Vito Pistoia

Mesenchymal stem cells in health and disease

Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells that can be isolated from many adult tissues. They can differentiate into cells of the mesodermal lineage, such as adipocytes, osteocytes and chondrocytes, as well as cells of other embryonic lineages. MSCs can interact with cells of both the innate and adaptive immune systems, leading to the modulation of several effector functions. After in vivo administration, MSCs induce peripheral tolerance and migrate to injured tissues, where they can inhibit the release of pro-inflammatory cytokines and promote the survival of damaged cells. This Review discusses the targets and mechanisms of MSC-mediated immunomodulation and the possible translation of MSCs to new therapeutic approaches.

Immunoregulatory function of mesenchymal stem cells

Mesenchymal stem cells (MSC) are a rare subset of stem cells residing in the bone marrow where they closely interact with hematopoietic stem cells and support their growth and differentiation. MSC can differentiate into multiple mesenchymal and non‐mesenchymal lineages, providing a promising tool for tissue repair. In addition, MSC suppress many T cell, B cell and NK cell functions and may affect also dendritic cell activities. Due to their limited immunogenicity, MSC are poorly recognized by HLA‐incompatible hosts. Based on these unique properties, MSC are currently under investigation for their possible use to treat immuno‐mediated diseases. However, both their condition of immunoprivilege and their immunosuppressive function have recently been challenged when analyzed under particular experimental conditions. Thus, it is likely that MSC effects on the immune system may be deeply influenced not only by cell‐to‐cell interactions, but also by environmental factors shaping their phenotype and functions.

HUMAN MESENCHYMAL STEM CELLS INHIBIT NEUTROPHIL APOPTOSIS: A MODEL FOR NEUTROPHIL PRESERVATION IN THE BONE MARROW NICHE

Mesenchymal stem cells (MSC) establish close interactions with bone marrow sinusoids in a putative perivascular niche. These vessels contain a large storage pool of mature nonproliferating neutrophils. Here, we have investigated the effects of human bone marrow MSC on neutrophil survival and effector functions. MSC from healthy donors, at very low MSC:neutrophil ratios (up to 1:500), significantly inhibited apoptosis of resting and interleukin (IL)‐8‐activated neutrophils and dampened N‐formyl‐l‐methionin‐l‐leucyl‐l‐phenylalanine (f‐MLP)‐induced respiratory burst. The antiapoptotic activity of MSC did not require cell‐to‐cell contact, as shown by transwell experiments. Antibody neutralization experiments demonstrated that the key MSC‐derived soluble factor responsible for neutrophil protection from apoptosis was IL‐6, which signaled by activating STAT‐3 transcription factor. Furthermore, IL‐6 expression was detected in MSC by real‐time reverse transcription‐polymerase chain reaction and enzyme‐linked immunosorbent assay. Finally, recombinant IL‐6 was found to protect neutrophils from apoptosis in a dose‐dependent manner. MSC had no effect on neutrophil phagocytosis, expression of adhesion molecules, and chemotaxis in response to IL‐8, f‐MLP, or C5a. These results support the following conclusions: (a) in the bone marrow niche, MSC likely protect neutrophils of the storage pool from apoptosis, preserving their effector functions and preventing the excessive or inappropriate activation of the oxidative metabolism, and (b) a novel mechanism whereby the inflammatory potential of activated neutrophils is harnessed by inhibition of apoptosis and reactive oxygen species production without impairing phagocytosis and chemotaxis has been identified.

Increased level of extracellular ATP at tumor sites: In vivo imaging with plasma membrane luciferase

Background There is growing awareness that tumour cells build up a “self-advantageous” microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP. Methodology/Principal Findings Measurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours. Conclusions/Significance Our results show that ATP in the tumour interstitium is in the hundrends micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.

Fasting Cycles Retard Growth of Tumors and Sensitize a Range of Cancer Cell Types to Chemotherapy

