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CD28/CD154 blockade prevents autoimmune diabetes by inducing nondeletional tolerance after effector t-cell inhibition and regulatory T-cell expansion.

Rigby MR, Trexler AM, Pearson TC, Larsen CP - Diabetes (2008)

Bottom Line: Blocking T-cell signaling is an effective means to prevent autoimmunity and allograft rejection in many animal models, yet the clinical translation of many of these approaches has not resulted in the success witnessed in experimental systems.Improved understanding of these approaches may assist in developing safe and effective means to treat disorders such as autoimmune diabetes.Immunotherapies that selectively suppress effector T-cells while permitting the development of natural regulatory mechanisms may have a unique role in establishing targeted long-standing immune protection and peripheral tolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA. mrigby@emory.edu

ABSTRACT

Objective: Blocking T-cell signaling is an effective means to prevent autoimmunity and allograft rejection in many animal models, yet the clinical translation of many of these approaches has not resulted in the success witnessed in experimental systems. Improved understanding of these approaches may assist in developing safe and effective means to treat disorders such as autoimmune diabetes.

Research design and methods: We studied the effect of anti-CD154 and CTLA4-Ig on diabetes development, and the requirements to induce tolerance in nod.scid mice after transfer of transgenic beta-cell reactive BDC2.5.NOD T-cells.

Results: Nod.scid recipients of diabetogenic BDC2.5.NOD cells were protected indefinitely from diabetes by a short course of combined costimulation blockade, despite the continued diabetogenic potential of their T-cells. The presence of pathogenic T-cells in the absence of disease indicates peripheral immune tolerance. T-cell maturation occurred in protected recipients, yet costimulation blockade temporarily blunted early T-cell proliferation in draining pancreatic nodes. Tolerance required preexisting regulatory T-cells (Tregs), and protected recipients had greater numbers of Tregs than diabetic recipients. Diabetes protection was successful in the presence of homeostatic expansion and high T-cell precursor frequency, both obstacles to tolerance induction in other models of antigen-specific immunity.

Conclusions: Immunotherapies that selectively suppress effector T-cells while permitting the development of natural regulatory mechanisms may have a unique role in establishing targeted long-standing immune protection and peripheral tolerance. Understanding the mechanism of these approaches may assist in the design and use of therapies for human conditions, such as type 1 diabetes.

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Proinflammatory cytokine expression in donor, diabetic, and tolerant adoptive transfer recipients. Lymphocytes were isolated from Nod.scid mice rendered diabetic, protected from diabetes with costimulation blockade (both 6 weeks after adoptive transfer), BDC.NOD mice, and NOD mice. After a 6 h in vitro incubation with 10 μg/ml BDC2.5 stimulatory peptide (RTRPLWVRME; 1040-63; 28), cells were stained for surface molecules fixed; permeabilized; stained for IL-2, TNF-α, and IFN-γ; and then evaluated by flow cytometry. During analysis, cells were gated on CD3+CD4+, which in adoptive transfer recipients usually is composed of >90–95% vβ 4+ cells. A representative sample of flow plots from three to four separate experiments is shown (A). Results are displayed in graphical form (B), where the P value of * vs. ** and * vs. *** is <0.05, and the P value of ** vs. *** is NS.
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f4: Proinflammatory cytokine expression in donor, diabetic, and tolerant adoptive transfer recipients. Lymphocytes were isolated from Nod.scid mice rendered diabetic, protected from diabetes with costimulation blockade (both 6 weeks after adoptive transfer), BDC.NOD mice, and NOD mice. After a 6 h in vitro incubation with 10 μg/ml BDC2.5 stimulatory peptide (RTRPLWVRME; 1040-63; 28), cells were stained for surface molecules fixed; permeabilized; stained for IL-2, TNF-α, and IFN-γ; and then evaluated by flow cytometry. During analysis, cells were gated on CD3+CD4+, which in adoptive transfer recipients usually is composed of >90–95% vβ 4+ cells. A representative sample of flow plots from three to four separate experiments is shown (A). Results are displayed in graphical form (B), where the P value of * vs. ** and * vs. *** is <0.05, and the P value of ** vs. *** is NS.

Mentions: T-cell functional and maturational status can often be inferred by the types of cytokines expressed and the tempo of production (34). T-cells in nondiabetic recipients of BDC.NOD cells are not pathogenic to the host, whereas cells in diabetic recipients are. One explanation could be that T-cells from protected mice have acquired a “less” proinflammatory profile than cells from protected mice. To investigate this, cytokine production after antigen encounter was evaluated. After in vitro antigen-specific stimulation, there is greater expression of IFN-γ, TNF-α, and IL-2 from either tolerant or diabetic mice compared with nascent BDC.NOD mice (Fig. 4A). There was also a nonsignificant trend for greater expression of these proinflammatory cytokines in cells from diabetic compared with tolerant mice. (Fig. 4B). Few cells (i.e., <5%) from either tolerant or diabetic mice expressed significant IL-4 or IL-10 (data not shown). This suggests that T-cells from all recipients, whether diabetic or protected, have transitioned from functionally naïve to mature memory T-cells with a highly proinflammatory profile. Therefore β-cell protection does not appear to be due to an overwhelming shift from Th1-type T-cells to T-cells that express abundant regulatory cytokines (i.e., Th2 or regulatory cells).


