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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

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 selfepitope derived from the a3 chain of type IV collagen (a3135-145)1-4. 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 a3135-145-specific T cells expand in patients with Goodpasture disease and, in a3135-145- immunized HLA-DR15 transgenic mice, a3135-145-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 a3135-145 epitope in different binding registers. HLA-DR15-a3135-145 tetramer+ T cells in HLA-DR15 transgenic mice exhibit a conventional T-cell phenotype (Tconv) that secretes pro-inflammatory cytokines. In contrast, HLADR1- a3135-145 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 a3135-145-specific T-cell antigen receptor usage, HLADR15- a3135-145 tetramer+ Foxp3- Tconv and HLA-DR1-a3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes. Moreover, patients with Goodpasture disease display a clonally expanded a3135-145-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 ab

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

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 selfepitope derived from the a3 chain of type IV collagen (a3135-145)1-4. 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 a3135-145-specific T cells expand in patients with Goodpasture disease and, in a3135-145- immunized HLA-DR15 transgenic mice, a3135-145-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 a3135-145 epitope in different binding registers. HLA-DR15-a3135-145 tetramer+ T cells in HLA-DR15 transgenic mice exhibit a conventional T-cell phenotype (Tconv) that secretes pro-inflammatory cytokines. In contrast, HLADR1- a3135-145 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 a3135-145-specific T-cell antigen receptor usage, HLADR15- a3135-145 tetramer+ Foxp3- Tconv and HLA-DR1-a3135-145 tetramer+ Foxp3+CD25hiCD127lo Treg dominant phenotypes. Moreover, patients with Goodpasture disease display a clonally expanded a3135-145-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 ab

T-cell repertoire is selected according to self peptide-MHC (major histocompatibility complex) complexes in the thymus. Although most peripheral T cells recognize specific pathogen-derived peptides complexed to self-MHC exclusively, some possess cross-reactivity to other self or foreign peptides presented by self-MHC molecules; a phenomenon often termed T-cell receptor (TCR) promiscuity or degeneracy. TCR promiscuity has been attributed to various autoimmune conditions. On the other hand, it is considered a mechanism for a relatively limited TCR repertoire to deal with a potentially much larger antigenic peptide repertoire. Such property has also been utilized to bypass self-tolerance for cancer vaccine development. Although many studies explored such degeneracy for peptide of the same length, few studies reported such properties for peptides of different length. In this study, we finely characterized the CD8 + T-cell response specific for a 11mer peptide derived from influenza A viral polymerase basic protein 2. The short-term T-cell line, despite possessing highly biased TCR, was able to react with multiple peptides of different length sharing the same core sequence. Out data clearly showed the importance of detailed and quantitative assessments for such T-cell specificity. Our data also emphasize the importance of biochemical demonstration of the naturally presented minimal peptide.Copyright © 2015 Australasian Society for Immunology Inc.

T-cell repertoire is selected according to self peptide-MHC (major histocompatibility complex) complexes in the thymus. Although most peripheral T cells recognize specific pathogen-derived peptides complexed to self-MHC exclusively, some possess cross-reactivity to other self or foreign peptides presented by self-MHC molecules; a phenomenon often termed T-cell receptor (TCR) promiscuity or degeneracy. TCR promiscuity has been attributed to various autoimmune conditions. On the other hand, it is considered a mechanism for a relatively limited TCR repertoire to deal with a potentially much larger antigenic peptide repertoire. Such property has also been utilized to bypass self-tolerance for cancer vaccine development. Although many studies explored such degeneracy for peptide of the same length, few studies reported such properties for peptides of different length. In this study, we finely characterized the CD8 + T-cell response specific for a 11mer peptide derived from influenza A viral polymerase basic protein 2. The short-term T-cell line, despite possessing highly biased TCR, was able to react with multiple peptides of different length sharing the same core sequence. Out data clearly showed the importance of detailed and quantitative assessments for such T-cell specificity. Our data also emphasize the importance of biochemical demonstration of the naturally presented minimal peptide.Copyright © 2015 Australasian Society for Immunology Inc.