MHC II tetramers visualize human CD4+ T cell responses to Epstein–Barr virus infection and demonstrate atypical kinetics of the nuclear antigen EBNA1 response

Virus-specific CD4+ T cells are key orchestrators of host responses to viral infection yet, compared with their CD8+ T cell counterparts, remain poorly characterized at the single cell level. Here we use nine MHC II–epitope peptide tetramers to visualize human CD4+ T cell responses to Epstein–Barr virus (EBV), the causative agent of infectious mononucleosis (IM), a disease associated with large virus-specific CD8+ T cell responses. We find that, while not approaching virus-specific CD8+ T cell expansions in magnitude, activated CD4+ T cells specific for epitopes in the latent antigen EBNA2 and four lytic cycle antigens are detected at high frequencies in acute IM blood. They then fall rapidly to values typical of life-long virus carriage where most tetramer-positive cells display conventional memory markers but some, unexpectedly, revert to a naive-like phenotype. In contrast CD4+ T cell responses to EBNA1 epitopes are greatly delayed in IM patients, in line with the well-known but hitherto unexplained delay in EBNA1 IgG antibody responses. We present evidence from an in vitro system that may explain these unusual kinetics. Unlike other EBNAs and lytic cycle proteins, EBNA1 is not naturally released from EBV-infected cells as a source of antigen for CD4+ T cell priming.