Angela Rita Elia

Transcriptome of Hypoxic Immature Dendritic Cells: Modulation of Chemokine/Receptor Expression

Hypoxia is a condition of low oxygen tension occurring in inflammatory tissues. Dendritic cells (DC) are professional antigen-presenting cells whose differentiation, migration, and activities are intrinsically linked to the microenvironment. DCs will home and migrate through pathologic tissues before reaching their final destination in the lymph node. We studied the differentiation of human monocytes into immature DCs (iDCs) in a hypoxic microenvironment. We generated iDC in vitro under normoxic (iDCs) or hypoxic (Hi-DCs) conditions and examined the hypoxia-responsive element in the promoter, gene expression, and biochemical KEGG pathways. Hi-DCs had an interesting phenotype represented by up-regulation of genes associated with cell movement/migration. In addition, the Hi-DC cytokine/receptor pathway showed a dichotomy between down-regulated chemokines and up-regulated chemokine receptor mRNA expression. We showed that CCR3, CX3CR1, and CCR2 are hypoxia-inducible genes and that CCL18, CCL23, CCL26, CCL24, and CCL14 are inhibited by hypoxia. A strong chemotactic response to CCR2 and CXCR4 agonists distinguished Hi-DCs from iDCs at a functional level. The hypoxic microenvironment promotes the differentiation of Hi-DCs, which differs from iDCs for gene expression profile and function. The most prominent characteristic of Hi-DCs is the expression of a mobility/migratory rather than inflammatory phenotype. We speculate that Hi-DCs have the tendency to leave the hypoxic tissue and follow the chemokine gradient toward normoxic areas where they can mature and contribute to the inflammatory process. (Mol Cancer Res 2008;6(2):175–85)

Human dendritic cells differentiated in hypoxia down-modulate antigen uptake and change their chemokine expression profile

Dendritic cells (DCs) are the most potent antigen‐presenting cells and fine‐tune the immune response. We have investigated hypoxias effects on the differentiation and maturation of DCs from human monocytes in vitro, and have shown that it affects DC functions. Hypoxic immature DCs (H‐iDCs) significantly fail to capture antigens through down‐modulation of the RhoA/Ezrin‐Radixin‐Moesin pathway and the expression of CD206. Moreover, H‐iDCs released higher levels of CXCL1, VEGF, CCL20, CXCL8, and CXCL10 but decreased levels of CCL2 and CCL18, which predict a different ability to recruit neutrophils rather than monocytes and create a proinflammatory and proangiogenic environment. By contrast, hypoxia has no effect on DC maturation. Hypoxic mature DCs display a mature phenotype and activate both allogeneic and specific T cells like normoxic mDCs. This study provides the first demonstration that hypoxia inhibits antigen uptake by DCs and profoundly changes the DC chemokine expression profile and may have a critical role in DC differentiation, adaptation, and activation in inflamed tissues.

Activin A induces Langerhans cell differentiation in vitro and in human skin explants.

Langerhans cells (LC) represent a well characterized subset of dendritic cells located in the epidermis of skin and mucosae. In vivo, they originate from resident and blood-borne precursors in the presence of keratinocyte-derived TGFβ. Ιn vitro, LC can be generated from monocytes in the presence of GM-CSF, IL-4 and TGFβ. However, the signals that induce LC during an inflammatory reaction are not fully investigated. Here we report that Activin A, a TGFβ family member induced by pro-inflammatory cytokines and involved in skin morphogenesis and wound healing, induces the differentiation of human monocytes into LC in the absence of TGFβ. Activin A-induced LC are Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ and possess typical APC functions. In human skin explants, intradermal injection of Activin A increased the number of CD1a+ and Langerin+ cells in both the epidermis and dermis by promoting the differentiation of resident precursor cells. High levels of Activin A were present in the upper epidermal layers and in the dermis of Lichen Planus biopsies in association with a marked infiltration of CD1a+ and Langerin+ cells. This study reports that Activin A induces the differentiation of circulating CD14+ cells into LC. Since Activin A is abundantly produced during inflammatory conditions which are also characterized by increased numbers of LC, we propose that this cytokine represents a new pathway, alternative to TGFβ, responsible for LC differentiation during inflammatory/autoimmune conditions.

