A rescue approach in refractory diffuse large B-cell lymphoma with obinutuzumab-redirected cytokine-induced killer cells: a first-in-human case report.

Not available.

Regulation of DNA repair pathway choice in S and G2 phases by the NHEJ inhibitor CYREN.

Classical non-homologous end joining (cNHEJ) and homologous recombination compete for the repair of double-stranded DNA breaks during the cell cycle. Homologous recombination is inhibited during the G1 phase of the cell cycle, but both pathways are active in the S and G2 phases. However, it is unclear why cNHEJ does not always outcompete homologous recombination during the S and G2 phases. Here we show that CYREN (cell cycle regulator of NHEJ) is a cell-cycle-specific inhibitor of cNHEJ. Suppression of CYREN allows cNHEJ to occur at telomeres and intrachromosomal breaks during the S and G2 phases, and cells lacking CYREN accumulate chromosomal aberrations upon damage induction, specifically outside the G1 phase. CYREN acts by binding to the Ku70/80 heterodimer and preferentially inhibits cNHEJ at breaks with overhangs by protecting them. We therefore propose that CYREN is a direct cell-cycle-dependent inhibitor of cNHEJ that promotes error-free repair by homologous recombination during cell cycle phases when sister chromatids are present.

Selective cell cycle arrest in glioblastoma cell lines by quantum molecular resonance alone or in combination with temozolomide.

BACKGROUND: Glioblastoma is the most aggressive form of brain cancer, characterised by high proliferation rates and cell invasiveness. Despite advances in surgery and radio-chemotherapy, patients continue to have poor prognoses, with a survival rate of 14-15 months. Thus, new therapeutic strategies are needed. Non-ionising electromagnetic fields represent an emerging option given the potential advantages of safety, low toxicity and the possibility to be combined with other therapies. METHODS: Here, the anticancer activity of quantum molecular resonance (QMR) was investigated. For this purpose, three glioblastoma cell lines were tested, and the QMR effect was evaluated on cancer cell proliferation rate and aggressiveness. To clarify the QMR mechanism of action, the proteomic asset after stimulation was delineated. Mesenchymal stromal cells and astrocytes were used as healthy controls. RESULTS: QMR affected cancer cell proliferation, inducing a significant arrest of cell cycle progression and reducing cancer tumorigenicity. These parameters were not altered in healthy control cells. Proteomic analysis suggested that QMR acts not only on DNA replication but also on the machinery involved in the mitotic spindle assembly and chromosome segregation. Moreover, in a combined therapy assessment, QMR significantly enhanced temozolomide efficacy. CONCLUSIONS: QMR technology appears to be a promising tool for glioblastoma treatment.

Logistics of an advanced therapy medicinal product during COVID-19 pandemic in Italy: successful delivery of mesenchymal stromal cells in dry ice.

BACKGROUND: During the coronavirus disease-2019 (COVID-19) pandemic, Italian hospitals faced the most daunting challenges of their recent history, and only essential therapeutic interventions were feasible. From March to April 2020, the Laboratory of Advanced Cellular Therapies (Vicenza, Italy) received requests to treat a patient with severe COVID-19 and a patient with acute graft-versus-host disease with umbilical cord-derived mesenchymal stromal cells (UC-MSCs). Access to clinics was restricted due to the risk of contagion. Transport of UC-MSCs in liquid nitrogen was unmanageable, leaving shipment in dry ice as the only option. METHODS: We assessed effects of the transition from liquid nitrogen to dry ice on cell viability; apoptosis; phenotype; proliferation; immunomodulation; and clonogenesis; and validated dry ice-based transport of UC-MSCs to clinics. RESULTS: Our results showed no differences in cell functionality related to the two storage conditions, and demonstrated the preservation of immunomodulatory and clonogenic potentials in dry ice. UC-MSCs were successfully delivered to points-of-care, enabling favourable clinical outcomes. CONCLUSIONS: This experience underscores the flexibility of a public cell factory in its adaptation of the logistics of an advanced therapy medicinal product during a public health crisis. Alternative supply chains should be evaluated for other cell products to guarantee delivery during catastrophes.

Functional avidity-driven activation-induced cell death shapes CTL immunodominance.

