Short-term inhibition of TERT induces telomere length-independent cell cycle arrest and apoptotic response in EBV-immortalized and transformed B cells

open7siBesides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumorigenesis through extra-telomeric functions. The possible therapeutic effects of BIBR1532 (BIBR), a powerful TERT inhibitor, have been evaluated in different cellular backgrounds, but no data are currently available regarding Epstein-Barr virus (EBV)-driven B-cell malignancies. Our aim was to characterize the biological effects of TERT inhibition by BIBR on EBV-immortalized lymphoblastoid cell lines (LCLs) and fully transformed Burkitt's lymphoma (BL) cell lines. We found that BIBR selectively inhibits telomerase activity in TERT-positive 4134/Late and 4134/TERT+ LCLs and EBV-negative BL41 and EBV-positive BL41/B95.8 BL cell lines. TERT inhibition led to decreased cell proliferation, accumulation of cells in the S-phase and ultimately to increased apoptosis, compared with mock-treated control cells. All these effects occurred within 72 h and were not observed in BIBR-treated TERT-negative 4134/TERT- and U2OS cells. The cell cycle arrest and apoptosis, consequent upon short-term TERT inhibition, were associated with and likely dependent on the activation of the DNA damage response (DDR), highlighted by the increased levels of γH2AX and activation of ATM and ATR pathways. Analyses of the mean and range of telomere lengths and telomere dysfunction-induced foci indicated that DDR after short-term TERT inhibition was not related to telomere dysfunction, thus suggesting that TERT, besides stabilizing telomere, may protect DNA via telomere-independent mechanisms. Notably, TERT-positive LCLs treated with BIBR in combination with fludarabine or cyclophosphamide showed a significant increase in the number of apoptotic cells with respect to those treated with chemotherapeutic agents alone. In conclusion, TERT inhibition impairs cell cycle progression and enhances the pro-apoptotic effects of chemotherapeutic agents in TERT-positive cells. These results support new therapeutic applications of TERT inhibitors in EBV-driven B-cell malignancies.openCeleghin, Andrea; Giunco, Silvia; Freguja, Riccardo; Zangrossi, Manuela; Nalio, Silvia; Dolcetti, Riccardo; De Rossi, AnitaCeleghin, Andrea; Giunco, Silvia; Freguja, Riccardo; Zangrossi, Manuela; Nalio, Silvia; Dolcetti, Riccardo; DE ROSSI, Anit

Impact of γ-chain cytokines on EBV-specific T cell cultures

<p>Abstract</p> <p>Background</p> <p>Recent preclinical adoptive immunotherapy studies in murine models prompt to employ "proper" rather than "as many as possible" antigen-specific T cells to gain better therapeutic results. Ideally, "proper" T cells are poorly differentiated <it>in vitro</it>, but retain the capacity to fully differentiate into effector cells <it>in vivo</it>, where they can undergo long-term survival and strong proliferation. Such requirements can be achieved by modifying culture conditions, namely using less "differentiating" cytokines than IL-2.</p> <p>Methods</p> <p>To evaluate this issue in human T cell cultures, we exploited a well characterized and clinical-grade protocol finalized at generating EBV-specific CTL for adoptive immunotherapy. In particular, we studied the impact of IL-7, IL-15 and IL-21 compared to IL-2 on different aspects of T cell functionality, namely growth kinetics, differentiation/activation marker expression, cytokine production, and short-term and long-term cytotoxicity.</p> <p>Results</p> <p>Results disclosed that the culture modifications we introduced in the standard protocol did not improve activity nor induce substantial changes in differentiation marker expression of EBV-specific CTL.</p> <p>Conclusions</p> <p>Our data indicated that the addition of γ-chain cytokines other than IL-2 for the generation of EBV-specific T cell cultures did not produce the improvements expected on the basis of recent published literature. This fact was likely due to the intrinsic differences between murine and human models and highlights the need to design <it>ad hoc </it>protocols rather than simply modify the cytokines added in culture.</p

Multiparametric flow cytometry to characterize vaccine-induced polyfunctional T cell responses and T cell/NK cell exhaustion and memory phenotypes in mouse immuno-oncology models

Suitable methods to assess in vivo immunogenicity and therapeutic efficacy of cancer vaccines in preclinical cancer models are critical to overcome current limitations of cancer vaccines and enhance the clinical applicability of this promising immunotherapeutic strategy. In particular, availability of methods allowing the characterization of T cell responses to endogenous tumor antigens is required to assess vaccine potency and improve the antigen formulation. Moreover, multiparametric assays to deeply characterize tumor-induced and therapy-induced immune modulation are relevant to design mechanism-based combination immunotherapies. Here we describe a versatile multiparametric flow cytometry method to assess the polyfunctionality of tumor antigen-specific CD4+ and CD8+ T cell responses based on their production of multiple cytokines after short-term ex vivo restimulation with relevant tumor epitopes of the most common mouse strains. We also report the development and application of two 21-color flow cytometry panels allowing a comprehensive characterization of T cell and natural killer cell exhaustion and memory phenotypes in mice with a particular focus on preclinical cancer models

