Mara Cirone

Consensus guidelines for the detection of immunogenic cell death

Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named “immunogenic cell death” (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death

The immunogenicity of malignant cells has recently been acknowledged as a critical determinant of efficacy in cancer therapy. Thus, besides developing direct immunostimulatory regimens, including dendritic cell-based vaccines, checkpoint-blocking therapies, and adoptive T-cell transfer, researchers have started to focus on the overall immunobiology of neoplastic cells. It is now clear that cancer cells can succumb to some anticancer therapies by undergoing a peculiar form of cell death that is characterized by an increased immunogenic potential, owing to the emission of the so-called “damage-associated molecular patterns” (DAMPs). The emission of DAMPs and other immunostimulatory factors by cells succumbing to immunogenic cell death (ICD) favors the establishment of a productive interface with the immune system. This results in the elicitation of tumor-targeting immune responses associated with the elimination of residual, treatment-resistant cancer cells, as well as with the establishment of immunological memory. Although ICD has been characterized with increased precision since its discovery, several questions remain to be addressed. Here, we summarize and tabulate the main molecular, immunological, preclinical, and clinical aspects of ICD, in an attempt to capture the essence of this phenomenon, and identify future challenges for this rapidly expanding field of investigation.

Epstein-Barr Virus Blocks the Autophagic Flux and Appropriates the Autophagic Machinery To Enhance Viral Replication

ABSTRACT Autophagy is a catabolic pathway that helps cells to survive under stressful conditions. Cells also use autophagy to clear microbiological infections, but microbes have learned how to manipulate the autophagic pathway for their own benefit. The experimental evidence obtained in this study suggests that the autophagic flux is blocked at the final steps during the reactivation of Epstein-Barr virus (EBV) from latency. This is indicated by the level of the lipidated form of LC3 that does not increase in the presence of bafilomycin and by the lack of colocalization of autophagosomes with lysosomes, which correlates with reduced Rab7 expression. Since the inhibition of the early phases of autophagy impaired EBV replication and viral particles were observed in autophagic vesicles in the cytoplasm of producing cells, we suggest that EBV exploits the autophagic machinery for its transportation in order to enhance viral production. The autophagic block was not mediated by ZEBRA, an immediate-early EBV lytic gene, whose transfection in Ramos, Akata, and 293 cells promoted a complete autophagic flux. The block occurred only when the complete set of EBV lytic genes was expressed. We suggest that the inhibition of the early autophagic steps or finding strategies to overcome the autophagic block, allowing viral degradation into the lysosomes, can be exploited to manipulate EBV replication. IMPORTANCE This study shows, for the first time, that autophagy is blocked at the final degradative steps during EBV replication in several cell types. Through this block, EBV hijacks the autophagic vesicles for its intracellular transportation and enhances viral production. A better understanding of virus-host interactions could help in the design of new therapeutic approaches against EBV-associated malignancies.

Primary Effusion Lymphoma Cell Death Induced by Bortezomib and AG 490 Activates Dendritic Cells through CD91

To understand how cytotoxic agent-induced cancer cell death affects the immune system is of fundamental importance to stimulate immune response to counteract the high mortality due to cancer. Here we compared the immunogenicity of Primary Effusion Lymphoma (PEL) cell death induced by anticancer drug Bortezomib (Velcade) and Tyrphostin AG 490, a Janus Activated Kinase 2/signal trasducer and activator of transcription-3 (JAK2/STAT3) inhibitor. We show that both treatments were able to induce PEL apoptosis with similar kinetics and promote dendritic cells (DC) maturation. The surface expression of molecules involved in immune activation, namely calreticulin (CRT), heat shock proteins (HSP) 90 and 70 increased in dying cells. This was correlated with DC activation. We found that PEL cell death induced by Bortezomib was more effective in inducing uptake by DC compared to AG 490 or combination of both drugs. However the DC activation induced by all treatments was completely inhibited when these cells were pretreated with a neutralizing antiboby directed against the HSP90/70 and CRT common receptor, CD91. The activation of DC by Bortezomib and AG 490 treated PEL cells, as seen in the present study, might have important implications for a combined chemo and immunotherapy in such patients.

