Investigation of the T helper cell response against Epstein-Barr virus

The Epstein-Barr virus (EBV) is associated with a number of human
malignancies. Following primary infection, the virus persists
lifelong in the infected host by latently infecting B cells and
occasional cycles of reactivation, virus production and
re-infection. Adoptively transferred EBV-specific T cells,
generated by repeated stimulation with autologous lymphoblastoid
cell lines (LCL) in vitro, are able to cure post-transplant
lymphoproliferative disease (PTLD). However, the generation of
these vaccines is labor and cost intensive precluding their general
availability for all patients at risk. Novel insights into the
mechanisms of protective antiviral immunity is expected to provide
a better understanding of the pathogenesis of EBV-associated
diseases and to facilitate the development of novel and generally
available immunotherapeutic options. The aim of this work was to
assess specificity and breadth of the EBV-specific T helper cell
response, using two different experimental strategies. To define
specificity, LCL-stimulated CD4+ T cell lines were established from
23 EBV-negative and -positive donors. The T cell lines generated
from EBV-negative donors responded poorly against LCL and failed to
show EBV-specificity. By contrast, all T cell lines established
from healthy virus carriers were EBV-specific. Half of the lines
from acutely EBV-infected patients with infectious mononucleosis
(IM) were also EBV-specific, while the other half recognized
EBV-positive and EBV-negative target cells. Unexpectedly, the
EBV-specific T cell lines did not recognize latent antigens of EBV
expressed in all LCL. Instead, these lines were specific for lytic
cycle antigens predominantly derived from virion proteins. Several
of the T cell lines recognized BNRF1, a viral tegument protein.
Most T cell lines, however, recognized different virion antigens,
suggesting that the family of virion proteins forms the
immunodominant targets of the EBV-specific T helper cell response.
Studies on the breadth of the EBV-specific T helper response
demonstrated that all healthy virus carriers maintain CD4+ T cell
memory to lytic cycle antigens. T cells specific for virion
antigens recognized EBV-positive cells directly and, surprisingly,
a much higher percentage of target cells than those expressing
lytic cycle proteins. Antigen was efficiently transferred to
bystander B cells by receptor-mediated uptake of released virions,
resulting in recognition of target cells incubated with less than
one virion per cell. T cell recognition did not require productive
infection and occurred early after virus entry before latency was
established. By secreting perforin and granzyme B upon antigen
recognition, virion-specific T helper cells inhibited proliferation
of LCLs and suppressed the outgrowth of LCLs following infection of
primary B cells with EBV. These results established a novel role
for virion-specific T helper cells in the control of EBV infection,
and identify virion proteins as important immune targets. The
findings have implications for the treatment of diseases associated
with EBV and potentially other coated viruses infecting MHC class
II-positive cells.