Short-term starvation increases the effectiveness of chemotherapy against a wide range of tumor cell types. Fasting: Good for You, Bad for Tumors Many promising cancer drugs being developed will require years to become approved by regulatory bodies and, in most cases, will only be effective for a fraction of patients with specific types of cancer. It is therefore important to develop broader, complementary strategies that can be translated rapidly into effective therapies. Two to 4 days of fasting before chemotherapy treatment is safe and protect animals, and possibly humans, against the side effects of chemotherapy. Here, cycles of fasting for 2 days in the absence of other treatments are shown to delay the progression of several tumor types in mice and, in some cases, to be as effective as toxic chemotherapy drugs. However, the combination of fasting and chemotherapy was much more effective than either alone and delayed the progression of a variety of tumors, including breast cancer and glioma, reduced the number of organs affected by melanoma metastases, and promoted long-term cancer-free survival in up to 40% of mice with neuroblastomas. In mice injected with human breast and ovarian cancer cells, fasting cycles promoted survival extension by protecting the mice from chemotherapy while causing a strong inhibition of tumor progression. Experiments in simple organisms, human cells, and mice indicated that these effects of fasting were caused by changes inside and outside cells that increased the death of tumor but not normal cells, a process termed differential stress sensitization. Although clinical trials testing the effect of fasting in cancer treatment are still in the early stages, they suggest that fasting cycles may boost the efficacy of chemotherapeutic agents and could be as effective as chemotherapy drugs in the killing of specific tumor cells. Short-term starvation (or fasting) protects normal cells, mice, and potentially humans from the harmful side effects of a variety of chemotherapy drugs. Here, we show that treatment with starvation conditions sensitized yeast cells (Saccharomyces cerevisiae) expressing the oncogene-like RAS2val19 to oxidative stress and 15 of 17 mammalian cancer cell lines to chemotherapeutic agents. Cycles of starvation were as effective as chemotherapeutic agents in delaying progression of different tumors and increased the effectiveness of these drugs against melanoma, glioma, and breast cancer cells. In mouse models of neuroblastoma, fasting cycles plus chemotherapy drugs—but not either treatment alone—resulted in long-term cancer-free survival. In 4T1 breast cancer cells, short-term starvation resulted in increased phosphorylation of the stress-sensitizing Akt and S6 kinases, increased oxidative stress, caspase-3 cleavage, DNA damage, and apoptosis. These studies suggest that multiple cycles of fasting promote differential stress sensitization in a wide range of tumors and could potentially replace or augment the efficacy of certain chemotherapy drugs in the treatment of various cancers.

Human Mesenchymal Stem Cells Promote Survival of T Cells in a Quiescent State

Mesenchymal stem cells (MSC) are part of the bone marrow that provides signals supporting survival and growth of bystander hematopoietic stem cells (HSC). MSC modulate also the immune response, as they inhibit proliferation of lymphocytes. In order to investigate whether MSC can support survival of T cells, we investigated MSC capacity of rescuing T lymphocytes from cell death induced by different mechanisms. We observed that MSC prolong survival of unstimulated T cells and apoptosis‐prone thymocytes cultured under starving conditions. MSC rescued T cells from activation induced cell death (AICD) by downregulation of Fas receptor and Fas ligand on T cell surface and inhibition of endogenous proteases involved in cell death. MSC dampened also Fas receptor mediated apoptosis of CD95 expressing Jurkat leukemic T cells. In contrast, rescue from AICD was not associated with a significant change of Bcl‐2, an inhibitor of apoptosis induced by cell stress. Accordingly, MSC exhibited a minimal capacity of rescuing Jurkat cells from chemically induced apoptosis, a process disrupting the mitochondrial membrane potential regulated by Bcl‐2. These results suggest that MSC interfere with the Fas receptor regulated process of programmed cell death. Overall, MSC can inhibit proliferation of activated T cells while supporting their survival in a quiescent state, providing a model of their activity inside the HSC niche.

Expression of P2X7 receptor increases in vivo tumor growth

The P2X7 receptor is an ATP-gated ion channel known for its cytotoxic activity. However, recent evidence suggests a role for P2X7 in cell proliferation. Here, we found that P2X7 exhibits significant growth-promoting effects in vivo. Human embryonic kidney cells expressing P2X7 exhibited a more tumorigenic and anaplastic phenotype than control cells in vivo, and the growth rate and size of these tumors were significantly reduced by intratumoral injection of the P2X7 inhibitor-oxidized ATP. The accelerated growth of P2X7-expressing tumors was characterized by increased proliferation, reduced apoptosis, and a high level of activated transcription factor NFATc1. These tumors also showed a more developed vascular network than control tumors and secreted elevated amounts of VEGF. The growth and neoangiogenesis of P2X7-expressing tumors was blocked by intratumoral injection of the VEGF-blocking antibody Avastin (bevacizumab), pharmacologic P2X7 blockade, or P2X7 silencing in vivo. Immunohistochemistry revealed strong P2X7 positivity in several human cancers. Together, our findings provide direct evidence that P2X7 promotes tumor growth in vivo.

