Immunoregulatory Effects of Mesenchymal Stem Cell-Derived Extracellular Vesicles on T Lymphocytes.

The immunomodulatory activity of mesenchymal stem cells (MSCs) is largely mediated by paracrine factors. We have recently shown that the immunosuppressive effects of MSCs on B lymphocytes in peripheral blood mononuclear cell (PBMC) culture can be reproduced by extracellular vesicles (EVs) isolated from MSC culture supernatants. Here we investigated the effect of bone marrow-derived MSC-EVs on T cells on PBMC cultures stimulated with anti-CD3/CD28 beads. Stimulation increased the number of proliferating CD3+ cells as well as of regulatory T cells (Tregs). Coculture with MSCs inhibited the proliferation of CD3+ cells, with no significant changes in apoptosis. Addition of MSC-EVs to PBMCs did not affect proliferation of CD3+ cells, but induced the apoptosis of CD3+ cells and of the CD4+ subpopulation and increased the proliferation and the apoptosis of Tregs. Moreover, MSC-EV treatment increased the Treg/Teff ratio and the immunosuppressive cytokine IL-10 concentration in culture medium. The activity of indoleamine 2,3-dioxygenase (IDO), an established mediator of MSC immunosuppressive effects, was increased in supernatants of PBMCs cocultured with MSCs, but was not affected by the presence of MSC-EVs. MSC-EVs demonstrate immunomodulatory effects on T cells in vitro. However, these effects and the underlying mechanisms appear to be different from those exhibited by their cells of origin

miR-221 and miR-222 Expression Affects the Proliferation Potential of Human Prostate Carcinoma Cell Lines by Targeting p27Kip1

MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of several types of cancers. Here we show that miR-221 and miR-222, encoded in tandem on chromosome X, are overexpressed in the PC3 cellular model of aggressive prostate carcinoma, as compared with LNCaP and 22Rv1 cell line models of slowly growing carcinomas. In all cell lines tested, we show an inverse relationship between the expression of miR-221 and miR-222 and the cell cycle inhibitor p27(Kip1). We recognize two target sites for the microRNAs in the 3' untranslated region of p27 mRNA, and we show that miR-221/222 ectopic overexpression directly results in p27 down-regulation in LNCaP cells. In those cells, we demonstrate that the ectopic overexpression of miR-221/222 strongly affects their growth potential by inducing a G(1) to S shift in the cell cycle and is sufficient to induce a powerful enhancement of their colony-forming potential in soft agar. Consistently, miR-221 and miR-222 knock-down through antisense LNA oligonucleotides increases p27(Kip1) in PC3 cells and strongly reduces their clonogenicity in vitro. Our results suggest that miR-221/222 can be regarded as a new family of oncogenes, directly targeting the tumor suppressor p27(Kip1), and that their overexpression might be one of the factors contributing to the oncogenesis and progression of prostate carcinoma through p27(Kip1) down-regulation

Activation of an endothelial Notch1-Jagged1 circuit induces VCAM1 expression, an effect amplified by interleukin-1β

The Notch1 and Notch4 signaling pathways regulate endothelial cell homeostasis. Inflammatory cytokines induce the expression of endothelial adhesion molecules, including VCAM1, partly by downregulating Notch4 signaling. We investigated the role of endothelial Notch1 in this IL-1β-mediated process. Brief treatment with IL-1β upregulated endothelial VCAM1 and Notch ligand Jagged1. IL-1β decreased Notch1 mRNA levels, but levels of the active Notch1ICD protein remained constant. IL-1β-mediated VCAM1 induction was downregulated in endothelial cells subjected to pretreatment with a pharmacological inhibitor of the γ-secretase, which activates Notch receptors, producing NotchICD. It was also downregulated in cells in which Notch1 and/or Jagged1 were silenced.Conversely, the forced expression of Notch1ICD in naïve endothelial cells upregulated VCAM1 per se and amplified IL-1β-mediated VCAM1 induction. Jagged1 levels increased and Notch4 signaling was downregulated in parallel. Finally, Notch1ICD and Jagged1 expression was upregulated in the endothelium of the liver in a model of chronic liver inflammation.In conclusion, we describe here a cell-autonomous, pro-inflammatory endothelial Notch1-Jagged1 circuit (i) triggering the expression of VCAM1 even in the absence of inflammatory cytokines and (ii) enhancing the effects of IL-1β. Thus, IL-1β regulates Notch1 and Notch4 activity in opposite directions, consistent with a selective targeting of Notch1 in inflamed endothelium