Exploiting the Interplay between Innate and Adaptive Immunity to Improve Immunotherapeutic Strategies for Epstein-Barr-Virus-Driven Disorders

The recent demonstration that immunotherapeutic approaches may be clinically effective for cancer patients has renewed the interest for this strategy of intervention. In particular, clinical trials using adoptive T-cell therapies disclosed encouraging results, particularly in the context of Epstein-Barr-virus- (EBV-) related tumors. Nevertheless, the rate of complete clinical responses is still limited, thus stimulating the development of more effective therapeutic protocols. Considering the relevance of innate immunity in controlling both infections and cancers, innovative immunotherapeutic approaches should take into account also this compartment to improve clinical efficacy. Evidence accumulated so far indicates that innate immunity effectors, particularly NK cells, can be exploited with therapeutic purposes and new targets have been recently identified. We herein review the complex interactions between EBV and innate immunity and summarize the therapeutic strategies involving both adaptive and innate immune system, in the light of a fruitful integration between these immunotherapeutic modalities for a better control of EBV-driven tumors

Reverse immunoediting: When immunity is edited by antigen

Immune selective pressure occurring during cancer immunoediting shapes tumor features revealed at clinical presentation. However, in the "Escape" phase, the tumor itself has the chance to influence the immunological response. Therefore, the capacity of the immune response to sculpt the tumor characteristics is only one side of the coin and even the opposite is likely true, i.e. that an antigen can shape the immune response in a sort of "reverse immunoediting". This reciprocal modeling probably occurs continuously, whenever the immune system encounters a tumor/foreign antigen, and can be operative in the pathogen/immune system interplay, thus possibly permeating the protective immunity as a whole. In line with this view, the characterization of a T cell response as well as the design of both active and passive immunotherapy strategies should also take into account all Ag features (type, load and presentation). Overall, we suggest that the "reverse immunoediting" hypothesis could help to dissect the complex interplay between antigens and the immune repertoire, and to improve the outcome of immunotherapeutic approaches, where T cell responses are manipulated and reprogrammed

Rituximab in patients with mucosal-associated lymphoid tissue-type lymphoma of the ocular adnexa

Eight patients with ocular adnexal mucosal-associated lymphpid tissue (MALT) lymphoma were treated with rituximab, at diagnosis (n=5) or relapse (n=3). All untreated patients achieved lymphoma regression, while relapsing patients had no benefit. Four responding patients experienced early relapse. The median time to progression was 5 months. The efficacy of rituximab in ocular adnexal lymphoma is lower than that reported for gastric MALT lymphomas

Multiple HLA-All-restricted cytotoxic T-lymphocyte epitopes of different immunogenicities in the EpsteinBarr virus-encoded nuclear antigen 4

Epstein-Barr virus (EBV), a ubiquitous herpesvirus, induces potent HLA class I-restricted cytotoxic T-lymphocyte (CTL) responses. Analyses of target antigen choice have shown that the very strong CTL responses which are often observed through the HLA All allele map are due almost entirely to a single transformation-associated EBV protein, the nuclear antigen EBNA4. Here, we sought to determine the number and relative immunogenicities of HLA All-restricted epitopes within this 938-amino-acid protein. An initial screening with a series of recombinant vaccinia virus vectors encoding progressively truncated forms of EBNA4 was followed by peptide sensitization experiments using overlapping 14-or 15-mers from the entire sequence. These two approaches allowed the identification of five epitope regions located between residues 101 and 115, 416 and 429, 396 and 410, 481 and 495, and 551 and 564 of the EBNA4 molecule. CTL preparations from all seven HLA All-positive donors tested had demonstrable reactivities against the 416-to-429 peptide, whereas reactivities against the other epitopes either tended to be lost on serial passage or, for some of the donors, were never detected. The immunodominance of the 416-to-429 epitope was further supported by peptide dilution assays using polyclonal effectors and by CTL cloning experiments. Analysis of the 416-to-429 region identified the nanomer 416-424 (IVTDFSVIK) as the cognate peptide. This peptide was able to sensitize targets to lysis by All-restricted CTL clones at concentrations as low as 5 x 10-14 M. Epstein-Barr virus (EBV) is a widespread lymphotropic herpesvirus which causes infectious mononucleosis and is strongly linked to at least three lymphoid malignancies: endemic Burkitt&apos;s lymphoma, immunoblastic B-cell lymphomas of immunosuppressed patients, and a subset of Hodgkin&apos;s lymphomas (7-9). Primary EBV infection of immunocompetent hosts is usually asymptomatic and leads to the establishment of a life-long carrier state, whereby the virus persists within the B-cell compartment of healthy carriers (5). These infected B lymphocytes can proliferate in vitro, giving rise to lymphoblastoid cell lines (LCLs) which express at least eight latency-associated viral antigens: the nuclear antigens EBNA1 to -6 and the membrane proteins LMP1 and -2 (reviewed in reference 13). The recent demonstration that the immunoblastic lymphomas occurring in immunosuppressed individuals represent the in vivo outgrowth of EBV-positive LCL-like cells (6) emphasizes the role of immune surveillance in controlling this potentially lymphomagenic virus. EBV induces long-lasting cytotoxic T-lymphocyte (CTL) memory in the infected host. Thus, EBV-specific CTL precursors can be reactivated in relatively large numbers from the T-cell pool of EBV-seropositive donors by challenging in vitro with autologous virus-infected B cell

