Interleukin-10-Producing DC-10 Is a Unique Tool to Promote Tolerance Via Antigen-Specific T Regulatory Type 1 Cells

The prominent role of tolerogenic dendritic cells (tolDCs) in promoting immune tolerance and the development of efficient methods to generate clinical grade products allow the application of tolDCs as cell-based approach to dampen antigen (Ag)-specific T cell responses in autoimmunity and transplantation. Interleukin (IL)-10 potently modulates the differentiation and functions of myeloid cells. Our group contributed to the identification of IL-10 as key factor in inducing a subset of human tolDCs, named dendritic cell (DC)-10, endowed with the ability to spontaneously release IL-10 and induce Ag-specific T regulatory type 1 (Tr1) cells. We will provide an overview on the role of IL-10 in modulating myeloid cells and in promoting DC-10. Moreover, we will discuss the clinical application of DC-10 as inducers of Ag-specific Tr1 cells for tailoring Tr1-based cell therapy, and as cell product for promoting and restoring tolerance in T-cell-mediated diseases

Hla-g expression levels influence the tolerogenic activity of human DC-10

Human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule with known immune-modulatory functions. Our group identified a subset of human dendritic cells, named DC-10, that induce adaptive interleukin-10-producing T regulatory type 1 (Tr1) cells via the interleukin-10-dependent HLA-G/ILT4 pathway. In this study we aimed at defining the role of HLA-G in DC-10-mediated Tr1 cell differentiation. We analyzed phenotype, functions, and genetic variations in the 3’ untranslated region of the HLA-G locus of in vitro-differentiated DC-10 from 67 healthy donors. We showed that HLA-G expression on DC-10 is donor-dependent. Functional studies demonstrated that DC-10, independently of HLA-G expression, secrete interleukin-10 and negligible levels of interleukin-12. Interestingly, DC-10 with high HLA-G promote allo-specific anergic T cells that contain a significantly higher frequency of Tr1 cells, defined as interleukin-10-producing (P=0.0121) or CD49b+LAG-3+(P=0.0031) T cells, compared to DC-10 with low HLA-G. We found that the HLA-G expression on DC-10 is genetically imprinted, being associated with specific variations in the 3’ untranslated region of the gene, and it may be finely tuned by microRNA-mediated post-transcriptional regulation. These data highlight the important role of HLA-G in boosting DC-10 tolerogenic activity and confirm that interleukin-10 production by DC-10 is necessary but not sufficient to promote Tr1 cells at high frequency. These new insights into the role of HLA-G in DC-10-mediated induction of Tr1 cells provide additional information for clinical use in Tr1-or DC-10-based cell therapy approaches

HLA-G Expression on Blasts and Tolerogenic Cells in Patients Affected by Acute Myeloid Leukemia

Human Leukocyte Antigen-G (HLA-G) contributes to cancer cell immune escape from host antitumor responses. The clinical relevance of HLA-G in several malignancies has been reported. However, the role of HLA-G expression and functions in Acute Myeloid Leukemia (AML) is still controversial. Our group identified a subset of tolerogenic dendritic cells, DC-10 that express HLA-G and secrete IL-10. DC-10 are present in the peripheral blood and are essential in promoting and maintaining tolerance via the induction of adaptive T regulatory (Treg) cells. We investigated HLA-G expression on blasts and the presence of HLA-G-expressing DC-10 and CD4+ T cells in the peripheral blood of AML patients at diagnosis. Moreover, we explored the possible influence of the 3′ untranslated region (3′UTR) of HLA-G, which has been associated with HLA-G expression, on AML susceptibility. Results showed that HLA-G-expressing DC-10 and CD4+ T cells are highly represented in AML patients with HLA-G positive blasts. None of the HLA-G variation sites evaluated was associated with AML susceptibility. This is the first report describing HLA-G-expressing DC-10 and CD4+ T cells in AML patients, suggesting that they may represent a strategy by which leukemic cells escape the host’s immune system. Further studies on larger populations are required to verify our findings

MALAT1: a druggable long non-coding RNA for targeted anti-cancer approaches

Abstract The deeper understanding of non-coding RNAs has recently changed the dogma of molecular biology assuming protein-coding genes as unique functional biological effectors, while non-coding genes as junk material of doubtful significance. In the last decade, an exciting boom of experimental research has brought to light the pivotal biological functions of long non-coding RNAs (lncRNAs), representing more than the half of the whole non-coding transcriptome, along with their dysregulation in many diseases, including cancer. In this review, we summarize the emerging insights on lncRNA expression and functional role in cancer, focusing on the evolutionary conserved and abundantly expressed metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) that currently represents the best characterized lncRNA. Altogether, literature data indicate aberrant expression and dysregulated activity of MALAT1 in human malignancies and envision MALAT1 targeting as a novel treatment strategy against cancer

The cellular amount of the common γ-chain influences spontaneous or induced cell proliferation

