Flow cytometry analysis (Figure 4a) revealed a reduction in the surface expression of MHC class II (I-a) on AE-pe-DCs isolated from AE-infected mice. This effect was more pronounced on AE-pe-DCs from the late stage than from the early stage of infection, as compared to naïve pe-DCs (from noninfected control mice). mRNA expression levels of different molecules implicated in the MHC class find more II (I-a) pathway and the formation of MHC
(I-a)–antigenic peptide complex [CIITA, Li, H-2Ma, I-aβ and Cat-S] as well as β-actin (as a housekeeping gene) in both naive pe-DCs and AE-pe-DCs were determined by semi-quantitative reverse-transcription PCR. Figure 4(b) shows the ratio of normalized integrated intensity values of the above-mentioned genes expressed by AE-pe-DCs vs. naive pe-DCs. The relative gene expression levels of the respective molecules (CIITA, Li, H-2Ma, I-aβ and Cat-S) appeared down-regulated in AE-pe-DCs when compared to naive pe-DCs. Consequently, the down-regulation of different gene expression levels contributed to the understanding of the very low level of MHC class II molecule
expression on the surface of AE-pe-DCs. Excretory/secretory products (E/S) and/or metacestode vesicular fluid (V/F) components were investigated for their putative involvement in the reduction of Ridaforolimus research buy functional MHC class II (Ia) in vitro. BMDCs were separately treated with E/S products and V/F for 2 h. Isolated membrane-associated proteins were investigated by Western blotting with anti-MHC class II Dipeptidyl peptidase antibodies (Figure 5). Both products reduced banding signals in comparison with that of mock-treated control BMDCs. These findings suggested that E/S products, and to a lesser extent also V/F, modified intact MHC class II (Ia) molecule expressed on the surface of BMDCs. The precedent
findings prompted us to investigate whether AE-pe-DCs (compared to naïve pe-DCs) affect differently a Con A-induced proliferative response of naïve CD4+ pe-T cells. These latter cells were Con A stimulated in the presence of increasing numbers of naive pe-DCs or AE-pe-DCs, respectively. Figure 6 showed that increasing numbers of naive pe-DCs enhanced a Con A-driven proliferation of naive CD4+ pe-T cells. Conversely, AE-pe-DCs failed to enhance such a proliferation, and even at relatively high numbers, we observed a decreased proliferation of naive CD4+ pe-T cells. Overall, it appears that AE-pe-DCs, characterized by a high level of TGF-β mRNA and a reduced surface expression of MHC class II molecules and co-stimulatory molecules (CD80 and CD86), displayed a suppressive effect on Con A-driven proliferation of naïve CD4+ pe-T cells. For the metazoan parasite E.