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Background Myeloperoxidase (MPO)-ANCA-associated GN is a significant cause of renal failure. Manipulating autoimmunity by inducing regulatory T cells is potentially a more specific and safer therapeutic option than conventional immunosuppression. Methods To generate MPO-specific regulatory T cells, we used a modified protein-conjugating compound, 1-ethyl-3-(3'dimethylaminopropyl)-carbodiimide (ECDI), to couple the immunodominant MPO peptide (MPO409-428) or a control ovalbumin peptide (OVA323-339) to splenocytes and induced apoptosis in the conjugated cells. We then administered MPO-and OVA-conjugated apoptotic splenocytes (MPO-Sps and OVA-Sps, respectively) to mice and compared their effects on development and severity of anti-MPO GN. We induced autoimmunity to MPO by immunizing mice with MPO in adjuvant; to trigger GN, we used low-dose antiglomerular basement membrane globulin, which transiently recruits neutro-phils that deposit MPO in glomeruli. We also compared the effects of transferring CD4+ T cells from mice treated with MPO-Sp or OVA-Sp to recipient mice with established anti-MPO autoimmunity. Results MPO-Sp but not OVA-Sp administration increased MPO-specific, peripherally derived CD4+ Foxp3- type 1 regulatory T cells and reduced anti-MPO autoimmunity and GN. However, in mice depleted of regulatory T cells, MPO-Sp administration did not protect from anti-MPO autoimmunity or GN. Mice with established anti-MPO autoimmunity that received CD4+ T cells transferred from mice treated with MPO-Sp (but not CD4+ T cells transferred from mice treated with OVA-Sp) were protected from anti-MPO autoimmunity and GN, confirming the induction of therapeutic antigen-specific regulatory T cells. Conclusions These findings in a mouse model indicate that administering apoptotic splenocytes conjugated with the immunodominant MPO peptide suppresses anti-MPO GN by inducing antigen-specific tolerance.Copyright © 2019 by the American Society of Nephrology.

Background: Myeloperoxidase (MPO)-ANCA-associated GN is a significant cause of renal failure. Manipulating autoimmunity by inducing regulatory T cells is potentially a more specific and safer therapeutic option than conventional immunosuppression. Method(s): To generate MPO-specific regulatory T cells, we used a modified protein-conjugating compound, 1-ethyl-3-(3'dimethylaminopropyl)-carbodiimide (ECDI), to couple the immunodominant MPO peptide (MPO409-428) or a control ovalbumin peptide (OVA323-339) to splenocytes and induced apoptosis in the conjugated cells. We then administered MPO- and OVA-conjugated apoptotic splenocytes (MPO-Sps and OVA-Sps, respectively) to mice and compared their effects on development and severity of anti-MPO GN. We induced autoimmunity to MPO by immunizing mice with MPO in adjuvant; to trigger GN, we used low-dose antiglomerular basement membrane globulin, which transiently recruits neutrophils that deposit MPO in glomeruli. We also compared the effects of transferring CD4+ T cells from mice treated with MPO-Sp or OVA-Sp to recipient mice with established anti-MPO autoimmunity. Result(s): MPO-Sp but not OVA-Sp administration increased MPO-specific, peripherally derived CD4+Foxp3- type 1 regulatory T cells and reduced anti-MPO autoimmunity and GN. However, in mice depleted of regulatory T cells, MPO-Sp administration did not protect from anti-MPO autoimmunity or GN. Mice with established anti-MPO autoimmunity that received CD4+ T cells transferred from mice treated with MPO-Sp (but not CD4+ T cells transferred from mice treated with OVA-Sp) were protected from anti-MPO autoimmunity and GN, confirming the induction of therapeutic antigen-specific regulatory T cells. Conclusion(s): These findings in a mouse model indicate that administering apoptotic splenocytes conjugated with the immunodominant MPO peptide suppresses anti-MPO GN by inducing antigen-specific tolerance.Copyright © 2019 by the American Society of Nephrology.