Immunomodulatory properties of mesenchymal stem cells: a review based on an interdisciplinary meeting held at the Kennedy Institute of Rheumatology Division, London, UK, 31 October 2005

Multipotent mesenchymal stromal cells isolated from bone marrow and other sites are currently being studied to determine their potential role in the pathogenesis and/or management of autoimmune diseases. In vitro studies have shown that they exhibit a dose-dependent antiproliferative effect on T and B lymphocytes, dendritic cells, natural killer cells and various B cell tumour lines – an effect that is both cell contact and soluble factor dependent. Animal models of autoimmune disease treated with multipotent mesenchymal stromal cells have mostly exhibited a positive clinical response, as have a limited number of patients suffering from acute graft versus host disease. This review summarizes the findings of a 1-day meeting devoted to the subject with the aim of coordinating efforts.

Mutation-Independent Anaplastic Lymphoma Kinase Overexpression in Poor Prognosis Neuroblastoma Patients

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified as a major oncogene associated with familial and sporadic neuroblastomas (NBL). Yet, a direct correlation between endogenous expression level of the ALK protein, oncogenic potential, and clinical outcome has not been established. We investigated ALK genetic mutations, protein expression/phosphorylation, and functional inhibition both in NBL-derived cell lines and in 34 localized and 48 advanced/metastatic NBL patients. ALK constitutive phosphorylation/activation was observed in high-ALK expressing cells, harboring either a mutated or a wild-type receptor. No activation was found in cell lines with low expression of wild-type ALK. After 72 hours of treatments, small molecule ALK inhibitor CEP-14083 (60 nmol/L) induced growth arrest and cell death in NBL cells overexpressing wild-type (viability: ALK(high) 12.8%, ALK(low) 73%, P = 0.0035; cell death: ALK(high) 56.4%, ALK(low) 16.2%, P = 0.0001) or mutated ALK. ALK protein expression was significantly up-regulated in advanced/metastatic compared with localized NBLs (ALK overexpressing patients: stage 1-2, 23.5%; stage 3-4, 77%; P < 0.0001). Interestingly, protein levels did not always correlate with ALK genetic alterations and/or mRNA abundance. Both mutated and wild-type ALK receptor can exert oncogenic activity in NBL cells. However, wild-type ALK receptor requires a critical threshold of expression to achieve oncogenic activation. Overexpression of either mutated or wild-type ALK defines poor prognosis patients. Alternative mechanisms other than direct mutations and/or gene amplification regulate the ALK level of expression in NBL cells. Wild-type ALK is a potential therapeutic target for advanced/metastatic NBLs.

IMMUNOGENICITY OF HUMAN MESENCHYMAL STEM CELLS IN HLA-CLASS I RESTRICTED T CELL RESPONSES AGAINST VIRAL OR TUMOR-ASSOCIATED ANTIGENS

Human mesenchymal stem cells (MSC) are immunosuppressive and poorly immunogenic but may act as antigen‐presenting cells (APC) for CD4+ T‐cell responses; here we have investigated their ability to serve as APC for in vitro CD8+ T‐cell responses. MSC pulsed with peptides from viral antigens evoked interferon (IFN)‐γ and Granzyme B secretion in specific cytotoxic T lymphocytes (CTL) and were lysed, although with low efficiency. MSC transfected with tumor mRNA or infected with a viral vector carrying the Hepatitis C virus NS3Ag gene induced cytokine release but were not killed by specific CTL, even following pretreatment with IFN‐γ. To investigate the mechanisms involved in MSC resistance to CTL‐mediated lysis, we analyzed expression of human leukocyte antigen (HLA) class I‐related antigen‐processing machinery (APM) components and of immunosuppressive HLA‐G molecules in MSC. The LMP7, LMP10, and ERp57 components were not expressed and the MB‐1 and zeta molecules were downregulated in MSC either unmanipulated or pretreated with IFN‐γ. Surface HLA‐G was constitutively expressed on MSC but was not involved in their protection from CTL‐mediated lysis. MSC supernatants containing soluble HLA‐G (sHLA‐G) inhibited CTL‐mediated lysis, whereas those lacking sHLA‐G did not. The role of sHLA‐G in such inhibition was unambiguously demonstrated by partial restoration of lysis following sHLA‐G depletion from MSC supernatants. In conclusion, human MSC can process and present HLA class I‐restricted viral or tumor antigens to specific CTL with a limited efficiency, likely because of some defects in APM components. However, they are protected from CTL‐mediated lysis through a mechanism that is partly sHLA‐G‐dependent.