Immunodominance is a complex phenomenon that relies on a mere numerical concept, while being potentially influenced at every step of the immune response. We investigated the mechanisms leading to the establishment of CTL immunodominance in a retroviral model and found that the previously defined subdominant Env-specific CD8(+) T cells are endowed with an unexpectedly higher functional avidity than is the immunodominant Gag-recognizing counterpart. This high avidity, along with the Env Ag overload, results in a supraoptimal TCR engagement. The overstimulation makes Env-specific T lymphocytes more susceptible to apoptosis, thus hampering their expansion and leading to an unintentional "immune kamikazing." Therefore, Ag-dependent, hyperactivation-induced cell death can be regarded as a novel mechanism in the establishment of the immunodominance that restrains and opposes the expansion of high-avidity T cells in favor of lower-affinity populations.

The Therapy and Management of Heart Failure with Preserved Ejection Fraction: New Insights on Treatment.

Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome characterised by the presence of diastolic dysfunction and elevated left ventricular filling pressure, in the setting of a left ventricular ejection fraction of at least 50%. Despite the epidemiological prevalence of HFpEF, a prompt diagnosis is challenging and many uncertainties exist. HFpEF is characterised by different phenotypes driven by various cardiac and non-cardiac comorbidities. This is probably the reason why several HFpEF clinical trials in the past did not reach strong outcomes to recommend a single therapy for this syndrome; however, this paradigm has recently changed, and the unmet clinical need for HFpEF treatment found a proper response as a result of a new class of drug, the sodium-glucose cotransporter 2 inhibitors, which beneficially act through the whole spectrum of left ventricular ejection fraction. The aim of this review was to focus on the therapeutic target of HFpEF, the role of new drugs and the potential role of new devices to manage the syndrome.

Right Ventricular Dysfunction before and after Cardiac Surgery: Prognostic Implications.

Right ventricular dysfunction is a prognostic factor for morbidity and mortality across a broad spectrum of cardiovascular diseases. While the role of the right ventricle in surgical patients has emerged, the prognostic impact of right ventricular dysfunction remains unclear in a large cardiac surgery population. We reviewed the existing literature about the role of right ventricular dysfunction in adults undergoing different kinds of cardiac surgery either present before or developed after surgery itself. Pre- and post-operative right ventricular dysfunction has demonstrated substantial prognostic implications. However, there remains a lack of consensus regarding its definition and diagnostic criteria. The available literature is limited to small-sized studies, underscoring the need for studies with larger populations.

Identification of TRPC6 as a possible candidate target gene within an amplicon at 11q21-q22.2 for migratory capacity in head and neck squamous cell carcinomas.

BACKGROUND: Cytogenetic and gene expression analyses in head and neck squamous cell carcinomas (HNSCC) have allowed identification of genomic aberrations that may contribute to cancer pathophysiology. Nevertheless, the molecular consequences of numerous genetic alterations still remain unclear. METHODS: To identify novel genes implicated in HNSCC pathogenesis, we analyzed the genomic alterations present in five HNSCC-derived cell lines by array CGH, and compared high level focal gene amplifications with gene expression levels to identify genes whose expression is directly impacted by these genetic events. Next, we knocked down TRPC6, one of the most highly amplified and over-expressed genes, to characterize the biological roles of TRPC6 in carcinogenesis. Finally, real time PCR was performed to determine TRPC6 gene dosage and mRNA levels in normal mucosa and human HNSCC tissues. RESULTS: The data showed that the HNSCC-derived cell lines carry most of the recurrent genomic abnormalities previously described in primary tumors. High-level genomic amplifications were found at four chromosomal sites (11q21-q22.2, 18p11.31-p11.21, 19p13.2-p13.13, and 21q11) with associated gene expression changes in selective candidate genes suggesting that they may play an important role in the malignant behavior of HNSCC. One of the most dramatic alterations of gene transcription involved the TRPC6 gene (located at 11q21-q22.2) which has been recently implicated in tumour invasiveness. siRNA-induced knockdown of TRPC6 expression in HNSCC-derived cells dramatically inhibited HNSCC-cell invasion but did not significantly alter cell proliferation. Importantly, amplification and concomitant overexpression of TRPC6 was also found in HNSCC tumour samples. CONCLUSIONS: Altogether, these data show that TRPC6 is likely to be a target for 11q21-22.2 amplification that confers enhanced invasive behavior to HNSCC cells. Therefore, TRPC6 may be a promising therapeutic target in the treatment of HNSCC.

Enalaprilat and losartan decrease erythroid precursors frequency in cells from patients with polycythemia vera.