CD28/CD154 blockade prevents autoimmune diabetes by inducing nondeletional tolerance after effector t-cell inhibition and regulatory T-cell expansion.

Rigby MR, Trexler AM, Pearson TC, Larsen CP - Diabetes (2008)

Proinflammatory cytokine expression in donor, diabetic, and tolerant adoptive transfer recipients. Lymphocytes were isolated from Nod.scid mice rendered diabetic, protected from diabetes with costimulation blockade (both 6 weeks after adoptive transfer), BDC.NOD mice, and NOD mice. After a 6 h in vitro incubation with 10 μg/ml BDC2.5 stimulatory peptide (RTRPLWVRME; 1040-63; 28), cells were stained for surface molecules fixed; permeabilized; stained for IL-2, TNF-α, and IFN-γ; and then evaluated by flow cytometry. During analysis, cells were gated on CD3+CD4+, which in adoptive transfer recipients usually is composed of >90–95% vβ 4+ cells. A representative sample of flow plots from three to four separate experiments is shown (A). Results are displayed in graphical form (B), where the P value of * vs. ** and * vs. *** is <0.05, and the P value of ** vs. *** is NS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2551677&req=5

f4: Proinflammatory cytokine expression in donor, diabetic, and tolerant adoptive transfer recipients. Lymphocytes were isolated from Nod.scid mice rendered diabetic, protected from diabetes with costimulation blockade (both 6 weeks after adoptive transfer), BDC.NOD mice, and NOD mice. After a 6 h in vitro incubation with 10 μg/ml BDC2.5 stimulatory peptide (RTRPLWVRME; 1040-63; 28), cells were stained for surface molecules fixed; permeabilized; stained for IL-2, TNF-α, and IFN-γ; and then evaluated by flow cytometry. During analysis, cells were gated on CD3+CD4+, which in adoptive transfer recipients usually is composed of >90–95% vβ 4+ cells. A representative sample of flow plots from three to four separate experiments is shown (A). Results are displayed in graphical form (B), where the P value of * vs. ** and * vs. *** is <0.05, and the P value of ** vs. *** is NS.
Mentions: T-cell functional and maturational status can often be inferred by the types of cytokines expressed and the tempo of production (34). T-cells in nondiabetic recipients of BDC.NOD cells are not pathogenic to the host, whereas cells in diabetic recipients are. One explanation could be that T-cells from protected mice have acquired a “less” proinflammatory profile than cells from protected mice. To investigate this, cytokine production after antigen encounter was evaluated. After in vitro antigen-specific stimulation, there is greater expression of IFN-γ, TNF-α, and IL-2 from either tolerant or diabetic mice compared with nascent BDC.NOD mice (Fig. 4A). There was also a nonsignificant trend for greater expression of these proinflammatory cytokines in cells from diabetic compared with tolerant mice. (Fig. 4B). Few cells (i.e., <5%) from either tolerant or diabetic mice expressed significant IL-4 or IL-10 (data not shown). This suggests that T-cells from all recipients, whether diabetic or protected, have transitioned from functionally naïve to mature memory T-cells with a highly proinflammatory profile. Therefore β-cell protection does not appear to be due to an overwhelming shift from Th1-type T-cells to T-cells that express abundant regulatory cytokines (i.e., Th2 or regulatory cells).

Bottom Line: Blocking T-cell signaling is an effective means to prevent autoimmunity and allograft rejection in many animal models, yet the clinical translation of many of these approaches has not resulted in the success witnessed in experimental systems.Improved understanding of these approaches may assist in developing safe and effective means to treat disorders such as autoimmune diabetes.Immunotherapies that selectively suppress effector T-cells while permitting the development of natural regulatory mechanisms may have a unique role in establishing targeted long-standing immune protection and peripheral tolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA. mrigby@emory.edu

ABSTRACT

Objective: Blocking T-cell signaling is an effective means to prevent autoimmunity and allograft rejection in many animal models, yet the clinical translation of many of these approaches has not resulted in the success witnessed in experimental systems. Improved understanding of these approaches may assist in developing safe and effective means to treat disorders such as autoimmune diabetes.

Research design and methods: We studied the effect of anti-CD154 and CTLA4-Ig on diabetes development, and the requirements to induce tolerance in nod.scid mice after transfer of transgenic beta-cell reactive BDC2.5.NOD T-cells.

Results: Nod.scid recipients of diabetogenic BDC2.5.NOD cells were protected indefinitely from diabetes by a short course of combined costimulation blockade, despite the continued diabetogenic potential of their T-cells. The presence of pathogenic T-cells in the absence of disease indicates peripheral immune tolerance. T-cell maturation occurred in protected recipients, yet costimulation blockade temporarily blunted early T-cell proliferation in draining pancreatic nodes. Tolerance required preexisting regulatory T-cells (Tregs), and protected recipients had greater numbers of Tregs than diabetic recipients. Diabetes protection was successful in the presence of homeostatic expansion and high T-cell precursor frequency, both obstacles to tolerance induction in other models of antigen-specific immunity.

Conclusions: Immunotherapies that selectively suppress effector T-cells while permitting the development of natural regulatory mechanisms may have a unique role in establishing targeted long-standing immune protection and peripheral tolerance. Understanding the mechanism of these approaches may assist in the design and use of therapies for human conditions, such as type 1 diabetes.

Show MeSH
Related in: MedlinePlus