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A novel clinically relevant strategy to abrogate autoimmunity and regulate alloimmunity in NOD mice.

Vergani A, D'Addio F, Jurewicz M, Petrelli A, Watanabe T, Liu K, Law K, Schuetz C, Carvello M, Orsenigo E, Deng S, Rodig SJ, Ansari JM, Staudacher C, Abdi R, Williams J, Markmann J, Atkinson M, Sayegh MH, Fiorina P - Diabetes (2010)

Bottom Line: Using allogeneic islet transplantation models as well as NOD mice with recent onset type 1 diabetes, we addressed the therapeutic efficacy and immunomodulatory mechanisms associated with a new immunoregulatory protocol based on prolonged low-dose mATG plus CTLA4-Ig.Immunologic analysis of mice receiving transplants revealed a complete abrogation of autoimmune responses and severe downregulation of alloimmunity in response to treatment.The capacity to regulate alloimmunity and to abrogate the autoimmune response in NOD mice in different settings confirmed that prolonged mATG+CTLA4-Ig treatment is a clinically relevant strategy to translate to humans with type 1 diabetes.

View Article: PubMed Central - PubMed

Affiliation: Transplantation Research Center, Children's Hospital and Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. paolo.fiorina@childrens.harvard.edu

ABSTRACT

Objective: To investigate a new clinically relevant immunoregulatory strategy based on treatment with murine Thymoglobulin mATG Genzyme and CTLA4-Ig in NOD mice to prevent allo- and autoimmune activation using a stringent model of islet transplantation and diabetes reversal.

Research design and methods: Using allogeneic islet transplantation models as well as NOD mice with recent onset type 1 diabetes, we addressed the therapeutic efficacy and immunomodulatory mechanisms associated with a new immunoregulatory protocol based on prolonged low-dose mATG plus CTLA4-Ig.

Results: BALB/c islets transplanted into hyperglycemic NOD mice under prolonged mATG+CTLA4-Ig treatment showed a pronounced delay in allograft rejection compared with untreated mice (mean survival time: 54 vs. 8 days, P < 0.0001). Immunologic analysis of mice receiving transplants revealed a complete abrogation of autoimmune responses and severe downregulation of alloimmunity in response to treatment. The striking effect on autoimmunity was confirmed by 100% diabetes reversal in newly hyperglycemic NOD mice and 100% indefinite survival of syngeneic islet transplantation (NOD.SCID into NOD mice).

Conclusions: The capacity to regulate alloimmunity and to abrogate the autoimmune response in NOD mice in different settings confirmed that prolonged mATG+CTLA4-Ig treatment is a clinically relevant strategy to translate to humans with type 1 diabetes.

