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

Bickham K, Goodman K, Paludan C, Nikiforow S, Tsang ML, Steinman RM, Münz C - J. Exp. Med. (2003)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021, USA.

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.

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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®.
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fig4: 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®.

Mentions: To evaluate the hypothesis that DCs, rather than EBV-infected B cells, are required to prime virus-specific T cells, we set up cocultures of T cells with EBV-infected or uninfected B cells (8) from four EBV-seronegative donors in the presence (B + DC + T) or absence (B + T) of mature DCs. After 12 d, the cultures were tested by ELISPOT for responses to a panel of EBV latency antigens (EBNA1, 2, 3A-C, LMP1, 2A) and the lytic antigen, BMLF1. Day 12 was chosen because keyhole limpet hemocyanin–pulsed DCs were capable of priming specific CD4+ T cell responses during this time period (32) and as shown in Fig. 4, this is the time when regression of EBV transformation is evident. To optimize ELISPOT assays, we used DCs infected with the respective recombinant vv as APCs. In all four EBV-seronegative donors, priming of T cells occurred only in cultures that contained DCs (Fig. 1, A–D) . Reactive T cells were of the Th1 type as demonstrated by secretion of IFNγ (Fig. 1, A–D) and no IL-4 (not depicted). The pattern of latency antigen recognition was similar to that seen in healthy carriers of EBV (i.e., reliable recognition of EBNA1 and EBNA3s but minimal recognition of LMP2A; reference 1). In all four donors, no reactivity to any EBV latency antigens was seen on day 0 or in the control cocultures of DCs with T cells and uninfected B cells (unpublished data). In the one seronegative donor from our laboratory, responses were similar in magnitude in four experiments (Fig. 1, A, G [donor 3], and H [donor 2]). The addition of CD14+ monocytes did not result in EBV-latent antigen-specific T cell priming (Fig. S1 B, available at http://www.jem.org/cgi/content/full/jem.20030646/DC1), but populations of immature DCs or mature DCs each could prime autologous LCL-specific T cells (Fig. S1 B). In contrast to the findings with cells from seronegative donors, DCs were not essential to expand EBV-specific T cells from seropositive donors beyond that seen with infected B and T cells (Fig. 1, E and F). However, DCs induced the expansion and/or survival of T cells for a broader panel of EBV latency antigens than B and T cell mixtures alone. The EBV-specific T cells again demonstrated a polarized Th1 phenotype with secretion of IFNγ and no IL-4 (unpublished data).


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

Bickham K, Goodman K, Paludan C, Nikiforow S, Tsang ML, Steinman RM, Münz C - J. Exp. Med. (2003)

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®.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2194129&req=5

fig4: 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®.
Mentions: To evaluate the hypothesis that DCs, rather than EBV-infected B cells, are required to prime virus-specific T cells, we set up cocultures of T cells with EBV-infected or uninfected B cells (8) from four EBV-seronegative donors in the presence (B + DC + T) or absence (B + T) of mature DCs. After 12 d, the cultures were tested by ELISPOT for responses to a panel of EBV latency antigens (EBNA1, 2, 3A-C, LMP1, 2A) and the lytic antigen, BMLF1. Day 12 was chosen because keyhole limpet hemocyanin–pulsed DCs were capable of priming specific CD4+ T cell responses during this time period (32) and as shown in Fig. 4, this is the time when regression of EBV transformation is evident. To optimize ELISPOT assays, we used DCs infected with the respective recombinant vv as APCs. In all four EBV-seronegative donors, priming of T cells occurred only in cultures that contained DCs (Fig. 1, A–D) . Reactive T cells were of the Th1 type as demonstrated by secretion of IFNγ (Fig. 1, A–D) and no IL-4 (not depicted). The pattern of latency antigen recognition was similar to that seen in healthy carriers of EBV (i.e., reliable recognition of EBNA1 and EBNA3s but minimal recognition of LMP2A; reference 1). In all four donors, no reactivity to any EBV latency antigens was seen on day 0 or in the control cocultures of DCs with T cells and uninfected B cells (unpublished data). In the one seronegative donor from our laboratory, responses were similar in magnitude in four experiments (Fig. 1, A, G [donor 3], and H [donor 2]). The addition of CD14+ monocytes did not result in EBV-latent antigen-specific T cell priming (Fig. S1 B, available at http://www.jem.org/cgi/content/full/jem.20030646/DC1), but populations of immature DCs or mature DCs each could prime autologous LCL-specific T cells (Fig. S1 B). In contrast to the findings with cells from seronegative donors, DCs were not essential to expand EBV-specific T cells from seropositive donors beyond that seen with infected B and T cells (Fig. 1, E and F). However, DCs induced the expansion and/or survival of T cells for a broader panel of EBV latency antigens than B and T cell mixtures alone. The EBV-specific T cells again demonstrated a polarized Th1 phenotype with secretion of IFNγ and no IL-4 (unpublished data).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021, USA.

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.

Show MeSH
Related in: MedlinePlus