Studies on Immune Regulation of Epstein-Barr Virus
McAulay, Karen A
Epstein-Barr virus (EBV) is a gammaherpes virus that infects >90% of the adult population worldwide. During childhood infection is generally sub-clinical, however if delayed until adolescence infectious mononucleosis (IM) may develop. The virus has also been aetiologically linked with a number of tumours including B-cell lymphoma following organ transplantation: post-transplant lymphoproliferative disease (PTLD). The symptoms of IM are caused by an expansion of immune cells in response to infection whilst in the transplant situation immunosuppressive drug therapy allows the outgrowth of the tumour. Understanding the immuno-regulatory mechanisms involved in such EBV-associated diseases is crucial for devising new treatment strategies. We undertook 3 separate studies (1-3) investigating different aspects of the immune response to EBV. In a recently reported phase II trial using allogenic, EBV-specific cytotoxic T-cell (CTL) to treat PTLD, tumour response was significantly increased with a high degree of donor/recipient HLA-allele matching suggesting that further refinement of the matching procedure may be important. In study 1 we investigated the epitope specificity and T-cell receptor (TCR) clonality of the infused CTL to identify potential areas for refinement. We found the protein specificity of the CTL to be polyclonal with dominant recognition of Epstein-Barr nuclear antigen-3 proteins and sub-dominant recognition of Latent membrane protein (LMP)-1 and LMP-2 proteins. Where possible, specificity was confirmed at the peptide level. No single TCR family was preferentially used by CTLs. The CTL epitope specificity did not differ between treatment responders and non-responders however the response was improved in those with several CTL HLA-restricted epitope matches and those infused with CTL containing polyclonal TCR families as opposed to monoclonal. CTL/recipient matching based on HLA matching alone was improved when also matched via HLA- restricted epitiope specificity. Therefore mapping CTL peptide epitope specificity prior to CTL infusions may enhance patient responses. In recent years, interest has developed in genetic variation within components of the immune system. Of particular interest are cytokine/cytokine receptor genes and genes of the human leukocyte antigen (HLA), both of which act to regulate the immune response. Variation within these genes could potentially alter the immune response leading to disease. In study 2 we investigated single nucleotide polymorphisms (SNPs) in several cytokine genes (TNF, IL-1, -6, -10) in both IM and PTLD cases and compared with relevant control groups. We found that the frequency of two TNF promoter alleles was significantly increased in PTLD patients compared to controls whilst the frequency of a TNF receptor II allele was increased in IM and EBV seropositive individuals, suggesting a role for this allele in susceptibility to EBV infection. The frequency of a second TNF receptor II allele was increased in both PTLD and IM subjects compared to controls highlighting the possible significance of TNF and its receptor in the development of EBV associated disease. In study 3 we analyzed two microsatellite markers and two SNPs located near the HLA class I locus in IM, PTLD and control subjects to further determine whether the HLA genes may affect development of EBV-associated diseases. Alleles of both microsatellite markers were significantly associated with development of IM. Specific alleles of the two SNPs were also more frequent in IM patients. Moreover IM cases possessing the associated microsatellite allele had significantly fewer lymphocytes, increased neutrophils, and displayed higher EBV titres and milder IM symptoms relative to IM cases lacking the allele. The results indicate that HLA class I polymorphisms may predispose patients to development of IM upon primary EBV infection and suggest that genetic variation in T cell responses can influence the course of EBV infection.