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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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Related in: MedlinePlus

BALB/c islets were transplanted into hyperglycemic NOD mice, and allograft survival was evaluated. All treatments significantly prolonged graft survival (P < 0.05 vs. untreated). The addition of CTLA4-Ig synergized with mATG treatment to delay rejection (graft survival: induction mATG vs. induction mATG+CTLA4-Ig, P < 0.05; prolonged mATG vs. prolonged mATG+CTLA4-Ig, *P < 0.05) (A). Prolonged mATG+CTLA4-Ig was shown to be more efficacious than induction mATG+CTLA4-Ig (**P < 0.05) (A). Immunocompetence was examined in prolonged mATG+CTLA4-Ig treated mice via evaluation of the capacity to mount an ovalbumin-specific response. The IFN-γ (B1)- and IL-4 (B2)-specific responses were higher in mice immunized during treatment compared with unimmunized untreated mice (*P < 0.05). Serum samples were collected after islet transplantation on days 7, 14, and 28, and peripheral cytokine profile was evaluated. The Th1-related cytokines (IFN-γ, IL-2) were increased by mATG treatment (P < 0.05 vs. hyper), whereas this increase was prevented by mATG combination with CTLA4-Ig (C). A similar pattern was observed for the immunoregulatory cytokine IL-10 and for the cytokines related to homeostatic proliferation (IL-7, IL-15) (C). The proinflammatory cytokine IL-6 increased after transplantation (P < 0.05 vs. hyper), whereas treatment with mATG completely prevented this increase (C). IL-1α and the Th2 cytokine IL-5 were not significantly affected (C).
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Figure 3: BALB/c islets were transplanted into hyperglycemic NOD mice, and allograft survival was evaluated. All treatments significantly prolonged graft survival (P < 0.05 vs. untreated). The addition of CTLA4-Ig synergized with mATG treatment to delay rejection (graft survival: induction mATG vs. induction mATG+CTLA4-Ig, P < 0.05; prolonged mATG vs. prolonged mATG+CTLA4-Ig, *P < 0.05) (A). Prolonged mATG+CTLA4-Ig was shown to be more efficacious than induction mATG+CTLA4-Ig (**P < 0.05) (A). Immunocompetence was examined in prolonged mATG+CTLA4-Ig treated mice via evaluation of the capacity to mount an ovalbumin-specific response. The IFN-γ (B1)- and IL-4 (B2)-specific responses were higher in mice immunized during treatment compared with unimmunized untreated mice (*P < 0.05). Serum samples were collected after islet transplantation on days 7, 14, and 28, and peripheral cytokine profile was evaluated. The Th1-related cytokines (IFN-γ, IL-2) were increased by mATG treatment (P < 0.05 vs. hyper), whereas this increase was prevented by mATG combination with CTLA4-Ig (C). A similar pattern was observed for the immunoregulatory cytokine IL-10 and for the cytokines related to homeostatic proliferation (IL-7, IL-15) (C). The proinflammatory cytokine IL-6 increased after transplantation (P < 0.05 vs. hyper), whereas treatment with mATG completely prevented this increase (C). IL-1α and the Th2 cytokine IL-5 were not significantly affected (C).

Mentions: We then compared the impact of mATG treatment alone or combined with CTLA4-Ig on islet allograft survival. Untreated NOD mice invariably rejected allografts (untreated: mean survival time [MST] of 8 days, n = 5). We then tested mATG as a single agent. Considering the potential of mATG treatment to cause reversal in newly hyperglycemic NOD mice (16), NOD mice were used as islet recipients after at least 14 consecutive days of glucose measurements >400 mg/dl. The mATG induction extended allograft survival (induction mATG: MST of 16.5 days, n = 10, P = 0.0001 vs. untreated) (Fig. 3A). Prolonged mATG treatment per se did not further increase graft survival (prolonged mATG: MST of 14 days, n = 5, NS vs. induction mATG) (Fig. 3A) with 1 of 5 mice reaching 40 days of survival. We therefore combined mATG with CTLA4-Ig, with the goal of reducing activation of the immune system in response to alloantigen and to counteract homeostatic proliferation, which follows T-cell depletion. CTLA4-Ig alone slightly prolonged graft survival (CTLA4-Ig: MST of 12.5 days, n = 4, P = 0.04 vs. control) (Fig. 3A) and synergized with mATG to further increase allograft survival (induction mATG+CTLA4-Ig: MST of 32 days, n = 9, P < 0.0001 vs. induction mATG) (Fig. 3A). Finally, the combination of prolonged mATG treatment and CTLA4-Ig significantly increased graft survival (prolonged ATG+CTLA4-Ig: MST of 54 days, n = 10, P = 0.007 vs. mATG+CTLA4) (Fig. 3A). Notably, in this stringent model of enhanced allo-/auto-immune responses, 30% of these mice retained graft function for more then 70 days. We also tested whether depletion of Tregs through treatment with anti-CD25-Ig at the time of transplantation (500 μg at days −6 and −1) was able to abrogate the effect of prolonged mATG+CTLA4-Ig. Although CD25-depleted untreated NOD mice rejected islet grafts rapidly (MST of 8 days), CD25-depleted NOD mice treated with prolonged mATG+CTLA4 showed a prolongation of islet graft survival (MST of 53 days), suggesting the capacity of our approach to reconstitute the Treg pool (data not shown).


