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Case Reports
. 2012 Sep;46(3):449-53.
doi: 10.1002/mus.23488.

Granulocyte macrophage colony-stimulating factor treatment of a patient in myasthenic crisis: effects on regulatory T cells

Julie Rowin  1 , Muthusamy Thiruppathi, Ebinehita Arhebamen, Jianrong Sheng, Bellur S Prabhakar, Matthew N Meriggioli
Affiliations
Case Reports

Granulocyte macrophage colony-stimulating factor treatment of a patient in myasthenic crisis: effects on regulatory T cells

Julie Rowin et al. Muscle Nerve. 2012 Sep.

Abstract

Introduction: In this study we describe a patient with a prolonged myasthenic crisis refractory to conventional immunomodulatory therapy who was treated with GM-CSF (granulocyte macrophage colony-stimulating factor, sargramostim).

Methods: T-regulatory cell (Treg) suppressive function and Foxp3 expression were evaluated before and after treatment with GM-CSF.

Results: Treatment with GM-CSF was associated with clinical improvement, expansion in the circulating numbers of Foxp3(+) cells, increase in Foxp3 expression levels in Tregs, early improvement in Treg suppressive capacity for AChR-α-induced T-cell proliferation, and subsequent enhancement in Treg suppression of polyclonal T-cell proliferation.

Conclusion: Although definitive conclusions cannot be drawn from a single case, the correlation with similar findings in GM-CSF-treated animals with experimental autoimmune myasthenia gravis suggests further exploration of the effects of GM-CSF in myasthenia gravis should be studied in a clinical trial setting.

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Figures

Figure 1
Figure 1
Manual muscle testing. Variation in manual muscle testing (MMT) in a patient in myasthenic crisis in response to GM-CSF after failure of conventional immunomodulatory treatments. The higher the MMT score, the weaker the subject. Of note, the patient was taking prednisone 60mg daily. Blood draws for analysis of Treg function were drawn pre-treatment (0), after 5 days of GM-CSF (1), 1 week after completion of 10days of GM-CSF (2), and 4 weeks after completion of GM-CSF (3).
Figure 2
Figure 2
Expression of Foxp3 in T cells. A) FACS analysis of the percentage of Foxp3-expressing cells within the CD4+ T cell subset. CD4+ T cells were sorted and isolated as described. The sorted cells were stained with AF488-anti-human Foxp3 and analyzed by flow cytometry. A plot from a healthy control subject is shown in addition to the results from the patient pre-treatment (MG), after 5 days of GM-CSF (MG + GM-CSF-1), 1 week after completion of 10 days of GM-CSF (MG + GM-CSF-2), and 4 weeks after completion of GM-CSF (MG + GMCSF-3). B) The mean fluorescent intensity (MFI) of Foxp3 expression was determined within the isolated CD4+CD25highCD127low Treg cells. The cells were sorted using APC-anti-human CD4, PE- anti-human CD25 and PE-Cy7 anti-human CD127 antibodies. The sorted cells were stained with AF488-anti-human Foxp3 and analyzed by flow cytometry. Results for a healthy control and the patient (pre- and post-treatment) are shown.
Figure 3
Figure 3
T regulatory cell proliferation/suppression studies pre and post-treatment with GM-CSF. CD4+CD25high CD127low Treg cells were isolated and cultured with CD4+CD25− Tresp (labeled with CFSE) at the indicated ratios in the presence of irradiated APC, and stimulated with anti-CD3 antibody and/or AChR-α peptides. After 4 days of in vitro culture, the proliferation of CFSE-labeled T cells was quantified, and expressed as a percentage of proliferating (CFSE low) cells. A). Relative ability of Tregs to suppress anti-CD3 induced T cell proliferation in the patient prior to treatment with GM-CSF (MG), after 5 days of treatment (MG + GM-CSF-1), 1 week after completion of treatment (MG + GM-CSF-2) and 4 weeks after completion of treatment (MG + GM-CSF-3) vs. control subjects: baseline proliferation - dark bars; proliferation after addition of Tregs - light bars. B). T cell proliferation/suppression studies stimulating with AChR-α 195–212 in the patient pre- and post-treatment with GM-CSF compared to control subjects. C). T cell proliferation/suppression studies stimulating with AChR-α 257–269 in the patient pre- and post-treatment compared to control subjects. For A, B, and C, control data is expressed as mean ± SEM of 14 subjects; percentage of proliferating cells were compared in Tresp alone and Tresp + Treg co-cultures, p <0.05.
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