Production and function of activin A in human dendritic cells

Activin A, a member of the transforming growth factor-beta superfamily, has a role in tissue repair and inflammation. In our previous studies, we identified by immunohistochemistry DC-SIGN(+) dendritic cells as a source of activin A in vivo. The present study was aimed at investigating activin A production by dendritic cells (DC) in vitro and its function. Here we demonstrate that monocyte-derived DC (Mo-DC) released abundant levels of activin A during the maturation process induced by TLR agonists, bacteria (B. henselae, S. thyphimurium), TNF and CD40L. Activin A was also induced in monocyte-derived Langerhans cells (LC) and in blood myeloid DC by LPS and/or CD40L stimulation, but not in blood plasmacytoid DC following stimulation with influenza virus. Activin A production by DC was selectively down-regulated by anti-inflammatory molecules such as dexamethasone or IL-10. Neutralization of endogenous activin A using its inhibitor follistatin, or the addition of exogenous activin A during LPS maturation did not affect Mo-DC maturation marker expression, cytokine release or allostimulatory function. However, Mo-DC matured with LPS in the presence of exogenous activin A displayed a higher FITC-dextran uptake, similar to that of immature DC. Moreover, activin A promoted monocyte differentiation to DC and reversed the inhibitory effects of IL-6 on DC differentiation of monocytes. These findings demonstrate that different subsets of DC release activin A, a cytokine that promotes DC generation, and affects the ability of mature DC to take up antigens (Ags).

miR-223 is a coordinator of breast cancer progression as revealed by bioinformatics predictions.

MicroRNAs are single-stranded non-coding RNAs that simultaneously down-modulate the expression of multiple genes post-transcriptionally by binding to the 3′UTRs of target mRNAs. Here we used computational methods to predict microRNAs relevant in breast cancer progression. Specifically, we applied different microRNA target prediction algorithms to various groups of differentially expressed protein-coding genes obtained from four breast cancer datasets. Six potential candidates were identified, among them miR-223, previously described to be highly expressed in the tumor microenvironment and known to be actively transferred into breast cancer cells. To investigate the function of miR-223 in tumorigenesis and to define its molecular mechanism, we overexpressed miR-223 in breast cancer cells in a transient or stable manner. Alternatively we overexpressed miR-223 in mouse embryonic fibroblasts or HEK293 cells and used their conditioned medium to treat tumor cells. With both approaches, we obtained elevated levels of miR-223 in tumor cells and observed decreased migration, increased cell death in anoikis conditions and augmented sensitivity to chemotherapy but no effect on adhesion and proliferation. The analysis of miR-223 predicted targets revealed enrichment in cell death and survival-related genes and in pathways frequently altered in breast cancer. Among these genes, we showed that protein levels for STAT5A, ITGA3 and NRAS were modulated by miR-223. In addition, we proved that STAT5A is a direct miR-223 target and highlighted a possible correlation between miR-223 and STAT5A in migration and chemotherapy response. Our investigation revealed that a computational analysis of cancer gene expression datasets can be a relevant tool to identify microRNAs involved in cancer progression and that miR-223 has a prominent role in breast malignancy that could potentially be exploited therapeutically.

T Cell Receptor (TCR) Gene Transfer with Lentiviral Vectors Allows Efficient Redirection of Tumor Specificity in Naive and Memory T Cells Without Prior Stimulation of Endogenous TCR

We investigated the possibility of introducing exogenous T cell receptor (TCR) genes into T cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor antigen specificity on recipient T cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T cells. Cytokine stimulation makes T cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high-affinity TCRs specific for self/tumor antigens. We selected, from a patient with vitiligo, a Mart1-specific TCR based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human peripheral blood mononuclear cells, preferential pairing of exogenous alpha and beta chains was observed, together with effective recognition of Mart1(+) melanoma cells. We tested transduction efficiency on various T cell subsets prestimulated with interleukin (IL)-2, IL-7, IL-15, and IL-21 (alone or in combination). Both naive and unfractionated CD8(+) T cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T cell differentiation (34 +/- 5% Mart1-TCR(+) cells in naive CD8(+) and 16 +/- 6% in unfractionated CD8(+)). Cytokine-prestimulated, Mart1-redirected naive and unfractionated CD8(+) cells expanded better than CD3-CD28-prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart1(+) melanoma cells. This strategy allows the generation of tumor-specific T cells encompassing truly naive T cells, endowed with an intact proliferative potential and a preserved differentiation stage.