p53 Activation Effect in the Balance of T Regulatory and Effector Cell Subsets in Patients With Thyroid Cancer and Autoimmunity

Carcinomas evade the host immune system by negatively modulating CD4+ and CD8+ T effector lymphocytes through forkhead box protein 3 (FOXP3) positive T regulatory cells' increased activity. Furthermore, interaction of the programmed cell death 1 (PD1) molecule and its ligand programmed cell death ligand 1 (PDL1) inhibits the antitumor activity of PD1+ T lymphocytes. Immunotherapy has become a powerful strategy for tailored cancer patients' treatment both in adult and pediatric patients aiming to generate potent antitumor responses. Nevertheless, immunotherapies can generate autoimmune responses. This study aimed to investigate the potential effect of the transformation-related protein 53 (p53) reactivation by a peptide-based inhibitor of the MDM2/MDM4 heterodimer (Pep3) on the immune response in a solid cancer, i.e., thyroid carcinoma frequently presenting with thyroid autoimmunity. In peripheral blood mononuclear cell of thyroid cancer patients, Pep3 treatment alters percentages of CD8+ and CD4+ T regulatory and CD8+ and CD4+ T effector cells and favors an anticancer immune response. Of note that reduced frequencies of activated CD8+ and CD4+ T effector cells do not support autoimmunity progression. In evaluating PD1 expression under p53 activation, a significant decrease of activated CD4+PD1+ cells was detected in thyroid cancer patients, suggesting a defective regulation in the initial activation stage, therefore generating a protective condition toward autoimmune progression

PYRROLO[1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) induce apoptosis in K562 cells

BACKGROUND: The objective of this study was to gain insight into the molecular mechanism of induced cell death (apoptosis) by PYRROLO [1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) series compounds, using human (K562) cells as a model. METHODS: We focused our attention on some members of the PBTDs family to test their potential apoptotic activity in K562 cells. Important apoptotic activity was demonstrated, as evidenced by the concentration and percentage of cell death quantified by measuring PI-uptake by flow cytometry, and DNA fragmentation analyzed by agarose gel electrophoresis, generating a characteristic ladder pattern of discontinuous DNA fragments. The expression of Bcl-2 family was tested using western blotting and transfection method. RESULTS: PBTDs-mediated suppression of K562 cell proliferation was induced by apoptosis characterized by the appearance of DNA fragmentation and was associated with the poly(ADP-ribose)polymerase (PARP) cleavage. PBTD-1 and -3 treatment resulted in caspase-3 activation through down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we used K562/vector and K562/bcl-2 cells, which were generated by transfection of the cDNA of the Bcl-2 gene. As compared with K562/vector, K562/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment with 10 muM PBTD-1 and -3 for 24 h produced morphological features of apoptosis and DNA fragmentation in K562/vector cells, respectively. In contrast, PBTD-1 and -3-induced caspase-3 activation and apoptosis were inhibited in K562/Bcl-2. Furthermore, Bcl-2 overexpressing cells exhibited less cytocrome c release during PBTDs-induced apoptosis. CONCLUSION: These results indicate that PBTDs effectively induce apoptosis of K562 leukemia cells through the activation of caspase cascades. In addition, these findings indicate that Bcl-2 inhibits PBTD-1 and -3 induced-apoptosis via a mechanism that interferes with cytocrome c release, and the activity of caspase-3, which is involved in the execution of apoptosis

