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. 2006 Apr 17;203(4):995-1006.
doi: 10.1084/jem.20051287. Epub 2006 Mar 20.

Control of Epstein-Barr virus infection in vitro by T helper cells specific for virion glycoproteins

Dinesh Adhikary  1 , Uta Behrends, Andreas Moosmann, Klaus Witter, Georg W Bornkamm, Josef Mautner
Affiliations

Control of Epstein-Barr virus infection in vitro by T helper cells specific for virion glycoproteins

Dinesh Adhikary et al. J Exp Med. .

Abstract

Epstein-Barr virus (EBV) establishes lifelong persistent infections in humans by latently infecting B cells, with occasional cycles of reactivation, virus production, and reinfection. Protective immunity against EBV is mediated by T cells, but the role of EBV-specific T helper (Th) cells is still poorly defined. Here, we study the Th response to the EBV lytic cycle proteins BLLF1 (gp350/220), BALF4 (gp110), and BZLF1 and show that glycoprotein-specific Th cells recognize EBV-positive cells directly; surprisingly, a much higher percentage of target cells than those expressing lytic cycle proteins were recognized. Antigen is efficiently transferred to bystander B cells by receptor-mediated uptake of released virions, resulting in recognition of target cells incubated with <1 virion/cell. T cell recognition does not require productive infection and occurs early after virus entry before latency is established. Glycoprotein-specific Th cells are cytolytic and inhibit proliferation of lymphoblastoid cell lines (LCL) and the outgrowth of LCL after infection of primary B cells with EBV. These results establish a novel role for glycoprotein-specific Th cells in the control of EBV infection and identify virion proteins as important immune targets. These findings have implications for the treatment of diseases associated with EBV and potentially other coated viruses infecting MHC class II-positive cells.

