The Antigen-Presenting Pathways In Dendritic Cells For Inactivated Foot-and-Mouth Disease Virus

Foot-and-mouth disease (FMD) is caused by the FMD virus(FMDV), which is a highly contagious vesicular disease of cloven-hoofed animals, such as pigs, cattle, and sheep. The mechanism that animals mount an immune response against FMDV antigens has not yet been profiled. It has been well-documented that initiation of adaptive immunity requires presentation of antigens by dendritic cells (DCs). DCs can cross-present antigens. And this leads to the conclusion that states DCs are the most powerful antigen-presenting cells in the body. However, the antigen-presenting pathways in DCs for inactivated FMDV are still unclear.To elucidate the antigen-presenting pathways in DCs for inactivated FMDV, the immature DCs which were pulsed with inactivated FMDV (FMDV~+DCs) cocultured with T cells, and the FMDV~+DCs as a control. T cells were stimulated for 9h, 12h, 24h, 36h and 48h and culture supernatants from in vitro inactivated FMDV-stimulated T lymphocytes were assayed for IFN-γcontents. The production of IFN-γwas found to increase in a T cell concentration-dependent manner, with less IFN-γbeing produced in the absence of T cells. At 9h and 48h post-coculturation, the content of IFN-γin the culture supernatants (9h: 13.235 6±0.003 4; 48h: 6.473 8±0.002 1) showed a significant difference to the control group(9h: 1.988±0.597 4; 48h: 1.254 5±0.135 7) (P<0.01), respectively. At 12h, 24h, and 36h post-coculturation, the content of IFN-γin the culture supernatants showed difference to the control group(P<0.05), respectively. In this system, the T cells were demonstrated to be an absolute requirement for IFN-γproduction. T cells comprise CD4~+T cells and CD8~+T cells. It was important to determine which subset of T cells was involved in making the determined IFN-γrelease. Then, CD4~+T cells and CD8~+T cells in cervical lymph nodes of BALB/c mice were obtained alternatively by adding anti-mouse CD4 antibody or anti- mouse CD8a antibody. The supernatants from in vitro co-culture of FMDV~+DCs and CD4~+ or CD8~+ T lymphocytes were assayed for IFN-γcontents using ELISA. The results show that both the CD4~+T cells and CD8~+T cells were involved in supporting the determined IFN-γrelease, indicating that MHC class II pathway and cross-presentation were uterlized during the presentation of inactivated FMDV in MoDCs. At 9h and 48h post-coculturation, the content of IFN-γin the CD4~+T cell-FMDV~+DCs culture supernatants showed a significant difference to the control group(P<0.01), respectively. At 24h and 36h post- coculturation, the content of IFN-γin the CD4~+T cell-FMDV~+DCs culture supernatants showed difference to the control group (P<0.05), respectively. Moreover, the production of IFN-γwas found to increase in a CD4~+T cell concentration-dependent manner, with less IFN-γbeing produced in the presence of CD8~+T cells. It is demonstrated that CD4~+T cells were an absolute requirement for IFN-γproduction, and this indicates that MHC class II pathway should be the primary pathway involved in the antigen-presenting pathways for inactivated FMDV antigens.To verify the function of proteasome and lysosome in the antigen processing, the immature DCs were treated with lactacystin or chloroquine respectively. These immature DCs were pulsed with inactivated FMDV and co-cutulred with CD8~+ cells or CD4~+ T cells, respectively. FMDV~+DCs were co-cutulred with CD8~+ cells or CD4~+ T cells as control. The production of the IFN-γwas analyzed at 9h, 12h, 24h, 36h, and 48h later by using ELISA kits according to manufacturer's instructions. Surprisingly, CD8~+T cells produced large amounts of IFN-γwhen DCs were first elicited with inactivated FMDV in the presence of lactacystin. But the production of IFN-γby CD4~+T cell was drastically blocked by chloroquine, a potent lysosome inhibitor. This supports the idea that inactivated FMDV presentation by DCs requires lysosome-dependent processing and the proteasome did not play a major role. This further indicates that MHC class II pathway should be the primary pathway involved in the antigen presention for inactivated FMDV antigens.Taking together, antigens of inactivated FMDV can be degraded either by proteasome or lysosome in DCs. Both CD4~+T cells and CD8~+T cells were involved in supporting the determined IFN-γrelease, while CD4~+T cells are the predominant resource of IFN-γ. These findings show that the antigen of inactivated FMDV is presented in DCs via both the classical MHC class II and cross-presentation passways, and the classical MHC class II pathway is the primary pathway.