Fatty acid metabolism complements glycolysis in th selective regulatory t cell expansion during tumor growth

The tumor microenvironment restrains conventional T cell (Tconv) activation while facilitating the expansion of Tregs. Here we showed that Tregs’ advantage in the tumor milieu relies on supplemental energetic routes involving lipid metabolism. In murine models, tumor-infiltrating Tregs displayed intracellular lipid accumulation, which was attributable to an increased rate of fatty acid (FA) synthesis. Since the relative advantage in glucose uptake may fuel FA synthesis in intratumoral Tregs, we demonstrated that both glycolytic and oxidative metabolism contribute to Tregs’ expansion. We corroborated our data in human tumors showing that Tregs displayed a gene signature oriented toward glycolysis and lipid synthesis. Our data support a model in which signals from the tumor microenvironment induce a circuitry of glycolysis, FA synthesis, and oxidation that confers a preferential proliferative advantage to Tregs, whose targeting might represent a strategy for cancer treatment

Inhibition of exosome biogenesis affects cell motility in heterogeneous sub-populations of paediatric-type diffuse high-grade gliomas

Background: Paediatric-type diffuse High-Grade Gliomas (PDHGG) are highly heterogeneous tumours which include distinct cell sub-populations co-existing within the same tumour mass. We have previously shown that primary patient-derived and optical barcoded single-cell-derived clones function as interconnected networks. Here, we investigated the role of exosomes as a route for inter-clonal communication mediating PDHGG migration and invasion. Results: A comprehensive characterisation of seven optical barcoded single-cell-derived clones obtained from two patient-derived cell lines was performed. These analyses highlighted extensive intra-tumour heterogeneity in terms of genetic and transcriptional profiles between clones as well as marked phenotypic differences including distinctive motility patterns. Live single-cell tracking analysis of 3D migration and invasion assays showed that the single-cell-derived clones display a higher speed and longer travelled distance when in co-culture compared to mono-culture conditions. To determine the role of exosomes in PDHGG inter-clonal cross-talks, we isolated exosomes released by different clones and characterised them in terms of marker expression, size and concentration. We demonstrated that exosomes are actively internalized by the cells and that the inhibition of their biogenesis, using the phospholipase inhibitor GW4689, significantly reduced the cell motility in mono-culture and more prominently when the cells from the clones were in co-culture. Analysis of the exosomal miRNAs, performed with a miRNome PCR panel, identified clone-specific miRNAs and a set of miRNA target genes involved in the regulation of cell motility/invasion/migration. These genes were found differentially expressed in co-culture versus mono-culture conditions and their expression levels were significantly modulated upon inhibition of exosome biogenesis. Conclusions: In conclusion, our study highlights for the first time a key role for exosomes in the inter-clonal communication in PDHGG and suggests that interfering with the exosome biogenesis pathway may be a valuable strategy to inhibit cell motility and dissemination for these specific diseases

Dysregulated miR-155 and miR-125b Are Related to Impaired B-cell Responses in Down Syndrome

Children with Down Syndrome (DS) suffer from immune deficiency with a severe reduction in switched memory B cells (MBCs) and poor response to vaccination. Chromosome 21 (HSA21) encodes two microRNAs (miRs), miR-125b, and miR-155, that regulate B-cell responses. We studied B- and T- cell subpopulations in tonsils of DS and age-matched healthy donors (HD) and found that the germinal center (GC) reaction was impaired in DS. GC size, numbers of GC B cells and Follicular Helper T cells (TFH) expressing BCL6 cells were severely reduced. The expression of miR-155 and miR-125b was increased in tonsillar memory B cells and miR-125b was also higher than expected in plasma cells (PCs). Activation-induced cytidine deaminase (AID) protein, a miR-155 target, was significantly reduced in MBCs of DS patients. Increased expression of miR-155 was also observed in vitro. MiR-155 was significantly overexpressed in PBMCs activated with CpG, whereas miR-125b was constitutively higher than normal. The increase of miR-155 and its functional consequences were blocked by antagomiRs in vitro. Our data show that the expression of HSA21-encoded miR-155 and miR-125b is altered in B cells of DS individuals both in vivo and in vitro. Because of HSA21-encoded miRs may play a role also in DS-associated dementia and leukemia, our study suggests that antagomiRs may represent pharmacological tools useful for the treatment of DS