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BALB/c skin was transplanted into NOD.SCID mice, which subsequently received (via adoptive transfer) 15 × 106 splenocytes from normoglycemic or hyperglycemic untransplanted mice or from normoglycemic (not rejecting) or hyperglycemic (rejecting) transplanted mice treated with prolonged mATG+CTLA4-Ig at day 56 after transplantation. Skin rejection was slowed when adoptive transfer was performed with splenocytes from treated transplanted mice compared with untreated untransplanted mice (normoglycemic vs. normoglycemic prolonged mATG+CTLA4-Ig P < 0.05; hyperglycemic vs. hyperglycemic prolonged mATG+CTLA4-Ig; P < 0.05) (A). Therefore, in the same NOD.SCID mice, we evaluated the onset of diabetes. Mice that received splenocytes by adoptive transfer from normoglycemic or hyperglycemic NOD mice developed diabetes, as expected. However, the ability to transfer diabetes by splenocytes was completely abrogated in NOD mice treated with prolonged mATG and CTLA4-Ig, either rejecting or not (untreated vs. prolonged mATG+CTLA4-Ig; P < 0.001) (B). BALB/c hearts were transplanted in NOD mice in a purely alloimmune setting; a prolongation of graft survival was observed in treated mice (untreated vs. prolonged mATG+CTLA4-Ig; P = 0.017), similar results were obtained in hyperglycemic recipients (P = 0.03) (C). NOD.SCID islets were transplanted into hyperglycemic NOD mice; indefinite survival was achieved in treated mice (untreated vs. prolonged mATG+CTLA4-Ig; P = 0.0018) (D). NOD.SCID islet grafts harvested 100 days after transplantation in treated mice revealed completely preserved islet allografts with very few infiltrating cells surrounding the islets (E1, H&E; E2, insulin; E3, glucagon; E4, B220; E5, CD3; E6, FoxP3). *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 6: BALB/c skin was transplanted into NOD.SCID mice, which subsequently received (via adoptive transfer) 15 × 106 splenocytes from normoglycemic or hyperglycemic untransplanted mice or from normoglycemic (not rejecting) or hyperglycemic (rejecting) transplanted mice treated with prolonged mATG+CTLA4-Ig at day 56 after transplantation. Skin rejection was slowed when adoptive transfer was performed with splenocytes from treated transplanted mice compared with untreated untransplanted mice (normoglycemic vs. normoglycemic prolonged mATG+CTLA4-Ig P < 0.05; hyperglycemic vs. hyperglycemic prolonged mATG+CTLA4-Ig; P < 0.05) (A). Therefore, in the same NOD.SCID mice, we evaluated the onset of diabetes. Mice that received splenocytes by adoptive transfer from normoglycemic or hyperglycemic NOD mice developed diabetes, as expected. However, the ability to transfer diabetes by splenocytes was completely abrogated in NOD mice treated with prolonged mATG and CTLA4-Ig, either rejecting or not (untreated vs. prolonged mATG+CTLA4-Ig; P < 0.001) (B). BALB/c hearts were transplanted in NOD mice in a purely alloimmune setting; a prolongation of graft survival was observed in treated mice (untreated vs. prolonged mATG+CTLA4-Ig; P = 0.017), similar results were obtained in hyperglycemic recipients (P = 0.03) (C). NOD.SCID islets were transplanted into hyperglycemic NOD mice; indefinite survival was achieved in treated mice (untreated vs. prolonged mATG+CTLA4-Ig; P = 0.0018) (D). NOD.SCID islet grafts harvested 100 days after transplantation in treated mice revealed completely preserved islet allografts with very few infiltrating cells surrounding the islets (E1, H&E; E2, insulin; E3, glucagon; E4, B220; E5, CD3; E6, FoxP3). *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: Interestingly, despite rejection, both allo- and autoimmune responses are controlled at day 56 in prolonged mATG+CTLA4-Ig-treated mice (Fig. 5A4–B4). To further challenge the allo- and autoimmune response potential in these animals, we performed crucial adoptive transfer experiments. BALB/c skin transplants were performed in NOD.SCID recipients, and 15 × 106 NOD splenocytes from prolonged mATG+CTLA4-Ig-treated mice at day 56 or from untransplanted mice were injected intraperitoneally the next day. To evaluate the transfer of alloimmunity, mice were followed for skin rejection; to evaluate the transfer of autoimmunity, the onset of diabetes was assessed. When splenocytes derived from untransplanted mice, either hyperglycemic or normoglycemic, were adoptively transferred, skin was promptly rejected (MST BALB/c skin: hyper, 13 days, and normo, 23 days, n = 4, P = 0.0067), and diabetes was transferred (diabetes onset: hyper 23 days; normo 49 days, n = 5, P = 0.0067) (Fig. 6A and B). When splenocytes obtained from prolonged mATG+CTLA4-Ig-treated mice (either hyperglycemic or normoglycemic) were adoptively transferred, skin rejection was significantly delayed; this was particularly evident when splenocytes were derived from normoglycemic mice (MST BALB/c skin: normoglycemic prolonged mATG+CTLA4 35 days, n = 5, P = 0.0067 vs. normo; hyperglycemic prolonged mATG+CTLA4-Ig 25 days, n = 4, P = 0.0011 vs. hyper) (Fig. 6A). Notably, when skin grafts from C57BL/6 mice, a 3rd party strain, were transplanted into NOD.SCID mice receiving adoptively transferred splenocytes from treated or untreated normoglycemic NOD mice, no significant differences were observed in graft survival (MST C57BL/6 skin: normo 23 days; normoglycemic prolonged mATG+CTLA4 29 days, n = 5, P = NS). Moreover, the adoptive transfer of splenocytes from either hyper- or normoglycemic treated mice did not cause diabetes onset in NOD.SCID mice (Fig. 6B).


