GM-CSF-induced regulatory T cells selectively inhibit anti-acetylcholine receptor-specific immune responses in experimental myasthenia gravis

Abstract

We and others have demonstrated the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) to suppress autoimmunity by increasing the number of CD4(+)CD25(+) regulatory T cells (Tregs). In the current study, we have explored the critical role of induced antigen specific Tregs in the therapeutic effects of GM-CSF in murine experimental autoimmune myasthenia gravis (EAMG). Specifically, we show that Tregs from GM-CSF treated EAMG mice (GM-CSF/AChR-induced-Tregs) adoptively transferred into animals with EAMG suppressed clinical disease more potently than equal numbers of Tregs from either GM-CSF untreated EAMG mice or healthy mice treated with GM-CSF. In addition, GM-CSF/AChR-induced-Tregs selectively suppressed antigen specific T cell proliferation induced by AChR relative to that induced by an irrelevant self antigen, (thyroglobulin) and failed to significantly alter T cell proliferation in response to an exogenous antigen (ovalbumin). These results are consistent with the hypothesized mechanism of action of GM-CSF involving the mobilization of tolerogenic dendritic cell precursors which, upon antigen (AChR) capture, suppress the anti-AChR immune response through the induction/expansion of AChR-specific Tregs.

Figure 1. Clinical Effects of GM-CSF

Clinical severity of EAMG in GM-CSF-treated, compared to PBS-treated, tAChR-immunized mice. Mice were evaluated as described in Materials and Methods beginning at the time of initiation of treatment (Day 0). A) Average clinical score during days 0–40. Day 0 corresponds to initiation of treatment (GM-CSF vs. PBS). All mice had received a priming immunization; an additional tAChR booster immunization was given on day 28. B) Muscle AChR content in GM-CSF-treated vs PBS-treated and healthy control animals. C) Serum anti-mouse AChR IgG and IgG2b subclasses for GM-CSF-treated, and PBS-treated mice. Serum was analyzed for anti -mouse AChR IgG Ab and IgG2b isotype by ELISA (n=10 per group) on day 0 and day 40. Statistical comparisons were made between IgG levels before and after treatment; *statistically significant (p < 0.05) differences are indicated.

Figure 2. Immunological effects of GM-CSF

A) Effect of GM-CSF treatment on DC phenotype in EAMG. Splenocytes were collected and stained with FITC-labeled anti-mouse CD11c and various other antibodies to cell surface proteins as described. DCs from GM-CSF treated-animals expressed lower levels of CD80, CD86, PDL-1, PDL-2 and CD40 and had an increase in the CD8α-negative population. B) Effects of GM-CSF on the population of FOXP3+ T cells. Representative plots indicating the percentage of FOXP3+ regulatory T cells in splenocytes are shown. C) T cell proliferative response to AChR stimulation. Responder CD4⁺ cells from GM-CSF-treated and untreated EAMG mice were labeled with CFSE and stimulated with AChR. The proliferative response was determined by CFSE dilution. D) B cell proliferative response to AChR stimulation. B cells were isolated based on expression of CD19 and cells from GM-CSF-treated and untreated mice were cultured and stimulated with AChR. The proliferative response was quantified by CFSE dilution as above. E. Effects of GM-CSF on cytokine profile in T cells. Mice were immunized with tAChR and treated with GM-CSF or PBS. CD4⁺ cells from mice immunized with tAChR and treated with GM-CSF or PBS were used to detect various cytokines by intra-cytoplasmic staining.

Figure 3. Adoptive transfer of GM-CSF/AChR-induced Tregs

A) Change (post-adoptive transfer score minus baseline score) in clinical severity in 4 groups of EAMG mice adoptively transferred with either Tregs (CD25⁺) from GM-CSF (◇) or PBS-treated (□) EAMG mice vs. non-Tregs (CD25⁻) from GM-CSF (△) or PBS-treated (X) EAMG mice. EAMG severity was significantly reduced in mice receiving Tregs from GM-CSF treated donors compared to Tregs from PBS-treated EAMG mice (*p < 0.05). B) Muscle AChR content (ng/ml) at the end of study in the four experimental groups. AChR content was more significantly preserved in mice receiving CD4⁺CD25⁺ (Tregs) from GM-CSF-treated EAMG donors when compared to mice receiving Tregs from untreated donors (p < 0.05).

Figure 4. Anti-AChR antibody levels in adoptively transferred EAMG mice

Bar graphs showing anti-AChR IgG (A) And IgG2b (B) antibody levels as determined in the four experimental groups by an ELISA assay. Significant reductions in both anti-AChR total IgG and IgG2b were observed only in mice receiving CD4⁺CD25⁺ (Tregs) from GM-CSF-treated EAMG donors. *Statistically significant value (p < 0.05).

Figure 5. Effects of Treg adoptive transfer on the splenic population of FOXP3⁺ T cells, and potency of GM-CSF-induced Tregs suppression of the AChR-stimulated T cell proliferative response

The percentage of FOXP3+ regulatory T cells in splenocytes are shown for the four experimental groups (A). No significant difference is apparent in the expansion of Foxp3-expressing splenocytes in mice receiving CD4+CD25+ cells. Despite this, GM-CSF-induced Tregs suppressed the AChR-induced proliferation of responder T cells more potently compared to Tregs from PBS-treated animals; GM-CSF-induced Tregs maintain their suppressive potency at lower Treg/Teff ratios (B). Results are representative of three separate experiments with each column representing the mean ± SEM (*p < 0.05).

Figure 6. Antigen-specific suppression of T cell proliferation

T cell proliferation in response to three different antigens (AChR, Tg (thyroglobulin), and ovalbumin (OVA). T responder (T_resp) cells were isolated from animals primed with the indicated antigen. A. Results from a representative experiment are shown. I. Baseline proliferation (without addition of Tregs) is shown in the top row; II. With the addition of Tregs from GM-CSF-treated, CFA-immunized mice; and III. With the addition of Tregs from GM-CSF-treated EAMG mice. B. Summary of T cell proliferation/suppression assays from three separate experiments, showing the mean ± SEM. *Statistically significant value (p < 0.05).

Figure 7. Adoptive transfer of GM-CSF expanded Tregs (exposed and not exposed to AChR)

Recipient mice were immunized and boosted with tAChR and divided into two treatment groups. Mice were adoptively transferred with Tregs (CD4⁺ CD25⁺ cells) from GM-CSF-treated EAMG donors (solid circles) or Tregs from GM-CSF-treated mice not exposed to AChR (solid squares). A. Serial determinations of mean clinical score are shown with day 0 corresponding to the day of adoptive transfer. B. Anti-AChR antibodies were determined using an ELISA assay at baseline (before adoptive transfer), prior to the tAChR boost (arrow) 30 days after adoptive transfer (After), and at the end of the study (post-boost). Mean OD values and standard errors are shown for three separate experiments. In each case, the post-treatment values were compared to the baseline value to determine significance (p < 0.05). *Statistically significant value (p < 0.05).