CC-chemokine ligand 16 induces a novel maturation program in human immature monocyte-derived dendritic cells

Dendritic cells (DCs) are indispensable for initiation of primary T cell responses and a host’s defense against infection. Many proinflammatory stimuli induce DCs to mature (mDCs), but little is known about the ability of chemokines to modulate their maturation. In the present study, we report that CCL16 is a potent maturation factor for monocyte-derived DCs (MoDCs) through differential use of its four receptors and an indirect regulator of Th cell differentiation. MoDCs induced to mature by CCL16 are characterized by increased expression of CD80 and CD86, MHC class II molecules, and ex novo expression of CD83 and CCR7. They produce many chemokines to attract monocytes and T cells and are also strong stimulators in activating allogeneic T cells to skew toward Th1 differentiation. Interestingly, they are still able to take up Ag and express chemokine receptors usually bound by inflammatory ligands and can be induced to migrate to different sites where they capture Ags. Our findings indicate that induction of MoDC maturation is an important property of CCL16 and suggest that chemokines may not only organize the migration of MoDCs, but also directly regulate their ability to prime T cell responses.

miR-214 and miR-148b targeting inhibits dissemination of melanoma and breast cancer

miR-214 and miR-148b have been proposed to antagonize the effects of each other in enabling or blocking metastasis, respectively. In this study, we provide evidence deepening their role and interrelationship in the process of metastatic dissemination. Depleting miR-214 or elevating miR-148b blocked the dissemination of melanoma or breast cancer cells, an effect that could be accentuated by dual alteration. Mechanistic investigations indicated that dual alteration suppressed passage of malignant cells through the blood vessel endothelium by reducing expression of the cell adhesion molecules ITGA5 and ALCAM. Notably, transendothelial migration in vitro and extravasation in vivo impaired by singly alternating miR-214 or miR-148b could be overridden by overexpression of ITGA5 or ALCAM in the same tumor cells. In clinical specimens of primary breast cancer or metastatic melanoma, we found a positive correlation between miR-214 and ITGA5 or ALCAM along with an inverse correlation of miR-214 and miR-148b in the same specimens. Our findings define an antagonistic relationship of miR-214 and miR-148b in determining the dissemination of cancer cells via tumor-endothelial cell interactions, with possible implications for microRNA-mediated therapeutic interventions aimed at blocking cancer extravasation. Cancer Res; 76(17); 5151-62. ©2016 AACR.

The molecular and functional characterization of clonally expanded CD8+ TCR BV T cells in eosinophilic granulomatosis with polyangiitis (EGPA)

In eosinophilic granulomatosis with polyangiitis (EGPA) clonally expanded T cells might concur in granuloma formation and vascular injury. The TCR β-variable (BV) chain repertoire and third complementarity determining region (CDR3) of peripheral CD4+ and CD8+ cells in EGPA patients and age-matched controls and the expression of cytokines and chemokine receptors were investigated. The CD8+ lymphocytes of EGPA patients showed an increased frequency of BV expansions with a skewed profile of BV CDR3 lengths, increased CCR5 and CXCR3 expression and increased INFγ and TNFα production. In two patients, the TCR CDR3 cDNA sequences of the expanded BV family were identified. The CD4+ lymphocytes of EGPA patients revealed a higher expression of CRTH2 and increased production of IL-5. In conclusion, CD4+ T cells display a Th2 profile and CD8+ T cells are clonally expanded in EGPA and have a proinflammatory phenotype, suggesting their pathogenic role in vasculitic damage.

Transient proteasome inhibition as a strategy to enhance lentiviral transduction of hematopoietic CD34(+) cells and T lymphocytes: implications for the use of low viral doses and large-size vectors.

The proteasome system restricts lentiviral transduction of stem cells. We exploited proteasome inhibition as a strategy to enhance transduction of both hematopoietic stem cells (HSC) and T lymphocytes with low dose or large-size lentiviral vectors (LV). HSC showed higher transduction efficiency if transiently exposed to proteasome inhibitor MG132 (41.8% vs 10.7%, p<0.0001). Treatment with MG132 (0.5 μM) retained its beneficial effect with 3 different LV of increasing size up to 10.9 Kb (p<0.01). We extended, for the first time, the application of proteasome inhibition to the transduction of T lymphocytes. A transient exposure to MG132 significantly improved lentiviral T-cell transduction. The mean percentage of transduced T cells progressively increased from 13.5% of untreated cells, to 21% (p=0.3), 30% (p=0.03) and 37% (p=0.01) of T lymphocytes that were pre-treated with MG132 at 0.1, 0.5 and 1 μM, respectively. MG132 did not affect viability or functionality of HSC or T cells, nor significantly increased the number of integrated vector copies. Transient proteasome inhibition appears as a new procedure to safely enhance lentiviral transduction of HSC and T lymphocytes with low viral doses. This approach could be useful in settings where the use of large size vectors may impair optimal viral production.