Japanese Encephalitis Virus Induce Immuno-Competency in Neural Stem/Progenitor Cells (original) (raw)
Figure 2
Increased surface expression of costimulatory molecules and MHC class I by NSPCs following JEV infection in vitro.
NSPCs isolated from the SVZ were cultured as free-floating neurospheres and infected with JEV at MOI = 5. Following infection, the cells were cultured and collected at different time points from 1 dpi to 4 dpi. Control and JEV infected NSPCs were stained with anti-CD40, anti-CD80, anti-CD86, and anti-MHC class I and isotype controls (IgG2aκ) antibodies, and analyzed using BD FACS Calibur system. The histograms were plotted and the mean fluorescent intensity (MFI) was calculated over all the existing peaks in each of different conditions and finally represented graphically as the averaged MFI for each condition (2A). Significant increase in surface expression of CD40 (2A i, v), CD80 (2A ii, vi) and CD86 (2A iii, viii) was noted at 3 dpi in JEV infected NSPCs. MHC class I (2A iv, viii) expression was upregulated significantly at both 2 and 3 dpi in the infected NSPCs compared to control NSPCs and JEV infected isotype. # indicates p<0.05 compared to control NSPCs and infected isotypes. Control and JEV infected neurospheres at 3 dpi were collected and allowed to adhere on PDL-coated chamber slides for 3 hours and immunostained for CD40, CD80, CD 86 and MHC class I molecules, as well as rat IgG2aκ isotype (2B). The nuclear counterstain was done with DAPI (blue). Increased surface expression of all the costimulatory molecules (CD40, 80, 86) was observed in infected neurospheres. Robust surface staining for MHC class I was noticed in the infected NSPCs compared to uninfected control. Scale bar corresponds to 20 microns.