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Objectives: In microscopic polyangiitis, autoreactivity to myeloperoxidase (MPO) can cause rapidly progressive glomerulonephritis. While it is unknown how tolerance to MPO is lost, infections have been implicated in the development of autoimmunity and disease. MPO409-428 is a promiscuous MPO CD4+ T-cell epitope overlapping a pathogenic B cell MPO epitope. This study tests the hypothesis that molecular mimicry can induce pathogenic anti-MPO autoimmunity. Method(s): BLAST searches were used to identify candidate microbial peptides with homology to MPO409-428. Anti-MPO T-cell responses were determined by immunizing C57BL/6 mice with candidate peptides, then measuring recall responses to MPO by 3[H]-T proliferation and IFN-g and IL-17A ELISPOTs. Anti-MPO antibodies were measured by ELISA, indirect immunofluorescence, and their activity by ex vivo ROS production and by neutrophil glomerular recruitment after passive antibody transfer. Disease was induced by immunizing mice with peptide or whole bacteria, followed by depositing MPO in the glomerulus using low-dose sheep anti-mouse basement membrane globulin. Result(s): Immunizing C57BL/6 mice with 4 different peptides from human pathogens most homologous to the core residues of MPO409-428 did not trigger autoreactivity to MPO or MPO409-428. However, immunization with a peptide derived from 6-phosphogluconate dehydrogenase (mimic-6-pgd), found only in some strains of Staphylococci, did induce T cell autoreactivity to MPO. The corresponding mammalian and closely related bacterial 6-pgd sequences did not induce anti-MPO autoimmunity, demonstrating the specificity of this sequence. Mimic-6-pgd peptide immunization induced MPOANCA that stained neutrophils in a p-ANCA pattern and were functionally active in vitro and in vivo. Mimic-6-pgd-induced MPO T cell autoreactivity resulted in anti-MPO glomerulonephritis in a T cell mediated model. Furthermore, when MPO was planted in glomeruli, mice immunized with whole killed Staphyloco

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

Anti-GBM disease is a form of small vessel vasculitis that manifests as rapidly progressive glomerulonephritis. It has a strong association with the HLA-DR15 allele (odds ratio: 8.5), and the immunodominant T cell epitope a3135-145 is restricted to HLA-DR15. A DR15-specific inhibitor, PV-267, has potential as a targeted therapy, as current treatment is broadly immunosuppressive and has detrimental side effects. This study aims to demonstrate that specific inhibition of HLA-DR15 prevents autoreactivity to the immunodominant T cell epitope and attenuates injury in experimental autoimmune anti-glomerular basement membrane (GBM) disease. HLA-DR15 transgenic mice (backcrossed onto an FcgRIIb-/-background) were used in this study. Autoreactivity to a3135-145 was determined by immunising mice with a3135-145 at day 0, while administering PV-267 daily (30mg/kg) from day-1 to day 10, then measuring recall responses ex vivo by [3H]-thymidine proliferation and IFNg and IL-17A ELISPOTs. Experimental autoimmune anti-GBM disease was induced in mice by immunisations of a3135-145 at days 0, 7, and 14. In a prevention model, mice were administered PV-267 every second day (30mg/kg) from day-1 to day 42. In a treatment model, PV-267 was administered daily (50mg/kg) from day 28 to day 42. Mice were culled at day 42. Functional and histological endpoints, as well as immune cell infiltration were determined. Compared with mice that received vehicle control, mice that received PV-267 had reduced a3135-145-specific proliferation (SI: 9.5+/-0.9 vs 2.2+/-0.5, P<0.001) as well as reduced numbers of IFNg and IL-17A spots. In the prevention model of disease, mice given PV-267 had fewer infiltrating glomerular CD4+ T-cells (0.90+/-0.12 vs 0.36+/-0.03 cells/glomerular cross section, P<0.01), reduced albuminuria (albumin/creatinine ratio: 0.215+/-0.056 vs 0.010+/-0.007 mg/mmol, P<0.01), and reduced glomerular segmental necrosis and crescents. In the treatment model, there was no difference in album

