Study of epstein-barr virus (EBV)-specific polyfunctional T cells responses in long term carriers and patients with infectious mononucleosis and haemophagocytic lymphohistiocytosis

Abstract

Effective control of chronic viral infections may require the generation of polyfunctional T cells (PFCs) which are capable of producing multiple cytokines and possess cytotoxic function. In this study, I investigate (i) whether PFCs play an important role in the long term immune control of a persistent human virus, Epstein-Barr virus (EBV), (ii) the relationship between the development of immunodominance and functionality during the evolution of the anti-viral T cell responses, and (iii) whether PFCs can be generated in patients with EBV-associated haemophagocytic lymphohistiocytosis (HLH). To tackle the first question, I established a 9-color flow cytometry assay to characterize the co-expression of four cytokines (interferon-f [IFN-[], macrophage inflammatory protein 1-] [MIP1-[], tumour necrosis factor-] [TNF- ] and interleukin-2 [IL-2]) and degranulation marker (CD107a) by both EBV lytic and latent peptide-specific CD4+ and CD8+ T cells in 20 healthy long term viral carriers. Two patients with EBV-associated post-transplant lymphoproliferative disorder (PTLD) were studied for comparison. Both CD4+ and CD8+ PFCs were readily generated in the long term carriers with the immunodominant EBV proteins apparently stimulating higher proportions of PFCs than the subdominant viral proteins. The PFCs producing greater amount of cytokines per cell than the single functional T cells. In contrast, EBV-specific PFCs were hardly generated in the patients with PTLD. To investigate the relationship between the development of immunodominance and functionality, I performed a longitudinal study of CD4+ and CD8+ T cell responses in 10 children with infectious mononucleosis (IM) and 4 asymptomatic individuals (AS) with primary EBV infection from the time of diagnosis to one year post-infection. Viral peptide-specific T cells were examined for the co-expression of three cytokines (IFN-t, TNF-T and IL-2), perforin and CD107a upon stimulation with overlapping peptide pools of lytic and latent proteins, respectively. PBMC viral loads were reduced gradually in both IM and AS subjects. Whilst lytic and latent peptide-specific PFCs were still detectable at the acute stage of infection, they showed an increase in functionality over time in response to peptide pools of immunodominant proteins. From acute to persistent infection stage, the CD8+ T cell responses shifted from reactivities against the lytic peptides to those against the latent EBNA3A and 3B peptides with concurrent increase in functionality. Change in CD4+ T cell responses is less obvious, apparently from reactivities towards broad range to EBNA1 peptides. Finally, we found that two patients with the life-threatening EBV-associated haemophagocytic lymphohistiocytosis (HLH) had very high viral loads at the onset of disease. The clinical symptoms improved and viral loads were gradually reduced by the immunosuppressive drug therapy. Interestingly, EBV lytic and latent peptide-specific PFCs could be subsequently generated post-treatment with sustained resolution of clinical symptoms and clearance of plasma viral loads . In conclusion, lytic and latent peptide-specific CD4+ and CD8+ PFCs may confer long term immune control to EBV. The PFCs may be generated concurrent with the establishment of immunodominance hierarchy during the evolution of viral-specific T cell responses. Long term polyfunctional T cell responses to EBV can be formed in patients with EBV-associated HLH.