OBJECTIVE: Polycythemia Vera (PV) is a myeloproliferative neoplasm characterized by the overproduction of red blood cells. First-line therapies are directed at lowering hematocrit levels. After the discovery of a mutation in the Janus kinase 2 (JAK2V617F), JAK2 inhibitors have been tested as second-line therapies. Despite these approaches, there is still the need for a major comprehension of the mechanisms involved in PV erythrocytosis and of more effective therapies. Angiotensin-converting enzyme (ACE) stimulates hematopoietic precursors proliferation and erythroid differentiation. We thus hypothesized that ACE inhibition could help in controlling erythrocytosis in PV. METHODS: We assessed the clonogenic potential by colony-forming unit (CFU) assay of mononuclear cells isolated from PV JAK2 positive or JAK2 negative patients with erythrocytosis treated with enalaprilat or losartan. RESULTS: Treatment with drugs led to a decrease of erythroid precursor frequency both in the presence and absence of JAK2 mutation, with a high extent in JAK2 positive cells and without affecting other types of precursors. No dose-dependent effect was observed. CONCLUSIONS: Our results demonstrate that ACE inhibition reduces erythroid precursor frequency, confirming the involvement of ACE in erythrocytosis despite the presence of JAK2 mutation and encouraging the hypothesis that ACE inhibitors and AT1R antagonists could help in directly managing erythrocytosis in PV.

Prognostic Value of sST2 in Heart Failure.

In recent years, there has been growing interest in the risk stratification for heart failure, and the use of multiple biomarkers to identify different pathophysiological processes associated with this condition. One such biomarker is soluble suppression of tumorigenicity-2 (sST2), which has shown some potential for integration into clinical practice. sST2 is produced by both cardiac fibroblasts and cardiomyocytes in response to myocardial stress. Other sources of sST2 are endothelial cells of the aorta and coronary arteries and immune cells such as T cells. Indeed, ST2 is also associated with inflammatory and immune processes. We aimed at reviewing the prognostic value of sST2 in both chronic and acute heart failure. In this setting, we also provide a flowchart about its potential use in clinical practice.

Virus-specific cytotoxic CD4+ T cells for the treatment of EBV-related tumors.

Although adoptive immunotherapy with CD8(+) CTL is providing clinically relevant results against EBV-driven malignancies, the effector role of CD4(+) T cells has been poorly investigated. We addressed this issue in a lymphoblastoid cell line-induced mouse model of posttransplant lymphoproliferative disease (PTLD) by comparing the therapeutic efficacy of EBV-specific CD4(+) and CD8(+) T cell lines upon adoptive transfer. CD4(+) T cells disclosed a long-lasting and stronger proliferative potential than CD8(+) T cells, had a similar activation and differentiation marker profile, efficiently killed their targets in a MHC class II-restricted manner, and displayed a lytic machinery comparable to that of cognate CD8(+) T cells. A detailed analysis of Ag specificity revealed that CD4(+) T cells potentially target EBV early lytic cycle proteins. Nonetheless, when assessed for the relative therapeutic impact after in vivo transfer, CD4(+) T cells showed a reduced activity compared with the CD8(+) CTL counterpart. This feature was apparently due to a strong and selective downmodulation of MHC class II expression on the tumor cells surface, a phenomenon that could be reverted by the demethylating agent 5-aza-2'-deoxycytidine, thus leading to restoration of lymphoblastoid cell line recognition and killing by CD4(+) T cells, as well as to a more pronounced therapeutic activity. Conversely, immunohistochemical analysis disclosed that HLA-II expression is fully retained in human PTLD samples. Our data indicate that EBV-specific cytotoxic CD4(+) T cells are therapeutic in mice bearing PTLD-like tumors, even in the absence of CD8(+) T cells. These findings pave the way to use cultures of pure CD4(+) T cells in immunotherapeutic approaches for EBV-related malignancies.

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.

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.

Differential expression of constitutive and inducible proteasome subunits in human monocyte-derived DC differentiated in the presence of IFN-alpha or IL-4.

