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Immunol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>In immunocompromised patients, Epstein-Barr virus (EBV) infection or reactivation is associated with increased morbidity and mortality, including the development of B-cell lymphomas. The first-line treatment consists of reduction of immunosuppression and administration of rituximab (anti-CD20 antibody). Furthermore, the presence of EBV-specific T cells against latent EBV proteins is crucial for the control of EBV-associated diseases. Therefore, in addition to effective treatment strategies, appropriate monitoring of T cells of high-risk patients is of great importance for improving clinical outcome. In this study, we hypothesized that rituximab-mediated lysis of malignant EBV-infected B cells leads to the release and presentation of EBV-associated antigens and results in an augmentation of EBV-specific effector memory T-cell responses.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>EBV-infected B lymphoblastoid cell lines (B-LCLs) were used as a model for EBV-associated lymphomas, which are capable of expressing latency stage II and III EBV proteins present in all known EBV-positive malignant cells. Rituximab was administered to obtain cell lysates containing EBV antigens (AC<jats:sup>EBV<\/jats:sup>). Efficiency of cross-presentation of EBV-antigen by B-LCLs compared to cross-presentation by professional antigen presenting cells (APCs) such as dendritic cells (DCs) and B cells was investigated by <jats:italic>in vitro<\/jats:italic> T-cell immunoassays. Deep T-cell profiling of the tumor-reactive EBV-specific T cells in terms of activation, exhaustion, target cell killing, and cytokine profile was performed, assessing the expression of T-cell differentiation and activation markers as well as regulatory and cytotoxic molecules by interferon-\u03b3 (IFN-\u03b3) EliSpot assay, multicolor flow cytometry, and multiplex analyses.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>By inhibiting parts of the cross-presentation pathway, B-LCLs were shown to cross-present obtained exogenous AC<jats:sup>EBV<\/jats:sup>-derived antigens mainly through major histocompatibility complex (MHC) class I molecules. This mechanism is comparable to that for DCs and B cells and resulted in a strong EBV-specific CD8<jats:sup>+<\/jats:sup> cytotoxic T-cell response. Stimulation with AC<jats:sup>EBV<\/jats:sup>-loaded APCs also led to the activation of CD4<jats:sup>+<\/jats:sup> T helper cells, suggesting that longer peptide fragments are processed <jats:italic>via<\/jats:italic> the classical MHC class II pathway. In addition, B-LCLs were also found to be able to take up exogenous antigens from surrounding cells by endocytosis leading to induction of EBV-specific T-cell responses although to a much lesser extent than cross-presentation of AC<jats:sup>EBV<\/jats:sup>-derived antigens. Increased expression of activation markers CD25, CD71 and CD137 were detected on EBV-specific T cells stimulated with AC<jats:sup>EBV<\/jats:sup>-loaded APCs, which showed high proliferative and cytotoxic capacity as indicated by enhanced EBV-specific frequencies and increased secretion levels of cytotoxic effector molecules (e.g. IFN-\u03b3, granzyme B, perforin, and granulysin). Expression of the regulatory proteins PD-1 and Tim-3 was induced but had no negative impact on effector T-cell functions.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>In this study, we showed for the first time that rituximab-mediated lysis of EBV-infected tumor cells can efficiently boost EBV-specific endogenous effector memory T-cell responses through cross-presentation of EBV-derived antigens. This promotes the restoration of antiviral cellular immunity and presents an efficient mechanism to improve the treatment of CD20<jats:sup>+<\/jats:sup> EBV-associated malignancies. This effect is also conceivable for other therapeutic antibodies or even for therapeutically applied unmodified or genetically modified T cells, which lead to the release of tumor antigens after specific cell lysis.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2023.878953","type":"journal-article","created":{"date-parts":[[2023,3,24]],"date-time":"2023-03-24T05:20:27Z","timestamp":1679635227000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":12,"title":["Reinforcement of cell-mediated immunity driven by tumor-associated Epstein-Barr virus (EBV)-specific T cells during targeted B-cell therapy with 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