Adoptive transfer of EBV specific CD8+ T cell clones can transiently control EBV infection in humanized mice

Epstein Barr virus (EBV) infection expands CD8+ T cells specific for lytic antigens to high frequencies during symptomatic primary infection, and maintains these at significant numbers during persistence. Despite this, the protective function of these lytic EBV antigen-specific cytotoxic CD8+ T cells remains unclear. Here we demonstrate that lytic EBV replication does not significantly contribute to virus-induced B cell proliferation in vitro and in vivo in a mouse model with reconstituted human immune system components (huNSG mice). However, we report a trend to reduction of EBV-induced lymphoproliferation outside of lymphoid organs upon diminished lytic replication. Moreover, we could demonstrate that CD8+ T cells against the lytic EBV antigen BMLF1 can eliminate lytically replicating EBV-transformed B cells from lymphoblastoid cell lines (LCLs) and in vivo, thereby transiently controlling high viremia after adoptive transfer into EBV infected huNSG mice. These findings suggest a protective function for lytic EBV antigen-specific CD8+ T cells against EBV infection and against virus-associated tumors in extra-lymphoid organs. These specificities should be explored for EBV-specific vaccine development

Membrane transfer from tumor cells overcomes deficient phagocytic ability of plasmacytoid dendritic cells for the acquisition and presentation of tumor antigens

The potential contribution of plasmacytoid dendritic cells (pDCs) in the presentation of tumor cell Ags remains unclear, and some controversies exist with regard to the ability of pDCs to phagocytose cell-derived particulate Ags and cross-present them to MHC class I-restricted T lymphocytes. In this study, we show that human pDCs, although inefficient in the internalization of cell membrane fragments by phagocytosis, can efficiently acquire membrane patches and associated molecules from cancer cells of different histotypes. The transfer of membrane patches to pDCs occurred in a very short time and required cell-to-cell contact. Membrane transfer also included intact HLA complexes, and the acquired Ags could be efficiently recognized on pDCs by tumor-specific CD8(+) T cells. Remarkably, pDCs isolated from human colon cancer tissues displayed a strong surface expression of epithelial cell adhesion molecule, indicating that the exchange of exogenous Ags between pDCs and tumor cells also can occur in vivo. These data demonstrate that pDCs are well suited to acquire membrane patches from contiguous tumor cells by a cell-to-cell contact-dependent mechanism that closely resembles "trogocytosis." This phenomenon may allow pDCs to proficiently present tumor cell-derived Ags, despite limited properties of endophagocytosis

CD8+ T cells retain protective functions despite sustained inhibitory receptor expression during Epstein-Barr virus infection in vivo.

Epstein Barr virus (EBV) is one of the most ubiquitous human pathogens in the world, persistently infecting more than 90% of the adult human population. It drives some of the strongest human CD8+ T cell responses, which can be observed during symptomatic primary infection known as infectious mononucleosis (IM). Despite high viral loads and prolonged CD8+ T cell stimulation during IM, EBV enters latency and is under lifelong immune control in most individuals that experience this disease. We investigated whether changes in T cell function, as frequently characterized by PD-1 up-regulation, occur during IM due to the prolonged exposure to high antigen levels. We readily detected the expansion of PD-1 positive CD8+ T cells together with high frequencies of Tim-3, 2B4, and KLRG1 expression during IM and in mice with reconstituted human immune system components (huNSG mice) that had been infected with a high dose of EBV. These PD-1 positive CD8+ T cells, however, retained proliferation, cytokine production, and cytotoxic abilities. Multiple subsets of CD8+ T cells expanded during EBV infection, including PD-1+Tim-3+KLRG1+ cells that express CXCR5 and TCF-1 germinal center homing and memory markers, and may also contain BATF3. Moreover, blocking the PD-1 axis compromised EBV specific immune control and resulted in virus-associated lymphomagenesis. Finally, PD-1+, Tim-3+, and KLRG1+ CD8+ T cell expansion coincided with declining viral loads during low dose EBV infection. These findings suggest that EBV infection primes PD-1 positive CD8+ T cell populations that rely on this receptor axis for the efficient immune control of this ubiquitous human tumor virus

Lytic EBV antigen specific CD8⁺ T cell clones can transiently control EBV infection in humanized mice.

<p>(<b>A</b>) Schematic diagram of adoptive transfer experiments. BMLF1- or LMP2-specific CD8⁺ T cell clones were transferred i.v. into humanized NSG-A2tg mice. The next day, mice were infected i.p. with WT EBV, ZKO EBV or PBS. Mice were bled weekly starting at week 2 post-infection and were euthanized at week 4 or 6 post-infection (n = 94). (<b>B</b>) Whole blood EBV loads for the second, third and fourth week post-infection of mice, which received BMLF1- or LMP2-specific CD8⁺ T cell clones prior to WT or ZKO EBV infection, is demonstrated for a representative experiment (cohort 2, n = 3–4 per group, total n = 21). Dotted line represents detection limit. (<b>C</b>) Representative figures and quantification of the ZEBRA⁺ cells in the spleen sections of WT EBV infected huNSG-A2tg mice, which received no T cells, BMLF1-specific T cell clones and LMP2-specific T cell clones for two initial experiments (n = 6–7 per group, total n = 20). Data represent the mean ± SEM (p _{no T vs BMLF1-T} <0.01, p _{BMLF1-T vs LMP2-T} = 0.07). Scale bars, 100 µm. (<b>D</b>) Whole blood EBV loads for the second, third and fourth week post-infection of mice from five independent adoptive transfer experiments (n = 94). Data are represented as the mean ± SEM. (<b>E</b>) Incidence of high blood viremia on the third week post-infection in the WT EBV infected mice, which received no T cells, BMLF1-specific T cell clone and LMP2-specific T cell clone (n.s. by one-way Chi-square). (<b>F</b>) Tumor incidence per organ is presented as the percentage of animals which developed visible tumors in the organs of the abdominal cavity (n = 92).</p

Lytic replication does not significantly contribute to EBV transformation <i>in vitro</i>.

<p>Bulk PBMCs were inoculated with WT EBV, ZKO EBV or mock treated in the presence of cyclosporine A. (<b>A</b>) Cells were harvested and absolute cell numbers per well were determined on indicated days. (<b>B</b>) Outgrowth of transformed CD19⁺ B cells was monitored using flow cytometry. (<b>C</b>) Proliferation of EBV transformed B cells was demonstrated by CFSE dilution and the subsequent increase in percentage of CFSE^low B cells. The experiments were performed in duplicate and data is presented as the mean ± SEM. Similar results were obtained in samples from two donors; the average of two experiments is presented (p _{mock vs WT/ZKO} <0.0001 by two-way ANOVA).</p