Dynamics of the Major Histocompatibility Complex Class I Processing and Presentation Pathway in the Course of Malaria Parasite Development in Human Hepatocytes: Implications for Vaccine Development
Figure 5
Effect of activated CTLs on the MHC class I pathway in infected hepatocytes.
(A) Expression of the genes involved in the MHC class I pathway was monitored by real-time PCR in uninfected, GFP+ and GFP- HC-04 cells in response to treatment with AS as described in the legend of Figure 2. HC-04 cells were either pre-incubated with AS for 6 hrs prior to infection with P. berghei ANKA GFP, or AS was introduced to the infected cultures 2 hrs post-infection. Data are shown as expression units relative to GAPDH housekeeping gene expression levels arbitrarily taken as 1 and represent means ± SD from 3 individual infection and cell sorting procedures. All p values are <0.0005 and indicate differences between control and treated uninfected (*), GFP negative (**) and GFP positive (***) cells. (B) Human CTLs were pre-activated on third party targets as described in Materials and Methods. HC-04 cultures infected with P. berghei ANKA GFP were either cultured in complete medium (untreated), exposed to AS or co-cultured with pre-activated CTLs (1:100 CTL to hepatocyte ratio). Additionally, each of the three cultures was incubated with IFNγ and TNFα blocking reagents (anti-TNF/anti-IFN) or left untreated (no blocking). Surface MHC class I staining was done on infected cultures at 48 hrs post-infection. Dot plots (GFP-) and contour plots (GFP+) represent MHC class staining with an APC-labeled MHC class I specific antibody or relevant isotype control antibody on infected and uninfected hepatocytes, respectively. One representative of 3 experiments is shown. The numbers within plots show MFI of MHC class I specific staining for the indicated culturing condition and cell subpopulation.