Control of epstein-barr virus infection by cytotoxic T lymphocytes

Sammanfattning: CONTROL OF EPSTEIN-BARR VIRUS INFECTION BY CYTOTOXIC T LYMPHOCYTES Pedro-Otavio de Campos-Lima Doctoral dissertation from the Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, SwedenCytotoxic T lymphocytes (CTL) play a critical role in response to Epstein-Barrvirus (EBV), a widespread human herpesvirus that persists in healthycarriers as a latent infection of B cells and is implicated in the pathogenesisof lymphoid and epithelial cell malignancies. Six virus encoded nuclearantigens, EBNA1-6, and three membrane proteins, LMP1, -2A and -2B, areregularly detected in EBV transformed lymphoblastoid cell lines (LCLs).Recombinant vaccinia viruses expressing these genes were used to infectEBV negative cells that were then tested as targets for polyclonal EBV-specific CTL cultures. Five HLA A11 restricted epitope regions were mappedin the EBNA4 protein using overlapping synthetic peptides in blastsensitization assays. The cognate peptides of the immunodominant andsubdominant epitopes were identified in residues 416-424 (IVTDFSVIK,designated IVT) and 399-408 (AVFDRKSDAK, designated AVF). The TCR aand beta chains of IVT- and AVF-specific clones derived from 4 donors werecloned and sequenced. The AVF specific responses was highly conservedwith virtually the same TCR detected in all donors. In contrast, there was nopreferential V(D)J usage and no obvious motif in the complementaritydetermining regions (CDR)3 loops of the IVT specific TCRs. Sequencevariations was reflected in unique fine specificity patterns detected by Alascanning mutagenesis. Analysis of TCR affinity as a function of sensitivity toCD8 blocking indicated that the diversity of the IVT specific repertoire doesnot reflect a compensatory expansion of low affinity clones due toelimination of a self reactive high affinity TCR. Escape from CTL mediatedrejection could play an important role for virus reactivation and spread andcould also contribute to the pathogenesis of EBV associated malignancies. Toaddress this question, the sequence of the IVT and AVF epitopes wereanalysed in virus strains isolated from populations with different HLAbackgrounds. Isolates from 33 Southeast Asian individuals (>50% HLA A11antigen frequency) had point mutations selectively affecting the codons forone of the IVT anchor residues in P2 or P9. In contrast only 4 of 15 isolatesfrom Caucasians (10-15% HLA-A11 antigen frequency) and none of 15isolates from Africans (HLA A11 practically absent) had mutations withinthe IVT sequence. Approximately half of the Southeast Asian isolatesisolates carried mutations affecting anchor residues of the AVF epitope.Key words: Epstein-Barr virus, Cytotoxic T lymphocytes, T cell receptor, CTLescape mutants