Immunoregulatory pathways controlling progression of autoimmunity in NOD mice - PubMed (original) (raw)

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Immunoregulatory pathways controlling progression of autoimmunity in NOD mice

Sylvaine You et al. Ann N Y Acad Sci. 2008 Dec.

Abstract

The activation, expansion, and survival of regulatory T cells (Tregs) as well as the expression of their suppressive capacities result from distinct signaling pathways involving various membrane receptors and cytokines. Multiple studies have shown that thymus-derived naturally occurring Tregs constitutively express the forkhead/winged helix transcription factor FoxP3 in addition to high levels of CD25, the negative co-stimulatory molecule CTLA-4, and the glucocorticoid-induced TNF receptor-related protein GITR. At variance, adaptive or induced Tregs acquire these phenotypic markers as they differentiate in the periphery, following adequate stimulation in the appropriate environment, together with their capacity to produce immunomodulatory cytokines (mainly, IL-4, IL-10 and TGF-beta) and to display regulatory capacities. However, none of these molecules but FoxP3 are restricted to Tregs since they may also be expressed and upregulated on activated effector T cells. This explains why different hypotheses were proposed to interpret interesting reports showing that in vivo abrogation of CTLA-4 signaling using neutralizing CTLA-4 antibodies triggers different autoimmune or immune-mediated manifestations. Thus, an effect on pathogenic T cell effectors and/or Tregs has been proposed. Here we present and discuss recent results we obtained in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes, arguing for a key role of CTLA-4 in the functional activity of Tregs. Moreover, data are presented that simultaneous blockade of CTLA4 and TGF-beta further impairs immunoregulatory circuits that control disease progression.

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Conflict of interest statement

Conflict of Interest

The authors declare no conflicts of interest.

Figures

Figure 1

Figure 1

Acceleration of diabetes in NOD mice after treatment with anti-CTLA4 and / or anti-TGF-β antibodies. Ten-day-old NOD mice (male or female) were treated with antibodies to CTLA-4, TGF-β, or both to CTLA-4 and TGF-β (cocktail). Unmanipulated NOD mice or mice treated with purified mouse IgGs were used as controls (purified mouse IgGs [Jackson Immunoresearch Laboratories]). The dose used was 0.8 mg/injection/mouse i.p. once a week on d10, dl7, and d24 of life. Glycosuria measurements were performed twice a week. The TGF-β antibody used was produced by the 2G.7 hybridoma (mouse IgG2b, specific for human TGF-β1, provided by C.J.M. Melief, Leiden University Medical Center, Leiden, the Netherlands). The anti-CTLA4 antibody used was produced by the UC10-4F10-11 hybridoma (hamster IgG, specific for mouse CTLA4). The two antibodies were produced in ascites fluid and purified by affinity chromatography. The occurrence of diabetes was plotted using the Kaplan–Meier method (i.e., a nonparametric cumulative survival plot). The statistical comparison between the curves was performed using the log-rank (Mantel–Cox) test. Incidence of diabetes was significantly accelerated after administration of CTLA-4 alone or in combination with anti-TGF-β (P < 0.0007 for female mice and P < 0.04 for male mice treated with anti-CTLA-4+anti-TGF-β antibodies versus controls, respectively).

Figure 2 Figure 2

Histologic analysis of pancreas from anti-CTLA-4- and/or anti-TGF-β antibody-treated NOD mice. Female and male NOD mice treated with antibodies to CTLA-4, TGF-β, or both to CTLA-4 and TGF-β (cocktail) on d10, d17, and d24 of life were culled at various ages for histopathologic analysis of pancreata. The proportion of islets massively infiltrated with mononuclear cells increased with age and according to the treatment in the following order: IgG < TGF-β

<ctla-4P < 0.0001) or anti-CTLA-4 alone (P < 0.008), respectively.</ctla-4

Figure 3

Figure 3

Polyautoimmune syndrome in NOD mice treated with anti-TGF-β, anti-CTLA-4, or both antibodies. (A) Histologic analysis of stomach, gut, and salivary gland of NOD mice having received antibodies against CTLA-4, TGF-β or CTLA-4 + TGF-β. Mice were killed at various ages and the severity of gastritis, colitis, and sialitis was measured and compared to control IgG-treated animals. (B) Anti-H+/K+ ATPase-specific autoantibody serum levels. The kinetics of anti-H+ / K+ ATPase autoantibody production in mice treated with anti-CTLA-4, anti-TGF-β antibodies, or control IgGs were determined by ELISA. The x axis details the antibody treatment received by the mice and the gender of the recipients. Values above 1 are considered as a marker of gastritis. Mean antibody levels detected in recipients injected with either a mixture of antibodies against CTLA-4 and TGF-β were significantly higher than those observed in other groups.

Figure 4

Figure 4

Anti-CTLA-4 antibodies preferentially target regulatory T cells in vivo. (A) Ten-day-old female NOD mice deficient for the CD28 molecule were treated with antibodies to CTLA-4 or to CTLA-4 and TGF-β (cocktail). As in conventional NOD mice, the dose used was 0.8 mg/injection/mouse i.p. once a week on d10, d17 and d24 of life. Glycosuria measurements were performed twice a week. Administration of anti-CTLA-4 antibodies alone did not accelerate diabetes incidence in CD28−/− NOD mice as opposed to combination of anti-CTLA-4 and anti-TGF-β (P < 0.007). (B) NOD-SCID mice were injected with 1 × 105 pathogenic CD25−CD62L− T splenocytes recovered from overtly diabetic mice. Cells were purified on the basis of CD62L, CD25, or CD4 expression using magnetic bead cell sorting (MACS; Miltenyi Biotech, Bergisch-Gladbach, Germany). When needed, recipients were treated with neutralizing monoclonal antibodies to CTLA-4 (1 mg/injection/mouse i.p. 3 times a week) starting 1 week after transfer and continuing for 5 consecutive weeks. (C) NOD-SCID recipients received diabetogenic cells alone (1 × 106 / recipient, Diab) or a mixture of diabetogenic cells with CD4+CD25+ T cells (1 × 106 / recipient) isolated from the spleen of 6 week-old NOD mice using magnetic bead cell sorting. Neutralizing monoclonal antibodies to CTLA-4 were injected (1 mg/injection/mouse i.p. 3 times a week) until all recipients injected with diabetogenic cells alone had become diabetic. Glycosuria was monitored twice a week. (D) NOD-SCID recipients received diabetogenic cells alone (1 × 106/recipient, Diab) or a mixture of diabetogenic cells with CD4+CD25+ T cells (1 × 106 / recipient) isolated from the thymus of 6 week-old NOD mice. Thymocyte suspensions were depleted of CD8+ T cells by magnetic bead cell sorting (Miltenyi Biotech), and total CD4+CD25+ T cells were purified by FACS sorting. When needed, recipients were treated with neutralizing monoclonal antibodies to CTLA-4 (1 mg / injection / mouse i.p. 3 times a week) as in (B).

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