Background Myeloperoxidase (MPO)-ANCA-associated GN is a significant cause of renal failure. Manipulating autoimmunity by inducing regulatory T cells is potentially a more specific and safer therapeutic option than conventional immunosuppression. Methods To generate MPO-specific regulatory T cells, we used a modified protein-conjugating compound, 1-ethyl-3-(3'dimethylaminopropyl)-carbodiimide (ECDI), to couple the immunodominant MPO peptide (MPO409-428) or a control ovalbumin peptide (OVA323-339) to splenocytes and induced apoptosis in the conjugated cells. We then administered MPO-and OVA-conjugated apoptotic splenocytes (MPO-Sps and OVA-Sps, respectively) to mice and compared their effects on development and severity of anti-MPO GN. We induced autoimmunity to MPO by immunizing mice with MPO in adjuvant; to trigger GN, we used low-dose antiglomerular basement membrane globulin, which transiently recruits neutro-phils that deposit MPO in glomeruli. We also compared the effects of transferring CD4+ T cells from mice treated with MPO-Sp or OVA-Sp to recipient mice with established anti-MPO autoimmunity. Results MPO-Sp but not OVA-Sp administration increased MPO-specific, peripherally derived CD4+ Foxp3- type 1 regulatory T cells and reduced anti-MPO autoimmunity and GN. However, in mice depleted of regulatory T cells, MPO-Sp administration did not protect from anti-MPO autoimmunity or GN. Mice with established anti-MPO autoimmunity that received CD4+ T cells transferred from mice treated with MPO-Sp (but not CD4+ T cells transferred from mice treated with OVA-Sp) were protected from anti-MPO autoimmunity and GN, confirming the induction of therapeutic antigen-specific regulatory T cells. Conclusions These findings in a mouse model indicate that administering apoptotic splenocytes conjugated with the immunodominant MPO peptide suppresses anti-MPO GN by inducing antigen-specific tolerance.Copyright © 2019 by the American Society of Nephrology.

Background: Myeloperoxidase (MPO)-ANCA-associated GN is a significant cause of renal failure. Manipulating autoimmunity by inducing regulatory T cells is potentially a more specific and safer therapeutic option than conventional immunosuppression. Method(s): To generate MPO-specific regulatory T cells, we used a modified protein-conjugating compound, 1-ethyl-3-(3'dimethylaminopropyl)-carbodiimide (ECDI), to couple the immunodominant MPO peptide (MPO409-428) or a control ovalbumin peptide (OVA323-339) to splenocytes and induced apoptosis in the conjugated cells. We then administered MPO- and OVA-conjugated apoptotic splenocytes (MPO-Sps and OVA-Sps, respectively) to mice and compared their effects on development and severity of anti-MPO GN. We induced autoimmunity to MPO by immunizing mice with MPO in adjuvant; to trigger GN, we used low-dose antiglomerular basement membrane globulin, which transiently recruits neutrophils that deposit MPO in glomeruli. We also compared the effects of transferring CD4+ T cells from mice treated with MPO-Sp or OVA-Sp to recipient mice with established anti-MPO autoimmunity. Result(s): MPO-Sp but not OVA-Sp administration increased MPO-specific, peripherally derived CD4+Foxp3- type 1 regulatory T cells and reduced anti-MPO autoimmunity and GN. However, in mice depleted of regulatory T cells, MPO-Sp administration did not protect from anti-MPO autoimmunity or GN. Mice with established anti-MPO autoimmunity that received CD4+ T cells transferred from mice treated with MPO-Sp (but not CD4+ T cells transferred from mice treated with OVA-Sp) were protected from anti-MPO autoimmunity and GN, confirming the induction of therapeutic antigen-specific regulatory T cells. Conclusion(s): These findings in a mouse model indicate that administering apoptotic splenocytes conjugated with the immunodominant MPO peptide suppresses anti-MPO GN by inducing antigen-specific tolerance.Copyright © 2019 by the American Society of Nephrology.