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Figures

Figure 1.
Figure 1.
CD4+ T cell memory to lytic cycle proteins of EBV in the peripheral blood of healthy virus carriers. 106/ml PBMCs from the latently EBV-infected donor JM were incubated in separate wells with purified recombinant BLLF1, BZLF1, and BALF4 proteins, and then used to stimulate autologous CD4+ T cells from peripheral blood. The specificity of the T cell lines was assayed by GM-CSF ELISA. After six rounds of stimulation (p6), the lines began to show reactivity to PBMCs pulsed with the stimulator protein, but not against control proteins.
Figure 2.
Figure 2.
Affinity of the T cell clones for their cognate antigen. Peptides at various concentrations were pulsed onto autologous PBMCs for 2 h at 37°C. Subsequently the cells were irradiated (40Gy), unbound peptide was removed by extensive washing, and the cells were used as stimulators for the T cells. IFN-γ secretion by the T cells was measured 24 h later by ELISA. As shown for three T cell clones, all T cells described in this work recognized their cognate peptide (closed symbols) at a minimum concentration of 1–3 nM, whereas no response against PBMCs pulsed with an irrelevant peptide (open symbols) was observed.
Figure 3.
Figure 3.
Recognition of EBV-positive target cells by lytic antigen-specific T cells. (A) Autologous LCL, allogeneic LCL sharing distinct MHC II alleles with donor JM (MA: DR8, DQ4; LA: DQ6, DP13; GB: DR13, DQ6, DP4), and MHC II–mismatched LCL (SM, DA) were cocultured with the T cell clones specific for lytic antigens for 24 h. Subsequently, IFN-γ secretion by T cells was measured by ELISA. T cells specific for the glycoproteins BALF4 and BLLF1, but not the immediate early protein BZLF1, recognized autologous and MHC II–matched LCL. Autologous PBMCs pulsed with the specific protein were used as specificity control and T cells were cultivated without target cells (T alone) were included as controls to detect nonspecific secretion of cytokines by the T cells. (B) The glycoprotein-specific T cells recognized EBV-positive BL cell lines expressing the restriction element of the T cell clones (Ag876: DP13; BL30: DR13; BL60: DQ4). BL30-B95.8 is an EBV-positive convertant of the parental, EBV-negative BL30 cell line. (C) The glycoprotein-specific T cells failed to recognize miniLCL generated by infection of B cells with an EBV mutant unable to enter the lytic cycle.
Figure 4.
Figure 4.
Antigen is transferred from lytically infected B cells to bystander B cells by virions. (A) Different numbers of autologous LCL, miniLCL, and MHC II–mismatched LCL were incubated with 105/well BLLF1-1H2 T cells. The number of target cells recognized by the T cells was determined by IFN-γ ELISPOT and are depicted as spot-forming cells (SFC). Approximately 20% of the autologous LCL were detected by the T cells, whereas miniLCL and MHC II–mismatched LCL gave background numbers of spots. (B) MiniLCL JM were cocultured with MHC II–mismatched LCL DA or miniLCL DA for 24 h and probed with lytic antigen-specific T cells. MiniLCL JM cocultured with LCL DA, but not miniLCL DA, were recognized by glycoprotein-specific, but not BZLF1-specific, T cells. MiniLCL JM incubated with the cognate antigen were included as specificity control. (C) MiniLCL JM were cocultured with allogeneic LCL or incubated with heat-inactivated purified virus (hi-EBV) supernatant for different periods of time up to 120 h. Subsequently, the cells were fixed with paraformaldehyde and probed with BLLF1- and BALF4-specific T cells. 12 h of coculture or incubation with heat-inactivated virus was sufficient for T cell recognition.
Figure 5.
Figure 5.
Efficient presentation of virion-derived antigens after receptor-mediated uptake. (A) MiniLCL JM were incubated with the anti-CD21 antibody FE8 or an isotype control (Iso1) antibody and pulsed with virus supernatant (EBV). In parallel, virus supernatant was incubated with the anti-BLLF1 antibody 72A1 or an isotype control antibody (Iso2) and pulsed onto miniLCL. After 24 h of incubation, virus-pulsed miniLCL were probed with the T cells. (B) MiniLCL, PBMCs, and dendritic cells (DC) were incubated with increasing amounts of purified virus for 24 h and probed with BLLF1-1H2 T cells. (C) The three types of APCs as in B were incubated with increasing amounts of purified BLLF1 mutant protein lacking the CD21 binding domain for 24 h and subsequently probed with the BLLF1-1H2 T cells. (D) PBMCs were separated into CD19+ and CD19− cell fractions by magnetic sorting and subsequently incubated for 24 h with increasing amounts of purified viral particles before addition of glycoprotein-specific T cells. (E) MiniLCL were pulsed for 24 h with increasing amounts of EBV geq and probed with BLLF1- and BALF4-specific T cells as indicated. (F) MiniLCL JM were incubated for 24 h with purified viral supernatant in the absence or presence of leupeptin or chloroquine. After the incubation period, unbound virus and inhibitors were removed by washing, and the cells were fixed and probed with the glycoprotein-specific T cells.
Figure 6.
Figure 6.
The amount of antigen released from lytically infected cells is insufficient for T cell recognition. MiniLCL JM (5 × 105/ml) and DC JM (5 × 105/ml) were cultured either alone or together with the MHC II–mismatched LCL DA (ratio 1:1) for 24 h. After the cocultures, the cells were probed with BLLF1-1H2 T cells and cytokine secretion determined 24 h later by ELISA. MiniLCL and DC pulsed with the cognate protein were included as controls.
Figure 7.
Figure 7.
Glycoprotein-specific CD4+ T cells are cytolytic and inhibit outgrowth and proliferation of LCL. (A) Decreasing numbers of LCL JM, miniLCL JM, or BL60 cells were either cultured alone or cocultured with BLLF1-1H2, BALF4-A9, BZLF1-3H11, or GFP-specific 3A2 T cells (10,000/well). Proliferation of the cells was followed over time and the minimum number of cells proliferating was determined after 4 wk. Coculture of LCL and BL60 cells, but not miniLCL, with glycoprotein-specific T cells increased the minimum number of cells necessary for proliferation 3–10-fold. (B) Purified B cells were infected with EBV and increasing cell numbers plated together with BLLF1-, BALF4-, BZLF1-, or irrelevant GFP-specific T cells (10,000/well). The minimum number of EBV-infected B cells required for LCL outgrowth was ∼10-fold higher in the presence of glycoprotein-specific T cells. (C) MiniLCL JM pulsed with the indicated peptides were cocultured with the BALF4-B5 T cells at different effector to target ratios. Upon antigen recognition, the glycoprotein-specific T cells efficiently lysed the target cells. (D) Serial dilutions of miniLCL JM pulsed with cognate or control peptides were cocultured with glycoprotein-specific T cells, and perforin secretion by the T cells was assayed by ELISPOT. (E) In addition to IFN-γ, all glycoprotein-specific T cell clones also secreted granzyme B in response to target cell recognition.
Figure 8.
Figure 8.
Healthy virus carriers are consistently positive for glycoprotein-specific CD4+ T cells. CD4+ T cells from peripheral blood of one EBV-seronegative (EBV−) and four EBV-seropositive (EBV+) healthy donors were repeatedly stimulated with protein-pulsed autologous PBMCs. After five to seven restimulations, the T cell lines from all healthy virus carriers, but not from the EBV-negative control donor, specifically responded against PBMCs pulsed with the protein used for stimulation.
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