Marta Coscia

Zoledronic acid repolarizes tumour-associated macrophages and inhibits mammary carcinogenesis by targeting the mevalonate pathway

It is unknown whether zoledronic acid (ZA) at clinically relevant doses is active against tumours not located in bone. Mice transgenic for the activated ErbB‐2 oncogene were treated with a cumulative number of doses equivalent to that recommended in human beings. A significant increase in tumour‐free and overall survival was observed in mice treated with ZA. At clinically compatible concentrations, ZA modulated the mevalonate pathway and affected protein prenylation in both tumour cells and macrophages. A marked reduction in the number of tumour‐associated macrophages was paralleled by a significant decrease in tumour vascularization. The local production of vascular endothelial growth factor and interleukin‐10 was drastically down‐regulated in favour of interferon‐γ production. Peritoneal macrophages and tumour‐associated macrophages of ZA‐treated mice recovered a full M1 antitumoral phenotype, as shown by nuclear translocation of nuclear factor kB, inducible nitric oxide synthase expression and nitric oxide production. These data indicate that clinically achievable doses of ZA inhibit spontaneous mammary cancerogenesis by targeting the local microenvironment, as shown by a decreased tumour vascularization, a reduced number of tumour‐associated macrophages and their reverted polarization from M2 to M1 phenotype.

Effector γδ T cells and tumor cells as immune targets of zoledronic acid in multiple myeloma

The aim of this study was to investigate the in vitro immunomodulatory effects of zoledronic acid (Zol) on peripheral blood Vγ9/Vδ2 (γδ) T cells of normal donors and multiple myeloma (MM) patients. γδ T cells were stimulated with Zol and low doses of interleukin-2 (IL-2), and then analyzed for proliferation, cytokine production, and generation of effector activity against myeloma cell lines and primary myeloma cells. Proliferation of γδ T cells was observed in 100% of normal donors and 50% of MM patients. γδ T cells produced IFN-γ, surface mobilized the CD107a and CD107b antigens, and exerted direct cell-to-cell antimyeloma activity irrespective of the ability to proliferate to Zol and IL-2. The memory phenotype was predominant in the MM γδ T cells that proliferated in response to Zol (responders), whereas effector cells were predominant in those that did not (nonresponders). Zol induced antimyeloma activity through the monocyte-dependent activation of γδ T cells and by enhancing the immunosensitivity of myeloma cells to γδ T cells. Mevastatin, a specific inhibitor of hydroxy-methylglutaryl-CoA reductase, completely abrogated this antimyeloma activity.

Molecular prediction of durable remission after first-line fludarabinecyclophosphamide-rituximab in chronic lymphocytic leukemia

Fludarabine, cyclophosphamide, and rituximab (FCR) has represented a significant treatment advancement in chronic lymphocytic leukemia (CLL). In the new scenario of targeted agents, there is an increasing interest in identifying patients who gain the maximum benefit from FCR. In this observational multicenter retrospective analysis of 404 CLL patients receiving frontline FCR, the combination of three biomarkers that are widely tested before treatment (IGHV mutation status, 11q deletion and 17p deletion; available in 80% of the study cohort) allowed to identify a very low-risk category of patients carrying mutated IGHV genes but neither 11q or 17p deletion that accounted for 28% of all cases. The majority of very low-risk patients (71%) remained free of progression after treatment and their hazard of relapse decreased after 4 years from FCR. The life expectancy of very low-risk patients (91% at 5 years) was superimposable to that observed in the matched normal general population, indicating that neither the disease nor complications of its treatment affected survival in this favorable CLL group. These findings need a prospective validation and may be helpful for the design of clinical trials aimed at comparing FCR to new targeted treatments of CLL, and, possibly, for optimized disease management.

The PD-1/PD-L1 axis contributes to T cell dysfunction in chronic lymphocytic leukemia