Differential regulation of miR-21 and miR-146a by Epstein–Barr virus-encoded EBNA2

The discovery of microRNA (miR) represents a novel paradigm in RNA-based regulation of gene expression and their dysregulation has become a hallmark of many a tumor. In virally associated cancers, the host–pathogen interaction could involve alteration in miR expression. Epstein–Barr virus (EBV)-encoded EBNA2 is indispensable for the capacity of the virus to transform B cells in vitro. Here, we studied how it affects cellular miRs. Extensive miR profiling of the virus-infected and EBNA2-transfected B lymphoma cells revealed that oncomiR miR-21 is positively regulated by this viral protein. Conversely, Burkitt’s lymphoma (BL) cell lines infected with EBNA2 lacking P3HR1 strain did not show any increase in miR-21. EBNA2 increased phosphorylation of AKT and this was directly correlated with increased miR-21. In contrast, miR-146a was downregulated by EBNA2 in B lymphoma cells. Low miR-146a expression correlates with an elevated level of IRAK1 and type I interferon in EBNA2 transfectants. Taken together, the present data suggest that EBNA2 might contribute to EBV-induced B-cell transformation by altering miR expression and in particular by increasing oncomiR-like miR-21 and by affecting the antiviral responses of the innate immune system through downregulation of its key regulator miR-146a.

Human herpesvirus 6 and multiple sclerosis: a study of T cell cross-reactivity to viral and myelin basic protein antigens.

Several reports have suggested an association of human herpesvirus 6 (HHV‐6) with multiple sclerosis. Autoreactive T lymphocytes directed against myelin components seem to contribute to the pathogenesis of the disease. It has been suggested that molecular mimicry between viral and self‐antigens might be one of the mechanisms that determine the onset of several autoimmune diseases. Following this hypothesis, the purpose of the present study was to evaluate if HHV‐6 could play a role in activating T cells capable of cross‐reaction with an important myelin component, the myelin basic protein. T cell lines were established from 22 multiple sclerosis patients and from 16 healthy controls, and their capability to react to both virus and myelin basic protein antigens was compared. The analysis of T cell cross‐reactivity in patients and controls did not show significant differences in the HHV‐6 ability to activate myelin basic protein‐reactive T cells. Similarly, the evaluation of the humoral immune response to HHV‐6 in patients and controls did not mirror any abnormality in the HHV‐6 status in multiple sclerosis patients. Therefore, although the findings of activity in vitro of T cell lines with dual specificity are consistent with the hypothesis of molecular mimicry, the lack of differences in cross‐reactivity between patients and controls do not support molecular mimicry as an important mechanism in the physiopathology of this disease. J. Med. Virol. 68: 268–272, 2002.

Epstein–Barr Virus Infection Induces Aberrant TLR Activation Pathway and Fibroblast–Myofibroblast Conversion in Scleroderma

Scleroderma (SSc) is a complex and heterogeneous connective tissue disease mainly characterized by autoimmunity, vascular damage, and fibrosis that mostly involve the skin and lungs. Epstein–Barr virus (EBV) is a lymphotropic γ-herpesvirus that has co-evolved with human species, infecting >95% of the adult population worldwide, and has been a leading candidate in triggering several autoimmune diseases. Here we show that EBV establishes infection in the majority of fibroblasts and endothelial cells in the skin of SSc patients, characterized by the expression of the EBV noncoding small RNAs (EBERs) and the increased expression of immediate-early lytic and latency mRNAs and proteins. We report that EBV is able to persistently infect human SSc fibroblasts in vitro, inducing an aberrant innate immune response in infected cells. EBV–Toll-like receptor (TLR) aberrant activation induces the expression of selected IFN-regulatory factors (IRFs), IFN-stimulated genes (ISGs), transforming growth factor-β1 (TGFβ1), and several markers of fibroblast activation, such as smooth muscle actin and Endothelin-1, and all of these genes play a key role in determining the profibrotic phenotype in SSc fibroblasts. These findings imply that EBV infection occurring in mesenchymal, endothelial, and immune cells of SSc patients may underlie the main pathological features of SSc including autoimmunity, vasculopathy, and fibrosis, and provide a unified disease mechanism represented by EBV reactivation.

