FoxP3+ regulatory T cells (Tregs) play a crucial role in the maintenance of immune homeostasis against self-antigens (Sakaguchi et al., 2004
). A prominent role for Tregs in the maintenance of immune tolerance has been emphasized recently (Brusko et al., 2008
), with evidence suggesting a reduced function of these cells in autoimmune diseases (Torgerson, 2006
; Venken et al., 2008
; Moes et al., 2010
), including MG (Huang et al., 2004
; Fattorossi et al., 2005
; Zhang et al., 2009
; Masayuki et al., 2010
). Tregs have been shown to be capable of suppressing Th cells that help autoantibody-producing B cells, thereby inhibiting antibody (and autoantibody) production (Yokoyama et al., 2011
). Several methods have been investigated to directly target Tregs for the treatment of autoimmune disease, including expansion and induction of Tregs in vitro
for subsequent adoptive immunotherapy (Hoffman et al., 2004
; Earle et al., 2005
). Despite experimental success with different methods of Treg induction and expansion, a safe, simple, and antigen-specific mode of inhibiting immune responses utilizing Tregs has not yet been achieved. Since Tregs make up only a very small percentage of the CD4+
T cell pool in both mice and humans, therapeutic applications have been limited by prohibitively low numbers of Tregs. Optimizing the expansion of Tregs in vivo
using agents like GM-CSF may therefore be a more viable approach to developing Treg therapies that augment functional, antigen-specific CD4+
We have shown in this study that adoptively transferred GM-CSF/AChR-induced Tregs are an effective treatment for EAMG, suppressing disease severity, preserving muscle AChR content, and reducing circulating levels of anti-AChR antibodies. GM-CSF/AChR-induced-Tregs from EAMG mice potently and specifically suppressed in vitro T cell proliferation induced by AChR, but did not alter T cell proliferation in response to an irrelevant self antigen (thyroglobulin) to any greater extent than Tregs obtained from GM-CSF-treated, non-AChR immunized mice. Perhaps even more importantly, GM-CSF/AChR-induced-Tregs did not significantly affect T cell proliferation in response to an exogenous antigen (OVA). This particular result may be very relevant from a translational point of view, suggesting that if effective in MG, GM-CSF treatment may not significantly affect the immune system’s ability to respond to exogenous (foreign) antigens (i.e., infections, malignancy, etc) unlike conventional immunosuppressant drugs.
While the adoptive transfer of Tregs at the time of experimental autoimmune disease induction has been consistently shown to decrease disease severity in a number of animal models (Brusko et al., 2008
; Langier et al., 2010
), this strategy has been reported to be less than successful in suppressing established, chronic autoimmune diseases, including EAMG (Nessi et al., 2010
). Our findings in this study are in sharp contrast to the above report, as we have shown significant suppression of ongoing EAMG after transfer of GM-CSF/AChR-induced Tregs, and a less potent (but still present) suppression of established disease using Tregs isolated from untreated EAMG mice. This differential result may be explained by a consideration of the effects of Treg subsets on antigen-specific immune responses.
Tregs can be divided into two principal subsets: 1) naturally-occurring thymus-derived natural CD4+
cells that express the α chain of the interleukin-2 receptor (CD25) (nTregs), and 2) adaptive CD4+
cells that are induced from CD25−
precursors in the peripheral lymphoid organs upon exposure to antigen (iTregs) (Horwitz et al., 2008
; Bluestone and Abbas, 2003
). Both nTregs and iTregs share a similar phenotype, expressing CTLA-4 (cytotoxic T lymphocyte 4), GITR (glucocorticoid-induced tumor necrosis factor receptor), CCR4 (chemokine receptor) and CD62L and requiring IL-2 and TGF-β (Bluestone and Tang, 2005
; Askenasy et al., 2008
). While our results suggested an AChR-selective effect of GM-CSF-induced Tregs, we did not know if this was explained by an expansion of the existing nTreg repertoire (only a small fraction of which are likely to be AchR specific) or if AChR-specific T cells were converted to iTregs. While it is difficult to differentiate nTregs from iTregs based on their in vitro
properties alone, since they have a similar phenotype and both can produce suppressor cytokines that inhibit Teff function in a non-antigen specific manner, only iTregs would be expected to produce selective inhibition of AChR-specific versus irrelevant Ag specific responses as we have shown for GM-CSF/AChR-induced Tregs ().
Previous studies (Nessi et al., 2010
) indicating that Tregs prevent induction of EAMG but do not improve established disease, utilized CD4+CD25+ T cells isolated from naïve animals (nTregs). The specificity of this type of Treg cell population would be anticipated to be polyclonal and likely contain a relatively small percentage of AChR-specific Tregs. It can be predicted that these Tregs might exert some suppression of an initial immune response (priming immunization), but would likely not significantly suppress an established antigen-specific immune process. Since antigenic stimulation is required for the conversion of Teff to iTregs, Tregs obtained from EAMG animals would, on the other hand, be hypothesized to have a larger proportion of iTregs with specificity for the AChR, and would therefore mediate a more efficient suppression of established EAMG. While nTregs are likely to be distributed more randomly, the iTregs, because of similar antigen specificities, are likely to co-localize with the effector T cells and thus be more effective in suppressing antigen specific immune response. Finally, larger numbers of these AChR-specific iTregs are likely to be induced in EAMG mice treated with GM-CSF, through interactions with increasing proportions of tolerogenic DCs, accounting for the enhanced potency and specificity of GM-CSF-induced Tregs. Our findings showing an enhanced in vivo
suppressive effect of Tregs from GM-CSF treated AChR-primed animals compared to Tregs from GM-CSF-treated, non-AChR immunized mice () provides support for this proposed mechanism.
We have also shown that adoptive transfer of GM-CSF/AChR-induced Tregs can potently suppress anti-AChR antibody production in vivo
( and ). This finding is in agreement with previous work demonstrating that depletion of Tregs in rodents can lead to dysregulated antibody production (Morgan et al., 2003
), and the adoptive transfer of Tregs into autoimmune animals can reduce pathogenic antibody responses (Seo et al, 2002
). We do not know if this suppression is primarily mediated through Treg effects on T-helper cells or possibly by a direct suppression of autoreactive B cells as has recently been reported in systemic lupus erythematosus (SLE) (Iikuni et al., 2009
). A direct effect on B cells in suggested by the suppression of B cell proliferation in GM-CSF-treated mice ().
Based on our previous work and the results of this study, we hypothesize that the mechanism of action of GM-CSF in EAMG involves the mobilization of “semi-mature or tolerogenic” bone-marrow derived DC precursors (Bhattacharya et al., 2011
) which, upon antigen (AChR) capture, initiate a suppression of the anti-AChR immune response, through the induction/expansion of AChR-specific Tregs. Our data indicate that a concomitant exposure to antigen (AChR) (as may be the case in active or recent onset MG) is required at the time of GM-CSF treatment for optimization of AChR-specific suppression. It is noteworthy that GM-CSF is currently used widely in the treatment of neutropenia in cancer patients receiving chemotherapy, and that its safety profile and toxicology/biodistribution are well-established (Arellano and Lonial, 2008
). Therefore, a clinical trial of systemically-administered GM-CSF in active or recent onset human MG is quite feasible, and is planned for the near future. Furthermore, GM-CSF could also be used to induce/expand autologous Tregs in vitro
for subsequent use as cellular immunotherapy of MG.