Chronic lymphocytic leukemia is marked by profound defects in T-cell function. Programmed death-1 is a receptor involved in tumor-mediated immunosuppression through binding of the PD-L1 ligand. Multiparametric flow cytometry and immunohistochemistry were used to study PD-1/PD-L1 expression. Functional assays were used to determine the involvement of the PD-1/PD-L1 axis in T-cell responses. PD-1 expression by CD4+ and CD8+ T lymphocytes was significantly higher in 117 chronic lymphocytic leukemia patients than in 33 donors of a comparable age. CD4+ and CD8+ T lymphocytes from chronic lymphocytic leukemia patients displayed increased numbers of effector memory and terminally differentiated cells, respectively, when compared to controls. The number of effector memory CD4+ and terminally differentiated CD8+ lymphocytes positively associated with a more advanced stage of disease, treatment requirements and unfavorable genomic aberrations. Furthermore, leukemic lymphocytes expressed higher levels of PD-L1 than circulating B lymphocytes from normal donors. PD-1 and PD-L1 surface expression spiked in proliferating T and B lymphocytes, suggesting that this interaction works efficiently in activated environments. Within chronic lymphocytic leukemia proliferation centers in the lymph node, CD4+/PD-1+ T lymphocytes were found to be in close contact with PD-L1+ chronic lymphocytic leukemia cells. Lastly, functional experiments using recombinant soluble PD-L1 and blocking antibodies indicated that this axis contributes to the inhibition of IFN-γ production by CD8+ T cells. These observations suggest that pharmacological manipulation of the PD-1/PD-L1 axis may contribute to restoring T-cell functions in the chronic lymphocytic leukemia microenvironment.

CD73-generated extracellular adenosine in chronic lymphocytic leukemia creates local conditions counteracting drug-induced cell death

Extracellular adenosine (ADO), generated from ATP or ADP through the concerted action of the ectoenzymes CD39 and CD73, elicits autocrine and paracrine effects mediated by type 1 purinergic receptors. We have tested whether the expression of CD39 and CD73 by chronic lymphocytic leukemia (CLL) cells activates an adenosinergic axis affecting growth and survival. By immunohistochemistry, CD39 is widely expressed in CLL lymph nodes, whereas CD73 is restricted to proliferation centers. CD73 expression is highest on Ki-67(+) CLL cells, adjacent to T lymphocytes, and is further localized to perivascular areas. CD39(+)/CD73(+) CLL cells generate ADO from ADP in a time- and concentration-dependent manner. In peripheral blood, CD73 expression occurs in 97/299 (32%) CLL patients and pairs with CD38 and ZAP-70 expression. CD73-generated extracellular ADO activates type 1 purinergic A2A receptors that are constitutively expressed by CLL cells and that are further elevated in proliferating neoplastic cells. Activation of the ADO receptors increases cytoplasmic cAMP levels, inhibiting chemotaxis and limiting spontaneous drug-induced apoptosis of CLL cells. These data are consistent with the existence of an autocrine adenosinergic loop, and support engraftment of leukemic cells in growth-favorable niches, while simultaneously protecting from the action of chemotherapeutic agents.

Exposure to myeloma cell lysates affects the immune competence of dendritic cells and favors the induction of Tr1-like regulatory T cells

The aim of this work was to investigate the interactions of tumor cells with dendritic cells (DC) in normal donors and patients with multiple myeloma (MM). Normal and MM DC internalized necrotic lysates derived from myeloma cell lines (MCL) with high efficiency, whereas necrotic lysates from primary myeloma cells (PMC) were internalized with significantly lower efficiency. A positive correlation was found between susceptibility to internalization and the ability to induce DC maturation. After PMC exposure, DC produced large amounts of IL‐10 and measurable amounts of IL‐4 but no detectable IL‐12. Two rounds of exposure to PMC‐treated DC generated autologous T cells with low proliferative capacity, decreased IFN‐γ production and increased IL‐10 production in the absence of IL‐4 production. These data indicate that myeloma cells can affect host immunity by priming DC towards a maturation state favoring the generation of T cells with a regulatory rather than an effector phenotype.

Long-term follow-up of idiotype vaccination in human myeloma as a maintenance therapy after high-dose chemotherapy

The aim of this work was to evaluate the long-term immunological and clinical impact of idiotype (Id) vaccination in multiple myeloma (MM) patients in first remission after high-dose chemotherapy. A total of 15 patients received a series of subcutaneous (s.c.) injections of autologous Id, conjugated to keyhole limpet hemocyanin (KLH) and in association with low doses of GM-CSF. The median duration of follow-up was 110 months from diagnosis. The vaccine induced immune responses that lasted almost 2 years after the end of treatment. Antibody responses included anti-KLH IgM and IgG (90% of patients), anti-KLH IgE (30%), anti-GM-CSF IgG (20%), anti-Id IgG (20%), and anti-Id IgE (30%). Id-specific delayed type hypersensitivity skin tests were positive in 85% of tested patients. Following vaccination, a progressive recovery of T-cell receptor (TCR) diversity was observed and the loss of oligoclonality was significantly correlated with the remission duration. Although Id/KLH conjugates did not eliminate the residual tumor burden, the median progression-free survival, and overall survival were 40 and 82 months, respectively. A retrospective case-matched analysis showed similar results in patients treated with IFN-α alone or in association with steroids. This vaccine formulation can overcome Id-specific immune tolerance by inducing clinical responses that are worthy of further investigation.