Mutations of the IL2RG encoding the common γ-chain (γc) lead to the X-linked SCID disease. Gene correction through ex vivo retroviral transduction restored the immunological impairment in the most of treated patients, although lymphoproliferative events occurred in five of them. Even though in two cases it was clearly documented an insertional mutagenesis in LMO2, it is conceivable that γc could have a role per se in malignant lymphoproliferation. The γc is a shared cytokine receptor subunit, involved also in growth hormone (GH) receptor signaling. Through short interfering RNA or using X-linked SCID B lympho-blastoid cell lines lacking γc, we demonstrate that self-sufficient growth was strongly dependent on γc expression. Furthermore, a correlation between γc amount and the extent of constitutive activation of JAK3 was found. The reduction of γc protein expression also reduced GH-induced proliferation and STAT5 nuclear translocation in B lymphoblastoid cell lines. Hence, our data demonstrate that γc plays a remarkable role in either spontaneous or GH-induced cell cycle progression depending on the amount of protein expression, suggesting a potential role as enhancing cofactor in lymphoproliferation. Copyright © 2009 by The American Association of Immunologists, Inc

Functional interaction of common gamma-chain and growth hormone receptor signaling apparatus

We previously reported on an X-linked SCID (X-SCID) patient, who also had peripheral growth hormone (GH) hyporesponsiveness and abnormalities of the protein phosphorylation events following GH receptor (GHR) stimulation. In the present study, we examined a potential role of common cytokine receptor gamma-chain (gammac) in GHR signaling using EBV-transformed lymphocytes from healthy subjects and gammac-negative X-SCID patients. We demonstrated that the proliferative response to GH stimulation of the B cell lines of gammac-negative patients was impaired despite a comparable cellular expression of GHR molecules to controls. In patients, after GH stimulation, no phosphorylation of STAT5 was observed. In addition, the molecule localization through confocal microscopy revealed that in B cell lines of patients no nuclear translocation of STAT5b following GH stimulation occurred differently from controls. Biochemical analysis of the nuclear extracts of gammac-negative cell lines provided further evidence that the amount of STAT5b and its phosphorylated form did not increase following GH stimulation. In patients, cells reconstituted with wild-type gammac abnormal biochemical and functional events were restored resulting in nuclear translocation of STAT5. Confocal experiments revealed that GHR and gammac were colocalized on the cell membrane. Our study demonstrates the existence of a previously unappreciated relationship between GHR-signaling pathway and gammac, which is required for the activation of STAT5b in B cell lines. These data also confirm that growth failure in X-SCID is primarily related to the genetic alteration of the IL2RG gene

Oleil Hydroxytyrosol (HTOL) Exerts Anti-Myeloma Activity by Antagonizing Key Survival Pathways in Malignant Plasma Cells

Polyphenols from olive oil are endowed with several biological activities. Chemical modifications have been recently applied to these compounds to improve their therapeutic activity in different pathological settings, including cancer. Herein, we describe the in vitro effects on multiple myeloma (MM) cells of oleil hydroxytyrosol (HTOL), a synthetic fatty ester of natural hydroxytyrosol with oleic acid. HTOL reduced the viability of various human MM cell lines (HMCLs), even when co-cultured with bone marrow stromal cells, triggering ER stress, UPR and apoptosis, while it was not cytotoxic against healthy peripheral blood mononuclear cells or B lymphocytes. Whole-transcriptome profiling of HTOL-treated MM cells, coupled with protein expression analyses, indicate that HTOL antagonizes key survival pathways for malignant plasma cells, including the undruggable IRF4–c-MYC oncogenic axis. Accordingly, c-MYC gain- and loss-of-function strategies demonstrate that HTOL anti-tumor activity was, at least in part, due to c-MYC targeting. Taken together, these findings underscore the anti-MM potential of HTOL, providing the molecular framework for further investigation of HTOL-based treatments as novel anti-cancer agents

Anti-tumor Activity and Epigenetic Impact of the Polyphenol Oleacein in Multiple Myeloma

Olive oil contains different biologically active polyphenols, among which oleacein, the most abundant secoiridoid, has recently emerged for its beneficial properties in various disease contexts. By using in vitro models of human multiple myeloma (MM), we here investigated the anti-tumor potential of oleacein and the underlying bio-molecular sequelae. Within a low micromolar range, oleacein reduced the viability of MM primary samples and cell lines even in the presence of bone marrow stromal cells (BMSCs), while sparing healthy peripheral blood mononuclear cells. We also demonstrated that oleacein inhibited MM cell clonogenicity, prompted cell cycle blockade and triggered apoptosis. We evaluated the epigenetic impact of oleacein on MM cells, and observed dose-dependent accumulation of both acetylated histones and α-tubulin, along with down-regulation of several class I/II histone deacetylases (HDACs) both at the mRNA and protein level, providing evidence of the HDAC inhibitory activity of this compound; conversely, no effect on global DNA methylation was found. Mechanistically, HDACs inhibition by oleacein was associated with down-regulation of Sp1, the major transactivator of HDACs promoter, via Caspase 8 activation. Of potential translational significance, oleacein synergistically enhanced the in vitro anti-MM activity of the proteasome inhibitor carfilzomib. Altogether, these results indicate that oleacein is endowed with HDAC inhibitory properties, which associate with significant anti-MM activity both as single agent or in combination with carfilzomib. These findings may pave the way to novel potential anti-MM epi-therapeutic approaches based on natural agents