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

Sammanfattning: PROCESSING OF LIVE AND HEAT INACTIVATED SENDAI VIRUS FOR PRESENTATIONON MHC CLASS I MOLECULES Tianmin Liu Doctoral dissertation from the Microbiology and Tumor Biology Center KarolinskaInstitute, Stockholm, Sweden The present thesis deals with processing of antigens from live and heat inactivatedSendai 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 transfectedwith H-2Kb), being defective in the expression of TAP peptide transporters, stillpresented Sendai virus antigen on MHC class I molecules to CTL. This presentationwas resistant to brefeldin A (BFA), suggesting that presentation of antigen was notdependent on newly synthesized MHC class I molecules. This indicated that Sendaivirus exhibited properties different from other viruses studied, in that antigensfrom this virus could still be presented on MHC class I molecules despite the lackof expression of a functional TAP1/2 complex. The results also suggested the existenceof a novel alternative pathway for presentation of viral antigens on MHC class Imolecules. In paper II we extended the analysis of this novel antigen processingpathway with studies that leading to the conclusion that the TAP independent BFA-resistantpathway was cell specific (T2 lineage), virus specific (Sendai virus), but not epitopespecific (SV NP325-332). In the course of the studies in paper I and paperII, it was also observed that not only live but also heat inactivated Sendaivirus could be presented on MHC class I molecules in T2 cells. The ability of T2cells to process and present heat inactivated Sendai virus antigens was studied inpaper III, in which a detailed comparison of the ability of T2 cells to presentlive and heat inactivated SV was performed. Heat inactivated virus with no fusionor hemaglutinin-neuraminidase activities behaved similarly to live SV in being processedin 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 IVis largely a continuation of paper III, suggesting that heat inactivated Sendaivirus is processed in an intracellular compartment with endosomal characteristicsin T2 cells. The data also suggest that this compartment contains class I moleculeswhich may be derived from the cell surface. Finally, Paper V is a continuationof the observation in paper III where it was observed that heat inactivatedSV could prime CTL in vivo. In paper V it was studied how normal splenic antigenpresenting cells process heat inactivated Sendai virus antigens for presentationon MHC class I molecules. Efficient processing and presentation of heat killed Sendaivirus 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 presentationwas seen on APC from TAPI/B2m -/- mice, which lack expression of MHC class 1. Presentationoccurred even in the absence of brefeldin A, but was blocked with cytochalasin Das well as chloroquine. B6 as well as TAPI -/- splenic APC, when loaded with heatkilled Sendai virus antigen in vitro, primed naive CD8+ T cells in vivo. This resultsuggested that a TAP-independent pathway for antigen presentation on MHC class Ialso existed in normal splenic APC and provided insights into the molecular basisfor the generation of the CTL cell responses observed after priming naive mice withheat killed Sendai virus. These results are discussed in relation to the events underlyingthe processing and presentation of exogenous antigen on MHC class 1, the molecularbasis for CD8+ T cell priming during viral infections, and prospects for vaccinedevelopment. Key words: antigen presentation, antigen processing, APC, CTL, MHC class1, peptides, Sendai virus, TAP ISBN 91-628-2735-9