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.

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.

In a mouse model of autoimmune Graves' disease, stimulatory anti-TSH receptor (TSHR) antibodies (TSAbs) slowly evolve upon repeated immunization with TSHR and lead to hyperthyroidism. Although all immunized mice developed high levels of TSH-binding inhibitory Ig (TBII), only a subset of these mice become hyperthyroid, suggesting that the generation of pathogenic antibodies (Abs) may require affinity maturation. We analyzed the complementarity-determining region 3 (CDR3) of IGHV1 and IGHV5 heavy chains from mice at different stages of disease development. Subcloned CDR3 PCR products were amplified from RNA isolated from enriched splenic B/plasma cells of a control mouse, and mice with low TBII and normal T(4) levels (LTNT(4)), high TBII and normal T(4) levels (HTNT(4)), and high TBII and high T(4) levels (HTHT(4)). Using statistical analyses, we correlated usage of D and J genes and the amino acid composition and length of and mutations within the CDR3 with different outcomes after TSHR immunization. CDR3 sequences from TSHR-immunized mice contained a higher frequency of D gene SP2.9 relative to control, whereas sequences from HTHT(4) contained a higher frequency of D gene Q52 compared with sequences from LTNT(4). Furthermore, HTHT(4) sequences also contained higher CDR3 replacement mutations, relative to LTNT(4) and HTNT(4) mice, that are indicative of somatic hypermutation. Collectively, our results suggest that higher somatic mutations within the CDR3 may correlate with pathogenic antibodies against the TSHR.

Autoimmune Graves' disease (GD), which is characterized by hyperthyroidism, is mediated by autoantibodies to the thyrotropin receptor (TSHR). Yersinia enterocolitica (Y.e.) has been shown to produce a lipoprotein (LP) that can cross-react with the TSHR and thus can act as a potential trigger of thyroid autoimmunity. In this study, to further characterize LP, we cloned the LP gene from Y. enterocolitica and expressed a recombinant LP. This recombinant LP was mitogenic for C3H/HeJ (LPS hyporesponsive) B cells and induced production and secretion of significant levels of IL-6 from splenocytes. A mouse antibody generated against the recombinant LP cross-reacted with TSHR as shown by western blot analysis. FACS analysis of splenocytes from mice immunized with LP revealed that LP could induce increased expression of B7.1 and B7.2. The immunomodulatory effects of LP including up-regulation of B7.1 and B7.2 coupled with its ability to induce antibodies that can cross-react with the TSHR showed several potential mechanisms by which it can cause breakdown of self-tolerance to TSHR.