Processing of live and heat inactivated Sendai virus for presentation on MHC class I molecules

Abstract

The present thesis deals with processing of antigens from live and heat inactivated Sendai virus and their loading on MHC class I molecules for presentation to CTL. In paper I it was observed surprisingly that T2 cells (T2 cells transfected with H-2Kb), being defective in the expression of TAP peptide transporters, still presented Sendai virus antigen on MHC class I molecules to CTL. This presentation was resistant to brefeldin A (BFA), suggesting that presentation of antigen was not dependent on newly synthesized MHC class I molecules. This indicated that Sendai virus exhibited properties different from other viruses studied, in that antigens from this virus could still be presented on MHC class I molecules despite the lack of expression of a functional TAP1/2 complex. The results also suggested the existence of a novel alternative pathway for presentation of viral antigens on MHC class I molecules.

In paper II we extended the analysis of this novel antigen processing pathway with studies that leading to the conclusion that the TAP independent BFA-resistant pathway was cell specific (T2 lineage), virus specific (Sendai virus), but not epitope specific (SV NP325-332). In the course of the studies in paper I and paper II, it was also observed that not only live but also heat inactivated Sendai virus could be presented on MHC class I molecules in T2 cells. The ability of T2 cells to process and present heat inactivated Sendai virus antigens was studied in paper III, in which a detailed comparison of the ability of T2 cells to present live and heat inactivated SV was performed. Heat inactivated virus with no fusion or hemaglutinin-neuraminidase activities behaved similarly to live SV in being processed in a TAP-independent and BFA-resistant manner in T2 cells. In addition to these observations, it was observed that heat inactivated SV could prime CTL in vivo. Paper IV is largely a continuation of paper III, suggesting that heat inactivated Sendai virus is processed in an intracellular compartment with endosomal characteristics in T2 cells. The data also suggest that this compartment contains class I molecules which may be derived from the cell surface.

Finally, Paper V is a continuation of the observation in paper III where it was observed that heat inactivated SV could prime CTL in vivo. In paper V it was studied how normal splenic antigen presenting cells process heat inactivated Sendai virus antigens for presentation on MHC class I molecules. Efficient processing and presentation of heat killed Sendai virus antigen on MHC class I molecules was observed with normal B6 as well as withTAPI -/- splenic APC. Presentation was MHC class I restricted since no presentation was seen on APC from TAPI/B2m -/- mice, which lack expression of MHC class 1. Presentation occurred even in the absence of brefeldin A, but was blocked with cytochalasin D as well as chloroquine. B6 as well as TAPI -/- splenic APC, when loaded with heat killed Sendai virus antigen in vitro, primed naive CD8+ T cells in vivo. This result suggested that a TAP-independent pathway for antigen presentation on MHC class I also existed in normal splenic APC and provided insights into the molecular basis for the generation of the CTL cell responses observed after priming naive mice with heat killed Sendai virus. These results are discussed in relation to the events underlying the processing and presentation of exogenous antigen on MHC class 1, the molecular basis for CD8+ T cell priming during viral infections, and prospects for vaccine development.