microRNA modified tumor-derived exosomes as novel tools for maturation of dendritic cells.

Tumor-derived exosomes (TEX) are known by their immune suppression effects as well as initiation mediators in cancer progression and metastasis. Meanwhile, they are appropriate sources to induce immunity against tumor cells, as consist of tumor specific and associated antigens. The aim of the current study is modifying TEX with microRNA miR-155, miR-142, and let-7i, to enhance their immune stimulation ability and induce potent dendritic cells (DC). For this, exosomes were isolated from mouse mammalian breast cancer cell line; 4T1, and subjected to miR-155, miR-142, and let-7i by electroporation. Immature DCs were generated from mouse bone marrow in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). To mature DCs, lipopolysaccharide (LPS), TEX, and modified TEX were used. The expression level of miRNAs and their target genes (IL-6, IL-17, IL-1b, TGFß, SOCS1, KLRK1, IFNγ, and TLR4) was determined. TEX were nanovesicles with spheroid morphology which expressed CD81, CD63, and TSG101, as exosome markers, at protein level. MHCII, CD80, and CD40 as maturation markers were assessed by flow cytometry. Overexpression of miRNAs were confirmed in exosomes and mDCs. Up and downregulation of target genes confirmed the gene network in DC maturation. We found that Let-7i could efficiently induce the DC maturation, as well as miR-142 and miR-155 have enhancing effects. These findings reveal that the modified TEX would be a hopeful cell-free vaccine for the cancer treatment.

The efficiency of CD40 down regulation by siRNA and antisense ODN: comparison of lipofectamine and FuGENE6.

BACKGROUND: Dendritic cells (DCs) are ideal accessory cells in the field of gene therapy. Delivery of DNA and siRNA into mammalian cells is a useful technique in treating various diseases caused by single gene defects. Selective gene silencing by small interfering RNAs (siRNAs) and antisense oligodeoxynucleotides (ODN)s is an efficient method for the manipulation of cellular functions. An efficient, functional delivery system with no toxicity problems would be attractive. OBJECTIVE: We compared two commercially available cationic lipids, Lipofectamine and FuGENE6, in the delivery of both siRNA and antisense ODNs into mice spleen-derived DCs. METHODS: Cellular uptake was measured by the means of fluorescein-labelled non-silencing siRNA and antisense ODNs as a model system using flow cytometry. Cytotoxicity of the two delivery systems was compared with propidium iodide and annexin-V staining, and quantified with flow cytometry. The efficiency of our oligonucleotide delivery systems was compared by measuring CD40 expression by flow cytometry. RESULTS: CD40 expression in DCs was 38%. After siRNA transfection by Lipofectamine, CD40 expression decreased to 13%, and after transfection by FuGENE6, it decreased to 18%. The difference was statistically significant. CD40 down regulation in DCs transfected with the two different antisense sequences by Lipofectamine was 21% and 23%, and down regulation after transfection by FuGENE6 was 19% and 18%, respectively. The differences were not statistically significant. The effects of siRNA and antisense ODNs were specific. CONCLUSION: Lipofectamine was a more potent delivery system in siRNA effect, followed by FuGENE6. There was no significant difference between Lipofectamine and FuGENE6 as a delivery system of antisense ODNs.

The efficient generation of immunocompetent dendritic cells from leukemic blasts in acute myeloid leukemia: a local experience.

Dendritic cells (DCs) are the most important antigen presenting cells with potentially useful applications in cancer immunotherapy. Leukemic cells of patients with acute myeloid leukemia (AML) could be differentiated to DC-like cells possessing the ability of stimulating anti-leukemic immune response. Despite obvious progress in DC-based immunotherapy, some discrepancies were reported in differentiation potential of AML blasts from all patients toward DC like cells. The present study, as a local experience, was set up to generate DCs from AML blasts of various subtypes. Leukemic Blasts from 16 Iranian AML patients were differentiated into functional DCs by culturing in the presence of rhGM-CSF, rhIL-4 and TNF-alpha for 8 days. The morphology, expression of key surface molecules and allostimulatory activity of resultant DCs were compared with primary blasts and cultured but cytokine untreated control groups. The pattern of angiotensin-converting enzyme (ACE) expression was used to approve the leukemic origin of generated DCs. Neo-expression or upregulation of DC-associated markers were occurred during culturing period in cytokine treated cells compared with primary blasts and cultured but cytokine untreated control groups: CD1a (63.22% vs. 3.22% and 11.79%), CD83 (41.27% vs. 0.11% and 0.70%), CD40 (15.17% vs. 0.00% and 0.04%), CD80 (49.96 vs. 0.02% and 0.32%), CD86 (56.49% vs. 0.50% and 5.71%) and HLA-DR (52.52% vs. 14.32% and 2.49%) respectively. The potency of generated DCs to induce allogeneic T cell proliferation increased significantly compared to pre and post culture control groups (27,533.4 +/- 2,548.3, 8,820.4 +/- 1,639.4 and 3,200.35 +/- 976 respectively). The expression pattern of ACE in AML-DCs, blast cells and DCs derived from normal monocytes (7.93%, 1.28% and 74.97% respectively) confirmed the leukemic origin of DCs. Our data confirmed the generation of sufficient AML-derived cells with the properties of DCs in all cases. This potency of AML blasts, offers a useful route for active immunotherapy of AML patients.

The efficient isolation of murine splenic dendritic cells and their cytochemical features.

Despite their importance in professional antigen presentation and their ubiquitous presence, dendritic cells (DCs) are usually found in such trace amounts in tissues that their isolation with high purity is a difficult task. Because of their scarcity, accurate determination of the purity of isolated dendritic cells is very important. In this study, we purified murine splenic dendritic cells by a three-step enrichment method and evaluated their morphological, cytochemical and functional characteristics. Purity of the isolated cells was determined by established methods such as flow cytometry (FC) and immunocytochemistry (ICC) using anti-CD11c monoclonal antibody. In order to test purified DC functional properties, we used in vivo antigen presentation assay. Our results showed that antigen-pulsed DCs are potent stimulators of antigen-specific lymphocyte proliferation. We studied myeloperoxidase (MPO) and non-specific esterase (NSE) activity in isolated cells to determine the purity of dendritic cells compared to more conventional methods. Our results showed that murine splenic dendritic cells were deficient in both MPO and NSE activity and the percentage of purity obtained by NSE staining on isolated cells was comparable to the results obtained by either FC or ICC. To our knowledge, this is the first report on using NSE activity for determination of the purity of isolated murine splenic dendritic cells. We, therefore, recommend that NSE activity be employed as a simple, inexpensive and yet accurate method for evaluation of the purity of isolated murine splenic dendritic cells.