Background: Glomerulonephritis, a major cause of end-stage renal failure, results from autoreactivity to the neutrophil derived enzyme myeloperoxidase (MPO). In most autoimmune conditions, treatment with apoptotic antigen-coupled splenocytes can induce peripheral T cell tolerance by facilitating host APCs to reprocess and present the Ag in a non-immunogenic manner. Recent discovery of immunodominant MPO peptides makes this approach relevant to anti-MPO glomerulonephritis. Aim(s): To assess whether antigen-coupled ethylenecarbodiimide (ECDI)-fixed splenocytes (Ag-Sp) can induce tolerance to MPO and suppress anti-MPO glomerulonephritis. Method(s): We compared disease between C57BL/6 (WT) mice recieving either MPO409-428-Sp or OVA323-339-Sp. A standard model of anti-MPO glomerulonephritis was used. Anti-MPO autoimmunity induced by MPO409-428 immunisations and glomerulonephritis triggered using low dose anti-glomerular basement membrane globulin. Result(s): Glomerulonephritis and autoimmunity was less severe in mice treated with MPO409-428-Sp, prior to the induction of autoimmunity than in control mice (segmental necrosis 43+/-5 vs 19+/-1%), glomerular leukocytes (macrophages; 0.53 +/- 0.08 vs 0.25 +/- 0.02 cells per glomerular cross section [c/gcs]; CD4+ cells 0.47 +/- 0.05 vs 0.24 +/- 0.02c/gcs) Autoimmunity, assessed by MPO induced DTH was reduced in MPO409-428-Sp treated mice versus control (0.11 +/- 0.02 vs 0.02 +/- 0.01DELTAmm). Furthermore transfer of CD4+ cells from mice treated with MPO409-428-Sp to mice with established anti MPO autoimmunity significantly decreased induced glomerulonephritis (segmental necrosis 45.25 +/- 4% vs 23.13 +/- 3%) compared to controls receiving CD4+ cells from OVA323-339-Sp treated mice. Conclusion(s): MPO409-428-Sp suppress anti-MPO glomerulonephritis by the induction of antigen specific tolerance mediated by Tregs and is relevant to human disease.

Background: Glomerulonephritis, a major cause of end-stage renal failure, results from autoreactivity to the neutrophil derived enzyme myeloperoxidase (MPO). In most autoimmune conditions, treatment with apoptotic antigen-coupled splenocytes can induce peripheral T cell tolerance by facilitating host APCs to reprocess and present the Ag in a non-immunogenic manner. Recent discovery of immunodominant MPO peptides makes this approach relevant to anti-MPO glomerulonephritis. Aim(s): To assess whether antigen-coupled ethylenecarbodiimide (ECDI)-fixed splenocytes (Ag-Sp) can induce tolerance to MPO and suppress anti-MPO glomerulonephritis. Method(s): We compared disease between C57BL/6 (WT) mice recieving either MPO409-428-Sp or OVA323-339-Sp. A standard model of anti-MPO glomerulonephritis was used. Anti-MPO autoimmunity induced by MPO409-428 immunisations and glomerulonephritis triggered using low dose anti-glomerular basement membrane globulin. Result(s): Glomerulonephritis and autoimmunity was less severe in mice treated with MPO409-428-Sp, prior to the induction of autoimmunity than in control mice (segmental necrosis 43+/-5 vs 19+/-1%), glomerular leukocytes (macrophages; 0.53 +/- 0.08 vs 0.25 +/- 0.02 cells per glomerular cross section [c/gcs]; CD4+ cells 0.47 +/- 0.05 vs 0.24 +/- 0.02c/gcs) Autoimmunity, assessed by MPO induced DTH was reduced in MPO409-428-Sp treated mice versus control (0.11 +/- 0.02 vs 0.02 +/- 0.01DELTAmm). Furthermore transfer of CD4+ cells from mice treated with MPO409-428-Sp to mice with established anti MPO autoimmunity significantly decreased induced glomerulonephritis (segmental necrosis 45.25 +/- 4% vs 23.13 +/- 3%) compared to controls receiving CD4+ cells from OVA323-339-Sp treated mice. Conclusion(s): MPO409-428-Sp suppress anti-MPO glomerulonephritis by the induction of antigen specific tolerance mediated by Tregs and is relevant to human disease.

