Epstein‐Barr virus nuclear antigen 1 evades direct immune recognition by CD4+ T helper cells

The Epstein‐Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein regularly expressed in EBV‐associated malignancies. Immune recognition of EBNA1 by CD8+ T cells is prevented by an internal glycine‐alanine repeat (GAr) which blocks proteasomal degradation. To test whether EBV‐infected cells could be recognized by T helper cells, human CD4+ T cell clones specific for EBNA1 were isolated from latently EBV‐infected individuals. These T cells, however, failed to recognize EBV‐positive target cells. To investigate whether endogenous presentation of EBNA1 epitopes on MHC class II was prevented by the GAr domain, a mutant EBV strain with an EBNA1 lacking the GAr (EBNA1ΔGA) was generated and used to establish an Epstein‐Barr virus‐immortalized lymphoblastoid B cell line (LCL). The EBNA1ΔGA LCL were not recognized by the EBNA1‐specific T cell clones either, indicating that the GAr domain does not mediate this effect. Immune recognition could be restored by overexpression of EBNA1, for which at least 60‐fold higher levels of both EBNA1 or EBNA1ΔGAr protein were required. These results demonstrate that EBNA1 evades direct recognition by CD4+ T helper cells, since its steady state level is below the threshold required for efficient presentation on MHC class II. These findings have important implications for the design of immunotherapeutic approaches to target EBV‐positive malignancies.