Role of skin surface lipids in UV-induced epidermal cell changes.

SummaryUltraviolet irradiation is capable of affecting skin surface lipids, especially squalene and cholesterol, both in vitro and in vivo, with generation of active lipoperoxides. The photodecomposition of the skin lipid component was carefully evaluated by capillary gas-chromatography. The effects of UV-induced lipoperoxides on human keratinocytes in culture and on guinea pig ear slices were compared with those of synthetic lipoperoxides, i.e. cumene hydroperoxide and 13-hydroperoxylinoleate. A time- and dose-dependent effect on protein synthesis and mitotic activity was observed. In cell culture low concentrations (0.05–5 Μg/ml) of peroxidated squalene and synthetic lipoperoxides stimulated the incorporation of radiolabelled thymidine and phenylalanine, while higher concentrations (>10 Μg/ml), or longer periods of treatment, induced cellular damage. In guinea pig ear slices, the lipoperoxides (5–50 Μg/ml) increased aminoacid incorporation and the number of epidermal pigment cells; higher concentrations (>100 Μg/ml) caused a derangement of epidermal structure. The results suggest that UV irradiation of skin generates lipoperoxides from the surface lipids which, in vitro, are capable of producing a number of changes in epidermal cells.

Suppression of dendritic cell differentiation through cytokines released by Primary Effusion Lymphoma cells

Functional impairment of dendritic cells (DC) appears to be one of the mechanisms responsible for tumor escape from the control of the immune system. DC isolated from tumor-bearing animals and cancer patients with solid or with hematological malignancies have phenotypic and functional abnormalities. In addition, supernatants from in vitro cultured tumor cells have been shown to interfere with DC differentiation from CD34+ and monocyte precursors. Primary effusion lymphoma (PEL) is a Human Herpesvirus-8 (HHV-8)-associated tumor, which releases several cytokines such as IL-6, IL-10 and VEGF and its growth seems to be dependent on them in vitro or in vivo. In the present study, we found that these cytokines released by PELs have also an important role in interfering with the in vitro differentiation of immature DC (iDC) from CD14+ monocytes. The iDC obtained in the presence of PEL supernatants showed reduction of FITC-dextran uptake, reduction of MLR allostimulatory activity and altered expression of surface molecules, suggesting that cytokines released by PEL adversely affect DC differentiation.

HSP70 inhibition by 2-phenylethynesulfonamide induces lysosomal cathepsin D release and immunogenic cell death in primary effusion lymphoma

Heat-shock protein (HSP) 70 is aberrantly expressed in different malignancies and has a cancer-specific cell-protective effect. As such, it has emerged as a promising target for anticancer therapy. In this study, the effect of the HSP70-specific inhibitor (PES), also Pifitrin-μ, on primary effusion lymphoma (PEL) cell viability was analyzed. PES treatment induced a dose- and time-dependent cytotoxic effect in BC3 and BCBL1 PEL cells by inducing lysosome membrane permeabilization, relocation of cathepsin D in the cytosol, Bid cleavage, mitochondrial depolarization with release and nuclear translocation of apoptosis-activating factor. The PES-induced cell death in PEL cells was characterized by the appearance of Annexin-V/propidium iodide double-positive cells from the early times of treatment, indicating the occurrence of an additional type of cell death other than apoptosis, which, accordingly, was not efficiently prevented by the pan-caspase inhibitor Z-VAD-fmk. Conversely, PES-induced cell death was robustly reduced by pepstatin A, which inhibits Bid and caspase 8 processing. In addition, PES was responsible for a block of the autophagic process in PEL cells. Finally, we found that PES-induced cell death has immunogenic potential being able to induce dendritic cell activation.