In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (T_HGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (T_HGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)γ or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and T_HGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into T_HGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the T_HGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFNα or IL-17. The predominant expression of GM-CSF by T_HGM cells was further confirmed by the analysis of the expression of GM-CSF, IFNα, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to T_HGM cells in vitro, which will be useful in the study of T_HGM cell biology.