, cells from HLA-typed healthy humans and from patients with GPD were used, with HLA-DR-alpha3135-145 tetramers, Xray crystallography, and in vivo/in vitro models of autoimmunity. Result(s): Autoreactive alpha3135-145-specific T cells clonally expanded in GPD patients. In alpha3135-145-immunized HLA-DR15 transgenic mice with GPD, alpha3135-145-specific T cells infiltrated the kidney. Structurally, HLA-DR15 and HLA-DR1 presented alpha3135-145 in different binding registers, resulting in differential T cell receptor (TCR) usage. HLA-DR15-alpha3135-145 tetramer+ CD4+ T cells in disease susceptible HLA-DR15 transgenic mice exhibited a conventional phenotype (Tconv), secreting pro-inflammatory cytokines. In contrast, HLA-DR1-alpha3135-145 tetramer+ cells in disease resistant HLA-DR1 and HLADR15/ DR1 transgenic mice were largely CD4+Foxp3+ regulatory T cells (Tregs) expressing tolerogenic cytokines. In HLA-DR15/DR1 transgenic mice, HLA-DR1-alpha3135-145 specific Tregs conferred protection to alpha3135-145-specific autoimmunity and protected from GPD (alpha3135-145-immunized, Treg intact vs Treg depleted: segmental necrosis 0+/-0 vs 50+/-13% P<0.01, serum urea 9+/-1 vs 31+/-10 P<0.001). HLA-DR15+ and HLA-DR1+ healthy human donors displayed altered alpha3135-145-specific TCRs, with HLA-DR15-alpha3135-145 tetramer+ Foxp3- Tconv and HLA-DR1-alpha3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes (alpha3135-145 tetramer+ Treg:Tconv ratios: DR15 0.04+/-0.00, DR1 9.61+/-1.79, P<0.001). Conclusio

Objectives: Anti-glomerular basement membrane (GBM) disease is a small vessel vasculitis caused by anti-a3(IV)NC1 T and B cell autoreactivity. An immunodominant T cell epitope, a3135-145, has been defined. HLA-DR15 confers an increased disease risk (OR 8.5) while HLA-DR1 is dominantly protective from this risk. These studies aimed to define the mechanisms by which different HLA-DR alleles lead to susceptibility or protection from disease. Method(s): a3135-145-specific CD4+ T cells were enumerated in anti-GBM patients, HLA-typed healthy humans and HLA-DR transgenic mice using HLA-DR-a3135-145 tetramers. The crystal structures of HLADR15-a3135-145 and HLA-DR1-a3135-145 were solved. a3135-145-specific T cell receptor (TCR) usage was determined by single cell sequencing. T cell phenotype was determined by flow cytometry. T cell responses were determined in vivo, ex vivo and by culturing naive T cells with a3135-145 in vitro. Experimental autoimmune anti-GBM disease was induced by a3135-145 immunization of HLA-DR transgenic mice (Treg intact or Treg depleted). Result(s): a3135-145-specific CD4+ T cells were expanded in anti-GBM patients and infiltrated kidneys of a3135-145-immunized HLA-DR15 transgenic mice. Mice transgenic for both HLA-DR15 and HLA-DR1 showed similar reactivity to most a3 peptides, but lacked proinflammatory responses to a3135-145. Structurally, HLA-DR15 and HLA-DR1 presented a3135-145 in different binding registers. Compared to the conformation of a3135-145 in HLA-DR15, in HLA-DR1 a3135-145 adopted a more pronounced conformation with a flipped backbone. These different HLA-peptide conformations altered a3135-145-specific TCR usage and T cell phenotypes. HLA-DR15-a3135-145-specific T cells exhibited a Foxp3-conventional T cell phenotype resulting in proinflammatory T cell autoreactivity, anti-GBM antibody production and disease. In contrast, HLA-DR1-a3135-145-specific T cells were predominantly anti-inflammatory Foxp3+ Tregs that proliferated and respon