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 islets were transplanted into hyperglycemic NOD mice, and allograft survival was evaluated. All treatments significantly prolonged graft survival (P < 0.05 vs. untreated). The addition of CTLA4-Ig synergized with mATG treatment to delay rejection (graft survival: induction mATG vs. induction mATG+CTLA4-Ig, P < 0.05; prolonged mATG vs. prolonged mATG+CTLA4-Ig, *P < 0.05) (A). Prolonged mATG+CTLA4-Ig was shown to be more efficacious than induction mATG+CTLA4-Ig (**P < 0.05) (A). Immunocompetence was examined in prolonged mATG+CTLA4-Ig treated mice via evaluation of the capacity to mount an ovalbumin-specific response. The IFN-γ (B1)- and IL-4 (B2)-specific responses were higher in mice immunized during treatment compared with unimmunized untreated mice (*P < 0.05). Serum samples were collected after islet transplantation on days 7, 14, and 28, and peripheral cytokine profile was evaluated. The Th1-related cytokines (IFN-γ, IL-2) were increased by mATG treatment (P < 0.05 vs. hyper), whereas this increase was prevented by mATG combination with CTLA4-Ig (C). A similar pattern was observed for the immunoregulatory cytokine IL-10 and for the cytokines related to homeostatic proliferation (IL-7, IL-15) (C). The proinflammatory cytokine IL-6 increased after transplantation (P < 0.05 vs. hyper), whereas treatment with mATG completely prevented this increase (C). IL-1α and the Th2 cytokine IL-5 were not significantly affected (C).
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Figure 3: BALB/c islets were transplanted into hyperglycemic NOD mice, and allograft survival was evaluated. All treatments significantly prolonged graft survival (P < 0.05 vs. untreated). The addition of CTLA4-Ig synergized with mATG treatment to delay rejection (graft survival: induction mATG vs. induction mATG+CTLA4-Ig, P < 0.05; prolonged mATG vs. prolonged mATG+CTLA4-Ig, *P < 0.05) (A). Prolonged mATG+CTLA4-Ig was shown to be more efficacious than induction mATG+CTLA4-Ig (**P < 0.05) (A). Immunocompetence was examined in prolonged mATG+CTLA4-Ig treated mice via evaluation of the capacity to mount an ovalbumin-specific response. The IFN-γ (B1)- and IL-4 (B2)-specific responses were higher in mice immunized during treatment compared with unimmunized untreated mice (*P < 0.05). Serum samples were collected after islet transplantation on days 7, 14, and 28, and peripheral cytokine profile was evaluated. The Th1-related cytokines (IFN-γ, IL-2) were increased by mATG treatment (P < 0.05 vs. hyper), whereas this increase was prevented by mATG combination with CTLA4-Ig (C). A similar pattern was observed for the immunoregulatory cytokine IL-10 and for the cytokines related to homeostatic proliferation (IL-7, IL-15) (C). The proinflammatory cytokine IL-6 increased after transplantation (P < 0.05 vs. hyper), whereas treatment with mATG completely prevented this increase (C). IL-1α and the Th2 cytokine IL-5 were not significantly affected (C).
Mentions: We then compared the impact of mATG treatment alone or combined with CTLA4-Ig on islet allograft survival. Untreated NOD mice invariably rejected allografts (untreated: mean survival time [MST] of 8 days, n = 5). We then tested mATG as a single agent. Considering the potential of mATG treatment to cause reversal in newly hyperglycemic NOD mice (16), NOD mice were used as islet recipients after at least 14 consecutive days of glucose measurements >400 mg/dl. The mATG induction extended allograft survival (induction mATG: MST of 16.5 days, n = 10, P = 0.0001 vs. untreated) (Fig. 3A). Prolonged mATG treatment per se did not further increase graft survival (prolonged mATG: MST of 14 days, n = 5, NS vs. induction mATG) (Fig. 3A) with 1 of 5 mice reaching 40 days of survival. We therefore combined mATG with CTLA4-Ig, with the goal of reducing activation of the immune system in response to alloantigen and to counteract homeostatic proliferation, which follows T-cell depletion. CTLA4-Ig alone slightly prolonged graft survival (CTLA4-Ig: MST of 12.5 days, n = 4, P = 0.04 vs. control) (Fig. 3A) and synergized with mATG to further increase allograft survival (induction mATG+CTLA4-Ig: MST of 32 days, n = 9, P < 0.0001 vs. induction mATG) (Fig. 3A). Finally, the combination of prolonged mATG treatment and CTLA4-Ig significantly increased graft survival (prolonged ATG+CTLA4-Ig: MST of 54 days, n = 10, P = 0.007 vs. mATG+CTLA4) (Fig. 3A). Notably, in this stringent model of enhanced allo-/auto-immune responses, 30% of these mice retained graft function for more then 70 days. We also tested whether depletion of Tregs through treatment with anti-CD25-Ig at the time of transplantation (500 μg at days −6 and −1) was able to abrogate the effect of prolonged mATG+CTLA4-Ig. Although CD25-depleted untreated NOD mice rejected islet grafts rapidly (MST of 8 days), CD25-depleted NOD mice treated with prolonged mATG+CTLA4 showed a prolongation of islet graft survival (MST of 53 days), suggesting the capacity of our approach to reconstitute the Treg pool (data not shown).

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