Several studies strongly suggest that DC differentiated in vitro in the presence of type I IFN acquire more potent immune stimulatory properties, compared with DC differentiated in vitro with IL-4. However, little is known about the molecular mechanisms underlying this phenomenon. To address this question, we compared the Ag-processing machinery (APM) profile in human DC grown in the presence of IFN-alpha ((IFN)DC) or IL-4 ((IL-4)DC). Using a panel of APM component-specific mAb in Western blot experiments, we found that (IFN)DC preferentially express inducible proteasome subunits (LMP2, LMP7, and MECL1) both at immature and mature stages. In contrast, immature (IL-4)DC co-express both constitutive (beta1, beta2, and beta5) and inducible subunits, as shown by Western blotting analysis. In addition, immature (IFN)DC express higher levels of TAP1, TAP2, calnexin, calreticulin, tapasin, and HLA class I molecules than (IL-4)DC. The different proteasome profiles of (IFN)DC and (IL-4)DC were associated with a greater ability of (IFN)DC to present an immunodominant epitope that requires LMP7 expression for its processing. In general, these data show the impact of cytokines on APM component expression and hence the Ag-processing ability of DC.

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

BACKGROUND: 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 in vitro, but retain the capacity to fully differentiate into effector cells in vivo, 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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 ad hoc protocols rather than simply modify the cytokines added in culture.

The interplay between Epstein-Barr virus and the immune system: a rationale for adoptive cell therapy of EBV-related disorders.

The Epstein-Barr virus has evolved a plethora of strategies to evade immune system recognition and to establish latent infection in memory B cells, where the virus resides lifelong without any consequence in the majority of individuals. However, some imbalances in the equilibrium between the inherent virus transforming properties and the host immune system can lead to the development of different tumors, such as lymphoproliferative disorders, Hodgkin's lymphoma, Burkitt's lymphoma, and nasopharyngeal carcinoma. The expression of viral antigens in malignant cells makes them suitable targets for immunotherapeutic approaches, which are mainly based on the ex vivo expansion of EBV-specific T cells. Indeed, the infusion of virus-specific cytotoxic T lymphocytes has proved not only to be safe and effective, but also capable of restoring or inducing a protective anti-virus immunity, which is lacking, albeit to a different extent, in every EBV-driven malignancy. The purpose of this review is to summarize the results of adoptive immunotherapy approaches for EBV-related malignancies, with particular emphasis on the immunological and virological aspects linked to the clinical responses obtained. Data collected confirm the clinical relevance of the use of EBV-specific cytotoxic T lymphocytes in the field of adoptive immunotherapy and suggest the increasing importance of this approach also against other tumors, concurrent with the increasing knowledge of the intimate and continuous interplay between the virus and the host immune system.

Fighting Viral Infections and Virus-Driven Tumors with Cytotoxic CD4+ T Cells.

CD4+ T cells have been and are still largely regarded as the orchestrators of immune responses, being able to differentiate into distinct T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions. Nonetheless, a growing body of evidence indicates that CD4+ T cells can also exert a direct effector activity, which depends on intrinsic cytotoxic properties acquired and carried out along with the evolution of several pathogenic infections. The relevant role of CD4+ T cell lytic features in the control of such infectious conditions also leads to their exploitation as a new immunotherapeutic approach. This review aims at summarizing currently available data about functional and therapeutic relevance of cytotoxic CD4+ T cells in the context of viral infections and virus-driven tumors.

A BARF1-specific mAb as a new immunotherapeutic tool for the management of EBV-related tumors.

The use of monoclonal antibodies (mAb) for the diagnosis and treatment of malignancies is acquiring an increasing clinical importance, thanks to their specificity, efficacy and relative easiness of use. However, in the context of Epstein-Barr virus (EBV)-related malignancies, only cancers of B-cell origin can benefit from therapeutic mAb targeting specific B-cell lineage antigens. To overcome this limitation, we generated a new mAb specific for BARF1, an EBV-encoded protein with transforming and immune-modulating properties. BARF1 is expressed as a latent protein in nasopharyngeal (NPC) and gastric carcinoma (GC), and also in neoplastic B cells mainly upon lytic cycle induction, thus representing a potential target for all EBV-related malignancies. Considering that BARF1 is largely but not exclusively secreted, the BARF1 mAb was selected on the basis of its ability to bind a domain of the protein retained at the cell surface of tumor cells. In vitro, the newly generated mAb recognized the target molecule in its native conformation, and was highly effective in mediating both ADCC and CDC against BARF1-positive tumor cells. In vivo, biodistribution analysis in mice engrafted with BARF1-positive and -negative tumor cells confirmed its high specificity for the target. More importantly, the mAb disclosed a relevant antitumor potential in preclinical models of NPC and lymphoma, as evaluated in terms of both reduction of tumor masses and long-term survival. Taken together, these data not only confirm BARF1 as a promising target for immunotherapeutic interventions, but also pave the way for a successful translation of this new mAb to the clinical use.