After co-culture with CII for 72 h, CD4+ T cells were isolated from SMNCs derived from

CII immunized mice and transcript levels of four Notch receptors, including Notch1, Notch2, Notch3 and Notch4, were assessed. We found that CII restimulation see more up-regulated Notch3 transcription significantly in CD4+ T cells. To further confirm the specific role of Notch3, we added specific neutralizing antibody to Notch3 to the SMNCs restimulation system and found that anti-Notch3 treatment reduced T cell proliferation and the frequency of Th1 and Th17 cells. These results indicate that Notch3 plays an important role in CII-specific T cell proliferation and expansion. Over-expression of the Notch3 intracellular domain in T cells has been reported to induce differentiation of IFN-γ-secreting Th1 but reduced IL-4-secreting Th2 cells. When Notch3 expression was inhibited with anti-sense-DNA, the Th1-type differentiation was also inhibited [17]. Our results were partly different from another research group, which explored the role of Notch signalling in myelin-reactive CD4+ T cells using the EAE model, and found that both Notch1 and Notch3 were up-regulated upon specific antigen restimulation, although Notch1 inhibition did not affect the proliferation and differentiation DMXAA of autoreactive

T cells [13]. These different data may result from the use of different antigens as well as different animal models. Nevertheless, we agree with the important role of Notch3 in antigen-specific Th1 and Th17 cell expansion other than Treg cells. Notch signalling is initiated by ligand–receptor interaction

between neighbouring cells. We next asked which Notch ligands are involved in CII-specific T cell proliferation and differentiation by the addition of Delta-like 1-Fc and Jagged1-Fc fusion proteins into SMNCs co-cultured with CII from CII immunized mice. Our results indicate that it should be Delta-like 1 rather than Jagged1 that promotes the collagen-specific Th1- and Th17-type expansion. In EAE, pathogenic Th1 and Th17 cells develop in the central nervous system, causing autoimmunity. Resminostat Specific antibodies against Delta-like 1, which attenuated EAE, have opposite effects to antibodies against Jagged1 which exacerbated EAE [18]. Maekawa et al. reported that Delta-like 1 interaction with Notch3 on CD4+ T cells promoted development towards the Th1 phenotype [17]. However, Delta-like 4-expressing dendritic cells (DCs), when activated with Toll-like receptor (TLR) ligands or Mycobacterium antigens, can promote the generation of Th17 cells through activation of the Th17 cell-specific transcription factor retinoic acid-related orphan receptor γ-T (RORγt) [19,20]. The specific interactions of Notch ligands and receptors on T cells may be regulated by the expression pattern of Notch ligands on neighbour cells [17].