Regression of Epstein-Barr Virus-Induced B-Cell Transformation In Vitro Involves Virus-Specific CD8⁺T Cells as the Principal Effectors and a Novel CD4⁺T-Cell Reactivity

Abstract

T-cell memory to Epstein-Barr virus (EBV) was first demonstrated through regression of EBV-induced B-cell transformation to lymphoblastoid cell lines (LCLs) in virus-infected peripheral blood mononuclear cell (PBMC)cultures. Here, using donors with virus-specific T-cell memory to well-defined CD4 and CD8 epitopes, we reexamine recent reports that the effector cells mediating regression are EBV latent antigen-specific CD4 ؉ and not, as previously assumed, CD8؉ T cells. In regressing cultures, we find that the reversal of CD23 ؉ B-cell proliferation was always coincident with an expansion of latent epitope-specific CD8؉ , but not CD4 ؉ , T cells; furthermore CD8؉ T-cell clones derived from regressing cultures were epitope specific and reproduced regression when cocultivated with EBV-infected autologous B cells. In cultures of CD4-depleted PBMCs, there was less efficient expansion of these epitope-specific CD8 ؉ T cells and correspondingly weaker regression. The data are consistent with an effector role for epitope-specific CD8 ؉ T cells in regression and an auxiliary role for CD4؉ T cells in expanding the CD8 response. However, we also occasionally observed late regression in CD8-depleted PBMC cultures, though again without any detectable expansion of preexisting epitope-specific CD4 ؉ T-cell memory. CD4 ؉ T-cell clones derived from such cultures were LCL specific in gamma interferon release assays but did not recognize any known EBV latent cycle protein or derived peptide. A subset of these clones was also cytolytic and could block LCL outgrowth. These novel effectors, whose antigen specificity remains to be determined, may also play a role in limiting virus-induced B-cell proliferation in vitro and in vivo.Epstein-Barr virus (EBV) is a human B lymphotropic herpesvirus that has cell growth transforming ability in vitro yet is carried by the great majority of immunocompetent individuals as a lifelong asymptomatic infection. The importance of T-cellmediated immune responses in maintaining asymptomatic persistence is emphasized by clinical observation. Thus, patients who are severely T-cell immunosuppressed either iatrogenically or through progressive human immunodeficiency virus (HIV) infection are at risk of developing fatal EBV-associated lymphoproliferative lesions (11,32,37). These lesions are largely composed of EBV-transformed B cells expressing the same panel of latent cycle proteins (the nuclear antigens EBNAs 1, 2, 3A, 3B, 3C, and LP and the latent membrane proteins [LMPs] 1 and 2), as do the lymphoblastoid cell lines (LCLs) that arise by EBV-induced transformation of normal resting B cells in vitro (8,12,54).The ready availability of such latently infected LCLs provided stimulator cells which, when cocultured at particular ratios with autologous peripheral blood mononuclear cells (PBMCs), were able to reactivate T-cell memory and generate EBV latent antigen-specific, interleukin 2 (IL-2)-dependent, T-cell lines (39, 51). These lines, which kill the autologous LCL in short-term cytotoxicity assays, tend to...

CD4+ T-cell clones recognizing human lymphoma-associated antigens: generation by in vitro stimulation with autologous Epstein-Barr virus–transformed B cells

“…When specified, PBMCs were depleted of CD56 ϩ natural killer (NK) cells and either CD8 ϩ or CD4 ϩ T cells with the use of magnetic-activated cell sorting beads (Miltenyi) or Dynabeads (Dynal) as described. 20 …”

“…All lines were cultured in standard medium with 8% fetal calf serum (FCS) or 5% HuS as specified. B lymphoblasts (B blasts) were generated by culturing PBMCs on CD40 ligand-expressing mouse L-cells in medium supplemented with interleukin-4 (IL-4), cyclosporin A, and either HuS or FCS as described 20,25 and maintained by twice-weekly transfer onto fresh L-cells. T lymphoblasts (T blasts) were generated from PBMCs cultured in 1 g/mL phytohemagglutinin (PHA) and expanded in IL-2-supplemented medium.…”

“…PBMCs, depleted of CD56 ϩ NK and either CD4 ϩ or CD8 ϩ T cells as described, 20 were tested in enzyme-linked immunospot (ELISpot) assays of IFN␥ release 22 at 40:1 responder-to-stimulator ratio against B95.8 or BZ k/o LCLs grown for at least 1 month in HuS. Spot-forming cells were counted with the use of an automated AID ELISpot reader (Autoimmun Diagnostika).…”

“…22,23 Second, clonal analysis of T cells harvested from EBV-infected PBMC cultures has identified CD4 ϩ T cells that recognize EBV-transformed but not mitogen-activated B cells yet appear not to map to any of the known EBV-encoded target epitopes. 20 However, because such reactivities were detected in cultures from 2 EBV-immune donors and not from the 1 EBV-naive donor tested, their target specificity remained an open question.…”

“…Two earlier findings cautioned against the assumption that such effectors are necessarily EBV specific. First, although EBVimmune persons possess CD4 ϩ T-cell memory to several defined EBV latent protein epitopes, LCL stimulation is a relatively inefficient means of reactivating these responses in vitro 20,21 because many such epitopes are poorly displayed on the LCL surface. 22,23 Second, clonal analysis of T cells harvested from EBV-infected PBMC cultures has identified CD4 ϩ T cells that recognize EBV-transformed but not mitogen-activated B cells yet appear not to map to any of the known EBV-encoded target epitopes.…”