Fig 1: GAD-specific CD4 T cells induced by GAD-alum immunisation are bifunctional. (a) Representative GAD-specific CD4 T cell clone stimulated with diluent DMSO or (b) PMA/ionomycin for 3 h and stained for intracellular expression of IL-13 and IFN-? shows the clone’s bifunctional potential. (c) GAD peptide-specific clone exhibits a bifunctionality when stimulated with cognate GAD65 555–567 peptide in vitro and analysed by dual-cytokine FluoroSpot. The control condition is stimulation with DMSO (the peptide diluent). The Th1/Th2 phenotype (IFN-?+/IL-13+) is shown in yellow. IFN-? responses are green and IL-13 are red (representative data from six clones). (d) Expression of cytokine and transcription factor mRNA by single sorted clone/line cells detected by PCR shows that a majority of GAD-alum induced bifunctional CD4 T cells co-express Th1 and Th2 differentiation markers TBX21 (encoding T-bet), IFNG and GATA3 as well as IL4 and/or IL13