Our earlier study showed that GM-CSF has the potential not only to prevent, but also to suppress, experimental autoimmune thyroiditis (EAT). GM-CSF-induced EAT suppression in mice was accompanied by an increase in the frequency of CD4+CD25+ regulatory T cells that could suppress mouse thyroglobulin (mTg)-specific T cell responses in vitro, but the underlying mechanism of this suppression was not elucidated. In this study we show that GM-CSF can induce dendritic cells (DCs) with a semimature phenotype, an important characteristic of DCs, which are known to play a critical role in the induction and maintenance of regulatory T cells. Adoptive transfer of CD4+CD25+ T cells from GM-CSF-treated and mTg-primed donors into untreated, but mTg-primed, recipients resulted in decreased mTg-specific T cell responses. Furthermore, lymphocytes obtained from these donors and recipients after adoptive transfer produced significantly higher levels of IL-10 compared with mTg-primed, untreated, control mice. Administration of anti-IL-10R Ab into GM-CSF-treated mice abrogated GM-CSF-induced suppression of EAT, as indicated by increased mTg-specific T cell responses, thyroid lymphocyte infiltration, and follicular destruction. Interestingly, in vivo blockade of IL-10R did not affect GM-CSF-induced expansion of CD4+CD25+ T cells. However, IL-10-induced immunosuppression was due to its direct effects on mTg-specific effector T cells. Taken together, these results indicated that IL-10, produced by CD4+CD25+ T cells that were probably induced by semimature DCs, is essential for disease suppression in GM-CSF-treated mice.

GM-CSF plays an essential role in the differentiation of dendritic cells (DCs). Our studies have shown that GM-CSF treatment can induce semi-mature DCs and CD4+CD25+ regulatory T cells (Tregs) and suppress ongoing autoimmunity in mouse models. In this study, we examined the differences in the potential of GM-CSF to exert tolerogenic function on CD8a+ and CD8a- sub-populations of DCs in vivo. We show that GM-CSF modulates CD8a-, but not CD8a+ DCs in vivo, by inhibiting the surface expression of activation markers MHC II and CD80 and production of inflammatory cytokines such as IL-12 and IL-1beta. Self-antigen [mouse thyroglobulin (mTg)] presentation by GM-CSF-exposed CD8a- DCs to T cells from mTg-primed mice induced a profound increase in the frequency of forkhead box P3 (FoxP3)-expressing T cells compared with antigen presentation by GM-CSF-exposed CD8a+ DCs and control CD8a+ and CD8a- DCs. This tolerogenic property of GM-CD8a- DCs was abrogated when IL-12 was added. GM-CSF-exposed CD8a- DCs could also induce secretion of significantly higher amounts of IL-10 by T cells from mTg-primed mice. Importantly, adoptive transfer of CD8a- DCs from GM-CSF-treated SCID mice, but not untreated mice, into wild-type CBA/J mice prevented the development of experimental autoimmune thyroiditis (EAT) in the recipient animals upon immunization with mTg. Collectively, our results show that GM-CSF renders CD8a- DCs tolerogenic, and these DCs induce Foxp3+ and IL-10+ Tregs.

Abnormalities in DC function are implicated in defective immune regulation that leads to type-1 diabetes (T1D) in NOD mice and humans. In this study, we used GM-CSF and Flt3-L to modulate DC function in NOD mice and observed the effects on T1D development. Treatment with either ligand at earlier stages of insulitis suppressed the development of T1D. Unlike Flt3-L, GM-CSF was more effective in suppressing T1D, even when administered at later stages of insulitis. In vitro studies and in vivo adoptive transfer experiments revealed that CD4+CD25+ T cells from GM-CSF-treated mice could suppress effector T cell response and T1D. This suppression is likely mediated through enhanced IL-10 and TGF-β1 production. Adoptive transfer of GM-CSF exposed DCs to naive mice resulted in an expansion of Foxp3+ T cells and a significant delay in T1D onset. Our results indicate that GM-CSF acted primarily on DCs and caused an expansion of Foxp3+ Tregs which delayed the onset of T1D in NOD mice.

Treatment of CBA/j mice with Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), a potent growth factor for dendritic cells can prevent and suppress ongoing experimental autoimmune thyroiditis (EAT). GM-CSF treatment causes expansion of CD11c+CD8a− myeloid dendritic cells (DCs) with a consequent increase in CD4+CD25+ T regulatory cells (Tregs) in an EAT model. In this study, we investigated the significance of the expansion of CD8a− myeloid DCs by GM-CSF and its direct role in inducing CD4+CD25+Foxp3+ T regs. Co-culture of CD4+ T cells with CD8a-ve DCs from GM-CSF treated mice in the presence of mouse thyroglobulin (mTg) induced an increase in the number of CD4+CD25+Foxp3+ T cells. These Foxp3+ T cells produced high levels of IL-10. These T regs could suppress the mTg specific proliferation of effector CD4+CD25− T cells, which was reversed upon addition of anti-IL10R antibodies. In contrast, GM-CSF treated CD8a+ lymphoid DCs failed to show a significant suppression of mTg specific T cell proliferation. In vivo experiments in a SCID mouse model revealed that GM-CSF treated SCID mice could induce increased numbers of Foxp3 and IL-10 expressing CD4+ T cells that were adoptively transferred from wild type CBA/j mice. These results indicate that GM-CSF acts directly on CD8a− myeloid DCs in the absence of T cells and renders them tolerogenic in that they cause expansion of CD4+Foxp3+ Tregs, which suppress mTg specific proliferation in an IL-10 dependent manner. NIH 1RO1AI058190

Impairment in dendritic cell (DC) functions is implicated in defective immune regulation that leads to type-1 diabetes (T1D) in both humans and the established non-obese diabetic (NOD) mouse model. In this study, we examined the roles of DC modulators such as GM-CSF and Flt3-L to affect DC function and prevent T1D development in NOD mice. Both GM-CSF and FLT3-L could suppress the development of T1D if treatment was initiated earlier in life prior to the development of clinical diabetes. However, unlike Flt3-L, GM-CSF was far more effective in suppressing T1D even when administered at later stages of insulitis. Ongoing periodic treatment of NOD mice with GM-CSF prevented the development of T1D and the animals remained healthy until 3 months after cessation of the treatment. Adoptive transfer of CD4+CD25+ T cells from GM-CSF-treated mice could suppress effector T cell response and T1D in NOD-SCID mice, and this suppression was associated with enhanced IL-10 and TGF-β1 production. Moreover, adoptive transfer of GM-CSF-exposed DCs to naive NOD mice resulted in an expansion of Foxp3+ T cells and a significant delay in T1D onset. These results indicate that primarily the GM-CSF renders DCs tolerogenic resulting in subsequent Treg induction and delayed onset of T1D in NOD mice. NIH 1RO1AI058190