Expression microarray analysis of mycobacteria-specific CD4 T cells

Live antigen-specific CD4 T cells are identified typically by tetramer staining or by staining of cytokines captured on the cell surface by bi-functional antibodies. We attempted identification of mycobacteria specific CD4 T cells using CD154 staining. In this method, splenocytes were stimulated with appropriate antigens in the presence of monensin and fluorochrome-conjugated anti-CD154 antibody. Antigen-specific cells will be stained positive for CD154. We performed microarrays with CD154 positive and negative CD4 T cells. Splencoytes from M. bovis BCG vaccinated mice were stimulated in vitro with M. tuberculoiss lysate in the presence of monensin and anti-CD154-PE antibody. Next, they were stained for dead cells, CD4, and CD44. All sorted cells were live and positive for CD4, and CD44 and two microarray groups were distinguished based on the status of CD154 staining (positive or negative).