Differential down-modulation of HLA class I and II molecule expression on human tumor cell lines upon in vivo transfer

Previous evidence from our laboratory showed that Epstein–Barr virus–immortalized lymphoblastoid B cells undergo a prominent down-modulation of HLA-II molecule expression when injected intraperitoneally in SCID mice, while HLA-I remains almost unaffected. Since this phenomenon can alter the experimental outcome of therapeutic protocols of adoptive cell therapy, we decided to evaluate the behavior of MHC antigens in a panel of cell lines belonging to the B- and T-cell lineages, as well as in epithelial tumor cell lines. Cells were administered in mice either intraperitoneally or subcutaneously and recovered 4 days later for HLA molecule expression analysis. Collected data showed a highly heterogeneous in vivo behavior of the various cell lines, which could alternatively down-modulate, completely abrogate or maintain unchanged the expression of either MHC-I or MHC-II molecules. Moreover, the site of injection impacted differentially on these aspects. Although such phenomena still lack a comprehensive clarification, epigenetic mechanisms are likely to be involved as epigenetic drugs could partially counteract MHC down-modulation in vivo. Nonetheless, it has to be pointed out that careful attention must be paid to the assessment of therapeutic efficacy of translational protocols of adoptive immunotherapy, as modulation of MHC molecules on human target cells when transferred in a mouse environment could readily interfere with the desired and expected therapeutic effects

Tracking of the origin of recurrent mutations of the BRCA1 and BRCA2 genes in the North-East of Italy and improved mutation analysis strategy

14noopenopenCini, Giulia; Mezzavilla, Massimo; Della Puppa, Lara; Cupelli, Elisa; Fornasin, Alessio; D'Elia, Angela Valentina; Dolcetti, Riccardo; Damante, Giuseppe; Bertok, Sara; Miolo, Gianmaria; Maestro, Roberta; de Paoli, Paolo; Amoroso, Antonio; Viel, AlessandraCini, Giulia; Mezzavilla, Massimo; Della Puppa, Lara; Cupelli, Elisa; Fornasin, Alessio; D'Elia, Angela Valentina; Dolcetti, Riccardo; Damante, Giuseppe; Bertok, Sara; Miolo, Gianmaria; Maestro, Roberta; de Paoli, Paolo; Amoroso, Antonio; Viel, Alessandr

High serum levels of soluble CD40-L in patients with undifferentiated nasopharyngeal carcinoma: pathogenic and clinical relevance

BACKGROUND: Engagement of CD40 promotes survival of undifferentiated nasopharyngeal carcinoma (UNPC) cells and similar effects are induced by the EBV oncoprotein LMP-1 that is expressed in a fraction of cases. Considering that CD40 may be activated also by the soluble isoform of CD40L (sCD40L), we investigated the serum levels of sCD40L in a series of 61 UNPC patients from Italy, a non-endemic area for this disease. RESULTS: At diagnosis, serum samples of UNPC patients contained significantly higher levels of sCD40L than age-matched healthy controls (p < 0.001). High levels of sCD40L (i.e., >18 ng/ml) were more frequently found in patients <40 years of age (p = 0.03) and with distant metastases at presentation (p = 0.03). Serum levels of sCD40L were inversely associated with the expression of the EBV oncoprotein LMP-1 (p = 0.03), which mimics a constitutively activated CD40. The amount of sCD40L decreased in a fraction of patients treated with local radiotherapy alone. Moreover, CD40L(+ )lymphoid cells admixed to neoplastic UNPC cells were detected in cases with high serum levels of sCD40L, suggesting that sCD40L is probably produced within the tumor mass. CONCLUSION: sCD40L may contribute to CD40 activation in UNPC cells, particularly of LMP-1-negative cases, further supporting the crucial role of CD40 signalling in the pathogenesis of UNPC. sCD40L levels may be useful to identify UNPC patients with occult distant metastases at presentation