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IntroductionIn 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.MethodsEBV-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 (ACEBV). 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 in vitro 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-γ (IFN-γ) EliSpot assay, multicolor flow cytometry, and multiplex analyses.ResultsBy inhibiting parts of the cross-presentation pathway, B-LCLs were shown to cross-present obtained exogenous ACEBV-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+ cytotoxic T-cell response. Stimulation with ACEBV-loaded APCs also led to the activation of CD4+ T helper cells, suggesting that longer peptide fragments are processed via 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 ACEBV-derived antigens. Increased expression of activation markers CD25, CD71 and CD137 were detected on EBV-specific T cells stimulated with ACEBV-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-γ, 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.ConclusionIn 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+ 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.

Additional file 3. Changes on standard blood counts and the cellular immune status after continuous and interval exercise. Peripheral blood samples of healthy donors (n = 12) were analysed by flow cytometry at different time points before and after a single 30 min continuous (CONT) or interval (HIT) exercise (before, directly after, 1 h after and 24 h after exercise). Determined frequencies of CD45+ leukocytes, CD14+ monocytes, CD19+ B cells, CD3+/CD56+ NKT cells, and CD3−/CD56+ NK cells with their bright and dim subsets are displayed as normalized frequencies. Data are mean ± SD.

Additional file 4. Changes in standard blood counts after continuous and interval exercise differentiating between moderately and highly fit donors. Peripheral blood samples of healthy donors (n = 12) were analysed using a haemocytometer at different time points before and after a single 30 min continuous (CONT) or interval (HIT) exercise (before, directly after, 1 h after and 24 h after exercise). Donors were grouped according to their fitness level (moderate, n = 8, and high, n = 4, tested with the International Physical Activity Questionnaire, IPAQ). (A) Lymphocyte, granulocyte, and monocyte frequency is shown as percentage of the total leukocyte count. (B) White blood cell (WBC), lymphocyte, granulocyte, and monocyte counts are shown as absolute counts (×103/µl blood). Data are shown as mean ± SD.

Additional file 7. Effects of a single continuous and interval exercise on CD8+ T-cell cholesterol and activation levels in a donor-related setting. Peripheral blood samples of healthy donors (n = 5) were analysed at different time points before and after a single 30 min continuous (CONT) or interval (HIT) exercise (before, directly after, 1 h after and 24 h after exercise) to (A) quantify the level of cholesterol (µM)/1 × 106 cells (Amplex Red Cholesterol Assay Kit) and (B) to obtain the expression of CD69 on CD8+ T cells (flow cytometry). Data are shown in a donor-related setting as mean ± SD. Statistically significant difference is indicated by (*p

Additional file 1. Representative flow cytometric plots. Representative flow cytometry plots illustrating the gating strategy used to analyse (A) the cellular immune status in whole blood sample on a single-cell platform using TruCount tubes, (B) the enrichment efficiency of IFN-γ+ VSTs selected from PBMCs using the IFN-γ cytokine secretion assay, and (C) the activation level of isolated CD8+ T cells using CD69 as specific marker before and after exercise.

Additional file 5. Effects of a single continuous and interval exercise on stress, proliferation markers, T-cell cytotoxicity and parameters of cellular age. Peripheral blood samples of healthy donors (n = 12) were analysed at different time points before and after a single 30 min continuous (CONT) or interval (HIT) exercise (before, directly after, 1 h after and 24 h after exercise) using real-time PCR for quantification of (A) HSP70- and Ki67-, IFN-γ-, and granzyme B-mRNA levels. Constitutively expressed GAPDH gene was used as the reference standard for normalization of mRNA levels. RQ values were calculated by the delta–delta CT method. (B) Blood samples were further analysed regarding p16 levels, telomere ratio and telomere base pairs. Results are displayed as RQ, with the value from before exercise being the base line, and shown as mean ± SD.

Additional file 6. Impact of a single continuous and interval exercise on antigen-specific T-cell responses. Peripheral blood samples of healthy donors (n = 12) were analysed separately with the two-sample t approach adjusting for period at different time points before and after a single 30 min continuous (CONT) or interval (HIT) exercise (before, directly after, 1 h after and 24 h after exercise). Isolated PBMCs were stimulated overnight with CMV-, EBV- and AdV-specific peptide pools (CMV pp65, CMV IE1, EBV EBNA1, and EBV Consensus, AdV5 Hexon, and AdV5 Penton) and frequencies of functional-active virus-specific T cells were determined by IFN-γ EliSpot assay as spots per 1000 CD3+ T cells

International audience; A functional Shaker potassium channel requires assembly of four α-subunits encoded by a single gene or various genes from the Shaker family. In Arabidopsis thaliana, AtKC1, a Shaker α-subunit that is silent when expressed alone, has been shown to regulate the activity of AKT1 by forming heteromeric AtKC1-AKT1 channels. Here, we investigated whether AtKC1 is a general regulator of channel activity. Co-expression in Xenopus oocytes of a dominant negative (pore-mutated) AtKC1 subunit with the inward Shaker channel subunits KAT1, KAT2 or AKT2, or the outward subunits SKOR or GORK, revealed that the three inward subunits functionally interact with AtKC1 while the outward ones cannot. Localization experiments in plant protoplasts showed that KAT2 was able to re-locate AtKC1 fused to GFP from endomembranes to the plasma membrane, indicating that heteromeric AtKC1-KAT2 channels are efficiently targeted to the plasma membrane. Functional properties of heteromeric channels involving AtKC1 and KAT1, KAT2 or AKT2 were analysed by voltage clamp after co-expression of the respective subunits in Xenopus oocytes. AtKC1 behaved as a regulatory subunit within the heterotetrameric channel, reducing the macroscopic conductance and negatively shifting the channel activation potential. Expression studies showed that AtKC1 and its identified Shaker partners have overlapping expression patterns, supporting the hypothesis of a general regulation of inward channel activity by AtKC1 in planta. Lastly, AtKC1 disruption appeared to reduce plant biomass production, showing that AtKC1-mediated channel activity regulation is required for normal plant growth.

Ectomycorrhizal fungi play an essential role in the ecology of boreal and temperate forests through the improvement of tree mineral nutrition. Potassium (K+ ) is an essential nutrient for plants and is needed in high amounts. We recently demonstrated that the ectomycorrhizal fungus Hebeloma cylindrosporum improves the K+ nutrition of Pinus pinaster under shortage conditions. Part of the transport systems involved in K+ uptake by the fungus has been deciphered, while the molecular players responsible for the transfer of this cation towards the plant remain totally unknown. Analysis of the genome of H. cylindrosporum revealed the presence of three putative tandem-pore outward-rectifying K+ (TOK) channels that could contribute to this transfer. Here, we report the functional characterization of these three channels through two-electrode voltage-clamp experiments in oocytes and yeast complementation assays. The expression pattern and physiological role of these channels were analysed in symbiotic interaction with P. pinaster. Pine seedlings colonized by fungal transformants overexpressing two of them displayed a larger accumulation of K+ in shoots. This study revealed that TOK channels have distinctive properties and functions in axenic and symbiotic conditions and suggested that HcTOK2.2 is implicated in the symbiotic transfer of K+ from the fungus towards the plant.