Murine β-defensin 2 promotes TLR-4/MyD88-mediated and NF-κB-dependent atypical death of APCs via activation of TNFR2

Mammalian antimicrobial peptides, including β‐defensins, represent an ancient arm of innate immunity designed to directly neutralize invading microbes. Previously, we demonstrated that murine β‐defensin 2 (mDF2β) also acted as an endogenous ligand for TLR‐4‐activating maturation of dendritic cells (DCs). Herein, we report that this TLR‐4 –dependent activation leads to induction of an atypical cell death that is unexpectedly exaggerated by the inhibition of caspases. Experiments using APCs with nonfunctional TNF‐α or its receptors suggest that this is a NF‐κB‐ and TNF‐α‐dependent process that does not require TNFR1. We demonstrate that mDF2β triggers a TNFR2‐mediated signaling cascade of “self‐destruction” through up‐regulation of membrane‐bound TNF‐α and TNFR2. This appears not to be an isolated phenomenon, as human synthetic β‐defenisn 3 was also able to activate and kill DCs. We propose that β‐defenins may play an important immunoregulatory role as controllers of the natural process of elimination of activated APCs.

Severe and long-lasting disruption of T-cell receptor diversity in human myeloma after high-dose chemotherapy and autologous peripheral blood progenitor cell infusion.

Vaccine‐based strategies are currently under investigation as a means of inducing tumour‐specific immune responses and improving the clinical outcome of multiple myeloma (MM) patients in remission after high‐dose chemotherapy and peripheral blood progenitor cell (PBPC) infusion. The immune competence of these patients was investigated by determining the overall diversity of the T‐cell receptor (TCR) repertoire in the peripheral blood (PB) and bone marrow (BM). The average time after transplantation was 13 months. The clonality and reciprocal usage of BV gene segments (TCRBV repertoire) was estimated at the cDNA level and membrane protein expression. The TCRBV repertoire of MM was severely disrupted compared with age‐matched normal donors. On average, one‐third of the total repertoire in both the PB and the BM consisted of T cells expressing oligoclonal TCRβ transcripts. Flow cytometry showed an increased frequency of abnormally expanded BV subfamilies at both sites. BV expansions were predominantly CD8+ and had the phenotype of antigen‐experienced memory T cells as well as T cells with the naive phenotype. Oligoclonality was not restricted to phenotypically expanded BV subfamilies, but also involved normally represented BV subfamilies. The TCR repertoire of MM in remission was then compared with monoclonal gammopathy of undetermined significance (MGUS) and MM patients at diagnosis. The degree of TCR diversity was similar in age‐matched normal donors and MGUS, but progressively decreased from MGUS to MM at diagnosis and then to MM in remission. These data indicate that: (1) there is a long‐lasting and severe disruption of TCR diversity after high‐dose chemotherapy and PBPC infusion, and (2) the extent of TCR disruption may affect the clinical outcome of vaccine‐based strategies delivered at the stage of minimal residual disease.

Immune Modulation by Zoledronic Acid in Human Myeloma: An Advantageous Cross-Talk between Vγ9Vδ2 T Cells, αβ CD8+ T Cells, Regulatory T Cells, and Dendritic Cells

Vγ9Vδ2 T cells play a major role as effector cells of innate immune responses against microbes, stressed cells, and tumor cells. They constitute <5% of PBLs but can be expanded by zoledronic acid (ZA)-treated monocytes or dendritic cells (DC). Much less is known about their ability to act as cellular adjuvants bridging innate and adaptive immunity, especially in patients with cancer. We have addressed this issue in multiple myeloma (MM), a prototypic disease with several immune dysfunctions that also affect γδ T cells and DC. ZA-treated MM DC were highly effective in activating autologous γδ T cells, even in patients refractory to stimulation with ZA-treated monocytes. ZA inhibited the mevalonate pathway of MM DC and induced the intracellular accumulation and release into the supernatant of isopentenyl pyrophosphate, a selective γδ T cell activator, in sufficient amounts to induce the proliferation of γδ T cells. Immune responses against the tumor-associated Ag survivin (SRV) by MHC-restricted, SRV-specific CD8+ αβ T cells were amplified by the concurrent activation of γδ T cells driven by autologous DC copulsed with ZA and SRV-derived peptides. Ancillary to the isopentenyl pyrophosphate-induced γδ T cell proliferation was the mevalonate-independent ZA ability to directly antagonize regulatory T cells and downregulate PD-L2 expression on the DC cell surface. In conclusion, ZA has multiple immune modulatory activities that allow MM DC to effectively handle the concurrent activation of γδ T cells and MHC-restricted CD8+ αβ antitumor effector T cells.