Long-term carriers generate Epstein-Barr virus (EBV)-specific CD4(+) and CD8(+) polyfunctional T-cell responses which show immunodominance hierarchies of EBV proteins

T cells simultaneously producing multiple cytokines and possessing cytotoxic capacity termed polyfunctional cells (PFCs) are increasingly recognized as the immune correlate of protection against pathogenic viruses. We investigated co‐expression of four cytokines (interferon‐γ, macrophage inflammatory protein 1‐α, tumour necrosis factor‐α and interleukin‐2) and degranulation capacity (CD107a surface expression) of Epstein–Barr virus (EBV) ‐specific CD4⁺ and CD8⁺ T cells upon stimulation by overlapping peptides of EBV lytic (BZLF1) and latent (EBNA1, EBNA3 and LMP2) proteins, in 20 healthy Chinese long‐term carriers. Two patients with post‐transplant lymphoproliferative disorder (PTLD), who had impaired T‐cell immunity, were studied for comparison. Both EBV‐specific CD4⁺ and CD8⁺ PFCs were readily generated in long‐term carriers and showed immunodominance hierarchies of latent proteins (EBNA1 > EBNA3/LMP2 and EBNA3 > LMP2 > EBNA1 for CD4⁺ and CD8⁺ T cells, respectively), as evidenced by a higher proportion of PFCs generated by immunodominant EBV proteins than by subdominant viral proteins. In contrast, the proportion of EBV‐specific PFCs was markedly decreased in patients with PTLD. The EBV‐specific PFCs produced more cytokine per cell than single‐functional T cells and comprised different subsets. Five‐functional CD4⁺ and CD8⁺ T cells were detected and four‐functional CD4⁺ T cells were mainly CD107a negative and expressed all four cytokines whereas four‐functional CD8⁺ T cells were mainly CD107a positive and expressed three of the four cytokines (interleukin‐2‐negative). We conclude that EBV‐specific PFCs are generated in much higher proportions in the long‐term carriers than in the patients with PTLD and maintain the immunodominant characteristics of the virus.