MPO-specific CD4+ and CD8+ T cells have the capacity to induce injury in experimental anti-MPO glomerulonephritis (GN) in mice. However, mechanisms by which CD8+ cells participate in disease in this model have not been elucidated. Patients with an unfavorable prognosis have a non-exhausted CD8+ phenotype associated with increased signaling through the IL-7Ra (CD127). Furthermore, the inhibitory receptor PD-1 is associated with reduced IL-7R signaling. We therefore investigated the contribution of the IL-7R to anti-MPO GN by blocking IL-7 signalling using a neutralizing but non-depleting anti-IL-7Ra antibody. Anti-MPO autoimmunity was induced by immunizing mice with recombinant mouse MPO (rmMPO) at day 0 and 7. GN was triggered using a subnephritogenic dose of anti-mouse basement membrane globulin at day 16 (this induces neutrophil recruitment and deposits MPO in glomeruli), and mice culled at day 20. We also used an extended model where immunization remained at days 0 and 7 (rmMPO or OVA), but the trigger was delayed until day 43 and mice were culled at day 47. Comparing CD8+ and CD4+ cells at days 20 and 47, a higher proportion of intrarenal and splenic CD8+ cells expressed CD127 at day 20 compared to day 47 in the MPO immunized groups. In contrast, a higher proportion of renal and splenic CD8+ cells expressed PD-1 at day 47 in MPO immunised mice, suggesting that the CD8+ cells are more exhausted at this time point. A similar pattern was also seen on CD4+ T cells, suggesting that the role of IL-7 signalling in this model may not be restricted to CD8+ T cells. Administration of anti-IL-7Ra antibodies after the induction of anti-MPO autoimmunity (commencing day 15) in the day 20 model attenuated anti-MPO GN with reduced albuminuria, segmental necrosis, dermal DTH, glomerular leukocytes and renal chemokines (CCL2, CXCL1, CXCL10 and CXCL20) compared with control antibody treatment. However, PD-1 expression in the kidney was not correspondingly increased in MPO immunize

Objectives: In microscopic polyangiitis, autoreactivity to myeloperoxidase (MPO) can cause rapidly progressive glomerulonephritis. While it is unknown how tolerance to MPO is lost, infections have been implicated in the development of autoimmunity and disease. MPO409-428 is a promiscuous MPO CD4+ T-cell epitope overlapping a pathogenic B cell MPO epitope. This study tests the hypothesis that molecular mimicry can induce pathogenic anti-MPO autoimmunity. Method(s): BLAST searches were used to identify candidate microbial peptides with homology to MPO409-428. Anti-MPO T-cell responses were determined by immunizing C57BL/6 mice with candidate peptides, then measuring recall responses to MPO by 3[H]-T proliferation and IFN-g and IL-17A ELISPOTs. Anti-MPO antibodies were measured by ELISA, indirect immunofluorescence, and their activity by ex vivo ROS production and by neutrophil glomerular recruitment after passive antibody transfer. Disease was induced by immunizing mice with peptide or whole bacteria, followed by depositing MPO in the glomerulus using low-dose sheep anti-mouse basement membrane globulin. Result(s): Immunizing C57BL/6 mice with 4 different peptides from human pathogens most homologous to the core residues of MPO409-428 did not trigger autoreactivity to MPO or MPO409-428. However, immunization with a peptide derived from 6-phosphogluconate dehydrogenase (mimic-6-pgd), found only in some strains of Staphylococci, did induce T cell autoreactivity to MPO. The corresponding mammalian and closely related bacterial 6-pgd sequences did not induce anti-MPO autoimmunity, demonstrating the specificity of this sequence. Mimic-6-pgd peptide immunization induced MPOANCA that stained neutrophils in a p-ANCA pattern and were functionally active in vitro and in vivo. Mimic-6-pgd-induced MPO T cell autoreactivity resulted in anti-MPO glomerulonephritis in a T cell mediated model. Furthermore, when MPO was planted in glomeruli, mice immunized with whole killed Staphyloco