Aim: To determine whether human amniotic epithelial stem cells (hAECs) can attenuate autoimmune anti-myeloperoxidase (MPO) glomerulonephritis. Background(s): hAECs are characteristically pluripotent, with low antigenicity and are highly anti-inflammatory, making them an attractive stem cell based therapeutic. Their use in anti-MPO glomerulonephritis has not been explored. Method(s): Anti-MPO glomerulonephritis was induced by immunisation with MPO/adjuvant (day [d] 0, 7) followed by disease trigger using low dose antiglomerular basement membrane globulin (d16). hAECs or vehicle were administered either 3d before each MPO/adjuvant injection or just before disease trigger (d14) and their effects on anti-MPO autoimmunity and GN assessed. hAECs were also co-cultured with splenocytes from MPO-immunised animals and their effect on MPO-specific T cell responses measured. Result(s): hAECs suppressed MPO-specific T cell responses when co-cultured with splenocytes in vitro (2.7 +/- 0.6 vs 7.5 +/- 0.8% bromodeoxyuridine+CD4+ cells, p < 0.01). In vivo, when used as a preventative, hAECs attenuated MPO-specific dermal delayed-type hypersensitivity (DTH; 0.1 +/- 0.03 vs 0.5 +/- 0.DELTAmm, p < 0.05), while enhancing proliferation of foxp3+CD25+ regulatory T cells (1.20 +/- 0.09 vs 0.84 +/- 0.13%, p < 0.05), CD4+ IL-10 producing cells (0.05 +/- 0.01 vs 0.26 +/- 0.09% p < 0.05) and surface expression of indoleamine 2,3 dioxygenase+ (0.027 +/- 0.004 vs 0.28 +/- 0.08%, p < 0.05), without affecting dendritic cells. Therapeutic administration of hAECs attenuated the development of glomerulonephritis (segmental necrosis: 15.6 +/- 2.2 vs 40.6 +/- 2.2%, p < 0.05), correlating with reduced accumulation of macrophages (0.2 +/- 0.04 vs 0.4 +/- 0.06 cells/ glomerular cross section [c/gcs], p < 0.05), CD4+ cells (0.3 +/- 0.03 vs 0.4 +/- 0.05 c/ gcs, p < 0.05) and neutrophils (0.2 +/- 0.03 vs 0.4 +/- 0.03 c/gcs, p < 0.01). Systemic anti-MPO autoimmunity was also reduced (DTH: 0.01 +/- 0.01 vs 0.4

Aim: To determine whether human amniotic epithelial stem cells (hAECs) can attenuate autoimmune anti-myeloperoxidase (MPO) glomerulonephritis. Background(s): hAECs are characteristically pluripotent, with low antigenicity and are highly anti-inflammatory, making them an attractive stem cell based therapeutic. Their use in anti-MPO glomerulonephritis has not been explored. Method(s): Anti-MPO glomerulonephritis was induced by immunisation with MPO/adjuvant (day [d] 0, 7) followed by disease trigger using low dose antiglomerular basement membrane globulin (d16). hAECs or vehicle were administered either 3d before each MPO/adjuvant injection or just before disease trigger (d14) and their effects on anti-MPO autoimmunity and GN assessed. hAECs were also co-cultured with splenocytes from MPO-immunised animals and their effect on MPO-specific T cell responses measured. Result(s): hAECs suppressed MPO-specific T cell responses when co-cultured with splenocytes in vitro (2.7 +/- 0.6 vs 7.5 +/- 0.8% bromodeoxyuridine+CD4+ cells, p < 0.01). In vivo, when used as a preventative, hAECs attenuated MPO-specific dermal delayed-type hypersensitivity (DTH; 0.1 +/- 0.03 vs 0.5 +/- 0.DELTAmm, p < 0.05), while enhancing proliferation of foxp3+CD25+ regulatory T cells (1.20 +/- 0.09 vs 0.84 +/- 0.13%, p < 0.05), CD4+ IL-10 producing cells (0.05 +/- 0.01 vs 0.26 +/- 0.09% p < 0.05) and surface expression of indoleamine 2,3 dioxygenase+ (0.027 +/- 0.004 vs 0.28 +/- 0.08%, p < 0.05), without affecting dendritic cells. Therapeutic administration of hAECs attenuated the development of glomerulonephritis (segmental necrosis: 15.6 +/- 2.2 vs 40.6 +/- 2.2%, p < 0.05), correlating with reduced accumulation of macrophages (0.2 +/- 0.04 vs 0.4 +/- 0.06 cells/ glomerular cross section [c/gcs], p < 0.05), CD4+ cells (0.3 +/- 0.03 vs 0.4 +/- 0.05 c/ gcs, p < 0.05) and neutrophils (0.2 +/- 0.03 vs 0.4 +/- 0.03 c/gcs, p < 0.01). Systemic anti-MPO autoimmunity was also reduced (DTH: 0.01 +/- 0.01 vs 0.4