A novel clinically relevant strategy to abrogate autoimmunity and regulate alloimmunity in NOD mice.

Vergani A, D'Addio F, Jurewicz M, Petrelli A, Watanabe T, Liu K, Law K, Schuetz C, Carvello M, Orsenigo E, Deng S, Rodig SJ, Ansari JM, Staudacher C, Abdi R, Williams J, Markmann J, Atkinson M, Sayegh MH, Fiorina P - Diabetes (2010)

BALB/c skin was transplanted into NOD.SCID mice, which subsequently received (via adoptive transfer) 15 × 106 splenocytes from normoglycemic or hyperglycemic untransplanted mice or from normoglycemic (not rejecting) or hyperglycemic (rejecting) transplanted mice treated with prolonged mATG+CTLA4-Ig at day 56 after transplantation. Skin rejection was slowed when adoptive transfer was performed with splenocytes from treated transplanted mice compared with untreated untransplanted mice (normoglycemic vs. normoglycemic prolonged mATG+CTLA4-Ig P < 0.05; hyperglycemic vs. hyperglycemic prolonged mATG+CTLA4-Ig; P < 0.05) (A). Therefore, in the same NOD.SCID mice, we evaluated the onset of diabetes. Mice that received splenocytes by adoptive transfer from normoglycemic or hyperglycemic NOD mice developed diabetes, as expected. However, the ability to transfer diabetes by splenocytes was completely abrogated in NOD mice treated with prolonged mATG and CTLA4-Ig, either rejecting or not (untreated vs. prolonged mATG+CTLA4-Ig; P < 0.001) (B). BALB/c hearts were transplanted in NOD mice in a purely alloimmune setting; a prolongation of graft survival was observed in treated mice (untreated vs. prolonged mATG+CTLA4-Ig; P = 0.017), similar results were obtained in hyperglycemic recipients (P = 0.03) (C). NOD.SCID islets were transplanted into hyperglycemic NOD mice; indefinite survival was achieved in treated mice (untreated vs. prolonged mATG+CTLA4-Ig; P = 0.0018) (D). NOD.SCID islet grafts harvested 100 days after transplantation in treated mice revealed completely preserved islet allografts with very few infiltrating cells surrounding the islets (E1, H&E; E2, insulin; E3, glucagon; E4, B220; E5, CD3; E6, FoxP3). *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 6: BALB/c skin was transplanted into NOD.SCID mice, which subsequently received (via adoptive transfer) 15 × 106 splenocytes from normoglycemic or hyperglycemic untransplanted mice or from normoglycemic (not rejecting) or hyperglycemic (rejecting) transplanted mice treated with prolonged mATG+CTLA4-Ig at day 56 after transplantation. Skin rejection was slowed when adoptive transfer was performed with splenocytes from treated transplanted mice compared with untreated untransplanted mice (normoglycemic vs. normoglycemic prolonged mATG+CTLA4-Ig P < 0.05; hyperglycemic vs. hyperglycemic prolonged mATG+CTLA4-Ig; P < 0.05) (A). Therefore, in the same NOD.SCID mice, we evaluated the onset of diabetes. Mice that received splenocytes by adoptive transfer from normoglycemic or hyperglycemic NOD mice developed diabetes, as expected. However, the ability to transfer diabetes by splenocytes was completely abrogated in NOD mice treated with prolonged mATG and CTLA4-Ig, either rejecting or not (untreated vs. prolonged mATG+CTLA4-Ig; P < 0.001) (B). BALB/c hearts were transplanted in NOD mice in a purely alloimmune setting; a prolongation of graft survival was observed in treated mice (untreated vs. prolonged mATG+CTLA4-Ig; P = 0.017), similar results were obtained in hyperglycemic recipients (P = 0.03) (C). NOD.SCID islets were transplanted into hyperglycemic NOD mice; indefinite survival was achieved in treated mice (untreated vs. prolonged mATG+CTLA4-Ig; P = 0.0018) (D). NOD.SCID islet grafts harvested 100 days after transplantation in treated mice revealed completely preserved islet allografts with very few infiltrating cells surrounding the islets (E1, H&E; E2, insulin; E3, glucagon; E4, B220; E5, CD3; E6, FoxP3). *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: Interestingly, despite rejection, both allo- and autoimmune responses are controlled at day 56 in prolonged mATG+CTLA4-Ig-treated mice (Fig. 5A4–B4). To further challenge the allo- and autoimmune response potential in these animals, we performed crucial adoptive transfer experiments. BALB/c skin transplants were performed in NOD.SCID recipients, and 15 × 106 NOD splenocytes from prolonged mATG+CTLA4-Ig-treated mice at day 56 or from untransplanted mice were injected intraperitoneally the next day. To evaluate the transfer of alloimmunity, mice were followed for skin rejection; to evaluate the transfer of autoimmunity, the onset of diabetes was assessed. When splenocytes derived from untransplanted mice, either hyperglycemic or normoglycemic, were adoptively transferred, skin was promptly rejected (MST BALB/c skin: hyper, 13 days, and normo, 23 days, n = 4, P = 0.0067), and diabetes was transferred (diabetes onset: hyper 23 days; normo 49 days, n = 5, P = 0.0067) (Fig. 6A and B). When splenocytes obtained from prolonged mATG+CTLA4-Ig-treated mice (either hyperglycemic or normoglycemic) were adoptively transferred, skin rejection was significantly delayed; this was particularly evident when splenocytes were derived from normoglycemic mice (MST BALB/c skin: normoglycemic prolonged mATG+CTLA4 35 days, n = 5, P = 0.0067 vs. normo; hyperglycemic prolonged mATG+CTLA4-Ig 25 days, n = 4, P = 0.0011 vs. hyper) (Fig. 6A). Notably, when skin grafts from C57BL/6 mice, a 3rd party strain, were transplanted into NOD.SCID mice receiving adoptively transferred splenocytes from treated or untreated normoglycemic NOD mice, no significant differences were observed in graft survival (MST C57BL/6 skin: normo 23 days; normoglycemic prolonged mATG+CTLA4 29 days, n = 5, P = NS). Moreover, the adoptive transfer of splenocytes from either hyper- or normoglycemic treated mice did not cause diabetes onset in NOD.SCID mice (Fig. 6B).