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

Anti-GBM disease is a form of small vessel vasculitis that manifests as rapidly progressive glomerulonephritis. It has a strong association with the HLA-DR15 allele (odds ratio: 8.5), and the immunodominant T cell epitope a3135-145 is restricted to HLA-DR15. A DR15-specific inhibitor, PV-267, has potential as a targeted therapy, as current treatment is broadly immunosuppressive and has detrimental side effects. This study aims to demonstrate that specific inhibition of HLA-DR15 prevents autoreactivity to the immunodominant T cell epitope and attenuates injury in experimental autoimmune anti-glomerular basement membrane (GBM) disease. HLA-DR15 transgenic mice (backcrossed onto an FcgRIIb-/-background) were used in this study. Autoreactivity to a3135-145 was determined by immunising mice with a3135-145 at day 0, while administering PV-267 daily (30mg/kg) from day-1 to day 10, then measuring recall responses ex vivo by [3H]-thymidine proliferation and IFNg and IL-17A ELISPOTs. Experimental autoimmune anti-GBM disease was induced in mice by immunisations of a3135-145 at days 0, 7, and 14. In a prevention model, mice were administered PV-267 every second day (30mg/kg) from day-1 to day 42. In a treatment model, PV-267 was administered daily (50mg/kg) from day 28 to day 42. Mice were culled at day 42. Functional and histological endpoints, as well as immune cell infiltration were determined. Compared with mice that received vehicle control, mice that received PV-267 had reduced a3135-145-specific proliferation (SI: 9.5+/-0.9 vs 2.2+/-0.5, P<0.001) as well as reduced numbers of IFNg and IL-17A spots. In the prevention model of disease, mice given PV-267 had fewer infiltrating glomerular CD4+ T-cells (0.90+/-0.12 vs 0.36+/-0.03 cells/glomerular cross section, P<0.01), reduced albuminuria (albumin/creatinine ratio: 0.215+/-0.056 vs 0.010+/-0.007 mg/mmol, P<0.01), and reduced glomerular segmental necrosis and crescents. In the treatment model, there was no difference in album

MPO-specific CD4+ and CD8+ T cells have the capacity to induce injury in experimental anti-MPO glomerulonephritis (GN) in mice. However, mechanisms by which CD8+ cells participate in disease in this model have not been elucidated. Patients with an unfavorable prognosis have a non-exhausted CD8+ phenotype associated with increased signaling through the IL-7Ra (CD127). Furthermore, the inhibitory receptor PD-1 is associated with reduced IL-7R signaling. We therefore investigated the contribution of the IL-7R to anti-MPO GN by blocking IL-7 signalling using a neutralizing but non-depleting anti-IL-7Ra antibody. Anti-MPO autoimmunity was induced by immunizing mice with recombinant mouse MPO (rmMPO) at day 0 and 7. GN was triggered using a subnephritogenic dose of anti-mouse basement membrane globulin at day 16 (this induces neutrophil recruitment and deposits MPO in glomeruli), and mice culled at day 20. We also used an extended model where immunization remained at days 0 and 7 (rmMPO or OVA), but the trigger was delayed until day 43 and mice were culled at day 47. Comparing CD8+ and CD4+ cells at days 20 and 47, a higher proportion of intrarenal and splenic CD8+ cells expressed CD127 at day 20 compared to day 47 in the MPO immunized groups. In contrast, a higher proportion of renal and splenic CD8+ cells expressed PD-1 at day 47 in MPO immunised mice, suggesting that the CD8+ cells are more exhausted at this time point. A similar pattern was also seen on CD4+ T cells, suggesting that the role of IL-7 signalling in this model may not be restricted to CD8+ T cells. Administration of anti-IL-7Ra antibodies after the induction of anti-MPO autoimmunity (commencing day 15) in the day 20 model attenuated anti-MPO GN with reduced albuminuria, segmental necrosis, dermal DTH, glomerular leukocytes and renal chemokines (CCL2, CXCL1, CXCL10 and CXCL20) compared with control antibody treatment. However, PD-1 expression in the kidney was not correspondingly increased in MPO immunize