Aim: To determine whether administration of antigen-coupled ethylenecarbodiimide (ECDI)-fixed splenocytes (Ag-Sp) can suppress autoimmune anti-myeloperoxidase (MPO) glomerulonephritis. Background(s): In some autoimmune conditions, treatment with apoptotic, ECDIinduced Ag-SP can induce peripheral T cell tolerance by facilitating host APCs to reprocess and present the Ag in a non-immunogenic manner. Recent discovery of immunodominant MPO peptides makes this approach relevant to anti-MPO glomerulonephritis. Method(s): We compared disease between C57BL/6 (WT) mice that received either no cells, MPO409-428-Sp or OVA323-339-Sp. A standard model of anti-MPO glomerulonephritis was used. Anti-MPO autoimmunity was induced by MPO409-428 immunisation and glomerulonephritis triggered using low dose antiglomerular basement membrane globulin. Result(s): Compared to mice with disease not receiving Ag-SP, glomerulonephritis was less severe in mice treated with MPO409-428-Sp prior to the induction of autoimmunity (glomerular segmental necrosis 23 +/- 3 vs 8 +/- 1% P < 0.05) and decreased glomerular leukocytes (macrophages; 1.7 +/- 0.03 vs 1.1 +/- 0.01 cells per glomerular cross section [c/gcs], P < 0.05; CD4+ cells 1.1 +/- 0.06 vs 0.54 +/- 0.06c/ gcs, P < 0.05). Systemic anti-MPO autoimmunity, assessed by MPO-induced dermal delayed type hypersensitivity (DTH) was also reduced in MPO409-428-Sp treated mice (0.14 +/- 0.04 vs 0.26 +/- 0.03DELTAmm, P < 0.05). To determine if the disease suppression by Ag-Sp is antigen specific, mice were either treated with OVA323-339-Sp or MPO409-428-Sp. Compared with administering MPO409-428-Sp, glomerular injury was more severe when mice were injected with OVA323-339-Sp before MPO immunization (segmental necrosis 43 +/- 5 vs 19 +/- 1%, P < 0.05). Administering MPO409-428-Sp to WT mice with established anti-MPO autoimmunity resulted in less glomerular disease (segmental necrosis 44 +/- 4 vs 31 +/- 4%, P < 0.05) and limited systemic anti-MPO responses (

Aim: To determine whether administration of antigen-coupled ethylenecarbodiimide (ECDI)-fixed splenocytes (Ag-Sp) can suppress autoimmune anti-myeloperoxidase (MPO) glomerulonephritis. Background(s): In some autoimmune conditions, treatment with apoptotic, ECDIinduced Ag-SP can induce peripheral T cell tolerance by facilitating host APCs to reprocess and present the Ag in a non-immunogenic manner. Recent discovery of immunodominant MPO peptides makes this approach relevant to anti-MPO glomerulonephritis. Method(s): We compared disease between C57BL/6 (WT) mice that received either no cells, MPO409-428-Sp or OVA323-339-Sp. A standard model of anti-MPO glomerulonephritis was used. Anti-MPO autoimmunity was induced by MPO409-428 immunisation and glomerulonephritis triggered using low dose antiglomerular basement membrane globulin. Result(s): Compared to mice with disease not receiving Ag-SP, glomerulonephritis was less severe in mice treated with MPO409-428-Sp prior to the induction of autoimmunity (glomerular segmental necrosis 23 +/- 3 vs 8 +/- 1% P < 0.05) and decreased glomerular leukocytes (macrophages; 1.7 +/- 0.03 vs 1.1 +/- 0.01 cells per glomerular cross section [c/gcs], P < 0.05; CD4+ cells 1.1 +/- 0.06 vs 0.54 +/- 0.06c/ gcs, P < 0.05). Systemic anti-MPO autoimmunity, assessed by MPO-induced dermal delayed type hypersensitivity (DTH) was also reduced in MPO409-428-Sp treated mice (0.14 +/- 0.04 vs 0.26 +/- 0.03DELTAmm, P < 0.05). To determine if the disease suppression by Ag-Sp is antigen specific, mice were either treated with OVA323-339-Sp or MPO409-428-Sp. Compared with administering MPO409-428-Sp, glomerular injury was more severe when mice were injected with OVA323-339-Sp before MPO immunization (segmental necrosis 43 +/- 5 vs 19 +/- 1%, P < 0.05). Administering MPO409-428-Sp to WT mice with established anti-MPO autoimmunity resulted in less glomerular disease (segmental necrosis 44 +/- 4 vs 31 +/- 4%, P < 0.05) and limited systemic anti-MPO responses (