Bottom Line: Using allogeneic islet transplantation models as well as NOD mice with recent onset type 1 diabetes, we addressed the therapeutic efficacy and immunomodulatory mechanisms associated with a new immunoregulatory protocol based on prolonged low-dose mATG plus CTLA4-Ig.Immunologic analysis of mice receiving transplants revealed a complete abrogation of autoimmune responses and severe downregulation of alloimmunity in response to treatment.The capacity to regulate alloimmunity and to abrogate the autoimmune response in NOD mice in different settings confirmed that prolonged mATG+CTLA4-Ig treatment is a clinically relevant strategy to translate to humans with type 1 diabetes.

View Article: PubMed Central - PubMed

Affiliation: Transplantation Research Center, Children's Hospital and Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA. paolo.fiorina@childrens.harvard.edu

ABSTRACT

Objective: To investigate a new clinically relevant immunoregulatory strategy based on treatment with murine Thymoglobulin mATG Genzyme and CTLA4-Ig in NOD mice to prevent allo- and autoimmune activation using a stringent model of islet transplantation and diabetes reversal.

Research design and methods: Using allogeneic islet transplantation models as well as NOD mice with recent onset type 1 diabetes, we addressed the therapeutic efficacy and immunomodulatory mechanisms associated with a new immunoregulatory protocol based on prolonged low-dose mATG plus CTLA4-Ig.

Results: BALB/c islets transplanted into hyperglycemic NOD mice under prolonged mATG+CTLA4-Ig treatment showed a pronounced delay in allograft rejection compared with untreated mice (mean survival time: 54 vs. 8 days, P < 0.0001). Immunologic analysis of mice receiving transplants revealed a complete abrogation of autoimmune responses and severe downregulation of alloimmunity in response to treatment. The striking effect on autoimmunity was confirmed by 100% diabetes reversal in newly hyperglycemic NOD mice and 100% indefinite survival of syngeneic islet transplantation (NOD.SCID into NOD mice).

Conclusions: The capacity to regulate alloimmunity and to abrogate the autoimmune response in NOD mice in different settings confirmed that prolonged mATG+CTLA4-Ig treatment is a clinically relevant strategy to translate to humans with type 1 diabetes.

Show MeSH
Related in: MedlinePlus