Objective: EBV-specific CD8+ T cells have been extensively studied in various settings, and appear to play a major role in the control of EBV-related malignancies. In contrast, it is still unclear whether EBV-specific CD4+ T cells play a role in vivo. To study this question, an assay was developed to measure the CD4+ T-cell response towards two EBV antigens, in both healthy (n=14) and HIV-infected subjects (n=23). In addition, both HAART-treated (n=12) and untreated HIV+ individuals (n=14) - including progressors to EBV-related lymphoma - were studied longitudinally.

Methods: EBV-specific CD4+ T cells were stimulated with peptide pools from latent protein EBNA1 and lytic protein BZLF1, and detected by measurement of IFNg-production.

Results: After direct ex vivo stimulation, EBNA1 or BZLF1-specific IFNg- (and/or IL2) producing CD4+ T cell numbers were low, and measurable in less than half of the subjects studied (either HIV- and HIV+). Therefore, PBMC were cultured for 12 days in the presence of peptides and IL2 (from day 3), and then restimulated with peptides, allowing specific and reproducible expansion of EBV-specific CD4+ T cells, independent of HLA type and ex vivo antigen processing. Interestingly, numbers of EBV-specific CD4+ T cells inversely correlated with EBV viral load, implying an important role for EBV-specific CD4+ T cells in the control of EBV in vivo. Untreated HIV-infected individuals had a lower CD4+ T cell response to EBNA1 and BZLF1 as compared to healthy EBV carriers and HAART-treated HIV+ subjects. In longitudinal samples, EBNA1-specific, but not BZLF1-specific T-cell numbers increased after HAART, while EBV load was not affected by treatment. In all the progressors to EBV-related lymphoma, EBV-specific CD4+ T cells were lost at least 24 months before lymphoma diagnosis.

Conclusions: Both cross-sectional and longitudinal data suggest an important role for EBV-specific CD4+ T cells in the control of EBV-related malignancies. Furthermore, it seems that HAART treatment leads to recovery of EBNA1-specific, but not BZLF1-specific CD4+ T-cell responses, implying changes in the latency pattern of EBV, despite an unaltered cell-associated EBV DNA load. Thus, early HAART treatment might prevent loss of specific CD4+ T-cell help and progression to NHL.

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