The Interplay between CD27dull and CD27bright B Cells Ensures the Flexibility, Stability, and Resilience of Human B Cell Memory

Summary: Memory B cells (MBCs) epitomize the adaptation of the immune system to the environment. We identify two MBC subsets in peripheral blood, CD27dull and CD27bright MBCs, whose frequency changes with age. Heavy chain variable region (VH) usage, somatic mutation frequency replacement-to-silent ratio, and CDR3 property changes, reflecting consecutive selection of highly antigen-specific, low cross-reactive antibody variants, all demonstrate that CD27dull and CD27bright MBCs represent sequential MBC developmental stages, and stringent antigen-driven pressure selects CD27dull into the CD27bright MBC pool. Dynamics of human MBCs are exploited in pregnancy, when 50% of maternal MBCs are lost and CD27dull MBCs transit to the more differentiated CD27bright stage. In the postpartum period, the maternal MBC pool is replenished by the expansion of persistent CD27dull clones. Thus, the stability and flexibility of human B cell memory is ensured by CD27dull MBCs that expand and differentiate in response to change. : Grimsholm et al. show that CD27dull and CD27bright represent sequential MBC developmental stages. T cell- and germinal center (GC)-independent CD27dull MBCs are the plastic source of strongly selected and GC-dependent CD27bright MBCs. CD27dull MBCs, able to expand and differentiate in response to change, ensure stability and flexibility of human B cell memory. Keywords: memory B cells, pregnancy, immunological memory, CD27, VH repertoire, immunodeficiency, aging, spleen, vaccine, germinal cente

Effects of exposure to gradient magnetic fields emitted by nuclear magnetic resonance devices on clonogenic potential and proliferation of human hematopoietic stem cells

This study investigates effects of gradient magnetic fields (GMFs) emitted by magnetic resonance imaging (MRI) devices on hematopoietic stem cells. Field measurements were performed to assess exposure to GMFs of staff working at 1.5 T and 3 T MRI units. Then an exposure system reproducing measured signals was realized to expose in vitro CD34+ cells to GMFs (1.5 T-protocol and 3 T-protocol). CD34+ cells were obtained by Fluorescence Activated Cell Sorting from six blood donors and three MRI-exposed workers. Blood donor CD34+ cells were exposed in vitro for 72 h to 1.5 T or 3 T-protocol and to sham procedure. Cells were then cultured and evaluated in colony forming unit (CFU)-assay up to 4 weeks after exposure. Results showed that in vitro GMF exposure did not affect cell proliferation but instead induced expansion of erythroid and monocytes progenitors soon after exposure and for the subsequent 3 weeks. No decrease of other clonogenic cell output (i.e., CFU-granulocyte/erythroid/macrophage/megakaryocyte and CFU-granulocyte/macrophage) was noticed, nor exposed CD34+ cells underwent the premature exhaustion of their clonogenic potential compared to sham-exposed controls. On the other hand, pilot experiments showed that CD34+ cells exposed in vivo to GMFs (i.e., samples from MRI workers) behaved in culture similarly to sham-exposed CD34+ cells, suggesting that other cells and/or microenvironment factors might prevent GMF effects on hematopoietic stem cells in vivo. Accordingly, GMFs did not affect the clonogenic potential of umbilical cord blood CD34+ cells exposed in vitro together with the whole mononuclear cell fraction