Dendritic cells initiate immune control of epstein-barr virus transformation of B lymphocytes in vitro

Abstract

The initiation of cell-mediated immunity to Epstein-Barr virus (EBV) has been analyzed with cells from EBV-seronegative blood donors in culture. The addition of dendritic cells (DCs) is essential to prime naive T cells that recognize EBV-latent antigens in enzyme-linked immunospot assays for interferon gamma secretion and eradicate transformed B cells in regression assays. In contrast, DCs are not required to control the outgrowth of EBV-transformed B lymphocytes from seropositive donors. Enriched CD4+ and CD8+ T cells mediate regression of EBV-transformed cells in seronegative and seropositive donors, but the kinetics of T-dependent regression occurs with much greater speed with seropositives. EBV infection of DCs cannot be detected by reverse transcription-polymerase chain reaction with primers specific for mRNA for the EBNA1 U and K exons. Instead, DCs capture B cell debris and generate T cells specific for EBV latency antigens. We suggest that the cross-presentation of EBV-latent antigens from infected B cells by DCs is required for the initiation of EBV-specific immune control in vivo and that future EBV vaccine strategies should target viral antigens to DCs.

Figure 1.

DCs are required for priming naive T cells to EBV antigens. T cells and EBV-infected B cells from EBV-seronegative (A–D) and -seropositive (E and F) donors were cultured in the presence (B + DC + T) or the absence (B + T) of mature DCs for 12–14 d. Results shown have the vector control, vvTK⁻, subtracted from the specific response, where vvTK⁻ responses were always <20 spots. A response was considered meaningful if it was at least twice that of the negative control (vvTK⁻) as well as 10 spots greater than the vvTK⁻ background and is indicated with an asterisk. (G) The seronegative B + DC + T cultures were also tested against autologous LCLs and the EBV-negative HD cell line HD-MY-Z by IFNγ ELISPOT. (H) Two sources of EBNA1 were compared for loading of DCs, recombinant vaccinia viruses (vv) leading to the expression of Gly-Ala–deficient EBNA1 (vvE1) or recombinant EBNA1 protein (rE1). DCs loaded with rPCNA (rP) or infected with vvTK⁻ were used as controls. The responses in A, G (donor 3), and H (donor 2) are representative of four experiments with the same seronegative donor.

Figure 2.

T cells primed by DCs in vitro control LCL outgrowth. T cells and EBV-infected B cells from EBV-seronegative (A–C) and -seropositive (D and E) donors were cocultured in the presence (B + DC + T) or absence (B + T) of mature DCs. After 18 d, cultures were collected and examined for outgrowth of LCLs as demonstrated by CD23⁺ and CD19⁺ double-positive cells quantified by FACS^® analysis. Cocultures of B cells and DCs alone (B + DC) were used as a control for B cell transformation. Gates were set on transformed B cells by forward and side scatter and to exclude autofluorescing cells in FL-3.

Figure 3.

EBV-transformed B cell regression is sensitive to cyclosporin A (CSA). EBV-infected B cells were cultured with autologous T cells from EBV-seropositive (top row) or -seronegative donors (bottom row) without (B + T) or with DCs (B + DC + T). To some of the cultures CSA was added to suppress T cell reactivity. CD19⁺CD23⁺ cells were evaluated by FACS^® staining 21 d after EBV infection and start of the coculture.

Figure 4.

Kinetics of EBV-mediated B cell transformation in culture. EBV-infected B cells were cultured with autologous T cells from EBV-seropositive (top two rows) or -seronegative donors (bottom two rows) without (B + T) or with DCs (B + DC + T). Aliquots of the cultures were taken at the indicated time points, and CD19⁺CD23⁺ cells were quantified by FACS^®.

Figure 5.

CD4⁺ and CD8⁺ T cells contribute to LCL regression. EBV-infected B cells and DCs were cultured with either CD4⁺ T cells (B + DC + CD4), CD8⁺ T cells (B + DC + CD8), or a mixture of both (B + DC + CD4 + CD8) from three EBV-seronegative donors (A–C). Cultures were analyzed by FACS^® after 18 d to identify LCLs as CD19⁺/CD23⁺ double-positive cells representing LCL outgrowth. Gates were set on transformed B cells by forward and side scatter and to exclude autofluorescing cells in FL-3.

Figure 6.

DCs phagocytize B cell fragments in the presence or absence of EBV infection. CD19⁺ cells were incubated overnight in the supernatant from the B95-8 EBV⁺ (+ virus) and Ramos EBV⁻ (− virus) cell lines. CD19⁺ cells were stained with PKH26 and added to autologous mature DCs at a ratio of 1:1. After the specified time periods at the indicated temperatures, cultures were collected and stained for CD11c and analyzed by FACS^® (A) and deconvolution microscopy (B). For FACS^® analysis, gates were set on large cells (forward scatter) and there was exclusion of autofluorescing cells in FL-3.

Figure 7.

DCs exposed to EBV do not express detectable levels of EBV latency antigens. CD19⁺ cells and mature DCs were cultured for 14 d in supernatant from the EBV⁻ cell line, Ramos (Mock), or in B95-8 supernatant. RT-PCR was performed on mRNA derived from the cells using primers spanning the U–K exon splice site of the mRNA for the EBNA1 latency protein (A). Primer pairs for G3PDH were used as a positive control for the reaction. PCR products were run on a 1% agarose gel. (B) Mature DCs were incubated for 24 h in media containing concentrated EBV viral stock (DC) or 1:2 with B cells infected with EBV for 24 h before DC/B coculture (B + DC). DCs were positively selected using anti-CD11c antibodies and cultured for 12 d with T cells from an EBV-seronegative (B, left) or -seropositive donor (B, right). DCs, infected with vaccinia vectors specific for the indicated EBV latency antigens, or EBV⁻ Ramos cells (negative control) and autologous LCLs were used to restimulate antigen specific cells in IFNγ ELISPOT assays. Results shown are representative of two EBV-seronegative donors (left) and four seropositive donors (right). Results shown have the vector control, vvTK⁻, subtracted from the specific response, and positive responses (*) were determined as in Fig. 1.