Figure. 4. IL-10 gene silenced DC results in moderately increased expression of co-stimulatory markers and generates a stronger CTL response against human melanoma associated antigenic epitope, MART-127−35.
(A). Dendritic cells treated with siRNA were matured and phenotyped for the surface expression of co-stimulatory markers CD80, CD86 and MHC classI (iDC- immature DC, mDC- mature DC, Si-mDC-mature siRNA treated DC). (B). IL-10 silenced DC results in generation of a robust CTL response against human melanoma associated antigenic epitope, MART-127−35. In-vitro co-cultures of peripheral blood derived CD8 +ve T cells with monocyte derived immature DC, matured DC and matured and siRNA treated DC were set up and 10 days following co-culture, the expanded populations was examined for MART-127−35 epitope specific T cells by staining with CD8 (X-axis) and MART-127−35 specific tetramer (Y-axis) (C-Baseline CTL precursor frequency, iDC- in-vitro co-culture of CD8 +ve T cells with immature DC, mDC- in-vitro co-culture of CD8 +ve T cells with mature DC, Si-mDC-in-vitro co-culture of CD8+ve T cells with mature IL-10 silenced DC). (C). Quantification of IFN-γ secretion by MART-127−35 epitope specific CTL. The in-vitro expanded populations generated in Fig.4B were examined for IFN-γ production by exposing them overnight with either MART-127−35 peptide or control MAGE-3 control peptide pulsed T2 cells and doing ELISA on culture supernatants (iDC-immature DC co-cultured CTL population, mDC-mature DC co-cultured CTL population, Si-mDC-IL-10 silenced mature DC co-cultured CTL population). These experiments were done twice with similar results.