Are interleukin-16 and thrombopoietin new tools for the in vitro generation of dendritic cells? (original) (raw)
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IL-16 Can Synergize With Early Acting Cytokines to Expand Ex Vivo CD34 + Isolated from Cord Blood
We previously reported that interleukin (IL)-16 can induce CD34 + hematopoietic cells to proliferate and differentiate in vitro into phenotypically and functionally mature dendritic cells. In this study, we investigated the effects of IL-16 on the expansion of CD34 + cells from human cord blood (CB). CD34 + CB cells were cultured for 14 days in medium containing a basal cocktail (BC) containing stem cell factor, Flt-3 ligand, thrombopoietin, IL-6, and IL-3 with and without IL-16 as a control. Interleukin-16 added to BC signifi cantly enhanced the expansion of CD34 + cells (66.47 ± 1.46-fold vs. 36.23 ± 1.67-fold), as well as CD34 + CD38 − early stem cells (106.67 ± 2.34-fold vs. 63.42 ± 1.89-fold) and progenitor cells [colony-forming unit (CFU)-mixed-(GEMM)] and multilineage-committed progenitors [burst-forming unit (BFU-E), CFU–granulocyte, macrophage (-GM), CFU-megakaryocyte (-MK)]. Interleukin-16 also signifi cantly increased long-term culture–initiating cells (160.8 ± 3.45-fold vs. 83 ± 2.89-fold with BC alone). Moreover, CD34 + cells expanded with IL-16 maintained the capacity to differentiate into the lymphoid-B and-NK lineage. The addition of IL-16 to BC increased the migratory capacity of expanded CD34 + cells compared to BC alone, leaving the expression of CXCR4 unaffected, and decreased the percentage of CD34 + CD4 + cells. We showed that IL-16 released endogenously affected the ex vivo expansion of CD34 + cells. Overall, this study suggests that IL-16 may have a new role in promoting the expansion of hematopoietic stem cells and may represent a new tool for the expansion of CD34 + cells for clinical applications.
Immunology, 1997
We report the generation of dendritic cells (DC) starting from CD34+ bone marrow (BM) progenitor cells, using a two-stage culture system in which, besides granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-a (TNF-a), stem-cell factor (SCF) was added during the first 5 days, while interleukin-4 (IL-4) and/or interferon-y (IFN-y) were added during the secondary culture period of 9 days. Addition of IL-4 favoured the outgrowth of CDla+, HLA-DR+, CD4+, CD40+, CD80+ but CD14cells with dendritic morphology and strong antigen-presenting capacity. Addition of IFN-y selectively induced HLA-DR and CD86 but did not up-regulate CDla expression or antigen-presenting capacity of the differentiated cells. An antagonism between IL-4 and IFN-y could further be confirmed in that, as compared with IL-4 alone, the simultaneous addition of IL-4 and IFN-y to GM-CSF plus TNF-a during maturation reduced both the phenotypical (CD la, CD4, CD40) and functional characteristics of DC. Using receptor-specific TNF-ax mutants, we investigated the relative involvement of TNF-ax receptors RI and R2 in the generation of DC. The induction of CDla and HLA-DR, as well as the increase in allostimulatory capacity were dependent on TNF-Rl triggering, whereas triggering through TNF-R2 had no measurable effect. We conclude first, that the expansion of DC from BM progenitors could most effectively be enhanced in a two-stage culture assay using SCF, GM-CSF, TNF-ac and IL-4; second, that the effect of TNF-a in DC generation involves signalling via the TNF-Rl receptor; and third, that IFN-y counteracts some of the effects of IL-4 in DC generation.
Immunology, 1997
We report the generation of dendritic cells (DC) starting from CD34+ bone marrow (BM) progenitor cells, using a two-stage culture system in which, besides granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-a (TNF-a), stem-cell factor (SCF) was added during the first 5 days, while interleukin-4 (IL-4) and/or interferon-y (IFN-y) were added during the secondary culture period of 9 days. Addition of IL-4 favoured the outgrowth of CDla+, HLA-DR+, CD4+, CD40+, CD80+ but CD14cells with dendritic morphology and strong antigen-presenting capacity. Addition of IFN-y selectively induced HLA-DR and CD86 but did not up-regulate CDla expression or antigen-presenting capacity of the differentiated cells. An antagonism between IL-4 and IFN-y could further be confirmed in that, as compared with IL-4 alone, the simultaneous addition of IL-4 and IFN-y to GM-CSF plus TNF-a during maturation reduced both the phenotypical (CD la, CD4, CD40) and functional characteristics of DC. Using receptor-specific TNF-ax mutants, we investigated the relative involvement of TNF-ax receptors RI and R2 in the generation of DC. The induction of CDla and HLA-DR, as well as the increase in allostimulatory capacity were dependent on TNF-Rl triggering, whereas triggering through TNF-R2 had no measurable effect. We conclude first, that the expansion of DC from BM progenitors could most effectively be enhanced in a two-stage culture assay using SCF, GM-CSF, TNF-ac and IL-4; second, that the effect of TNF-a in DC generation involves signalling via the TNF-Rl receptor; and third, that IFN-y counteracts some of the effects of IL-4 in DC generation.
Stem Cells, 2004
There is a growing interest in generating dendritic cells (DCs) for using as vaccines. Several cytokines, especially stem cell factor (SCF) and FLT3-ligand (FL), have been identified as essential to produce large numbers of myeloid precursors and even to increase DC yield obtained by the action of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-α). However, there are few studies on the effect of the early-acting cytokines, commonly used to expand CD34 + progenitor cells, on DC generation. We report here that in the absence of serum, SCF, FL, and thrombopoietin (TPO) plus interleukin-6 (IL-6) and SCF, FL, and TPO plus IL-3 were able to generate CD14 + CD1aand CD14-CD1a + myeloid DC precursors from CD34 + cells, but IL-6 had an inhibitory effect on the generation of CD14-CD1a + cells. Both DC precursors differentiated into mature DCs by GM-CSF, IL-4, and TNFα, and DCs obtained from both types of culture exhibited equal allostimulatory capacity. CD1a + DCs generated could be identified on the basis of DC-specific intracellular adhesion molecule-grabbing nonintegrin (DC-SIGN) expression, a novel C-type lectin receptor expressed on dermal DCs but not on Langerhans cells. In addition, the inclusion of IL-3 to the culture medium induced the appearance of CD13cells that differentiated into plasmacytoid DC (DC2) on the addition of TNF-α, allowing the identification of developmental stages of DC2. Like true plasmacytoid DCs, these cells secreted interferon-α after TLR9-specific stimulation with a specific CpG nucleotide.
Unexpected impairment of TNF-α-induced maturation of human dendritic cells in vitro by IL-4
Journal of Translational Medicine, 2016
Background: An efficient strategy for programing dendritic cells (DCs) for cancer immunotherapy is the optimization of their maturation so that they can efficiently stimulate cancer-specific T cell responses. Interleukin (IL)-4 has appeared as an essential cytokine, widely used in vitro with granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate monocytes into immature DCs (iDC) and to prevent macrophage formation. Conflicting data have been published regarding the effect of IL-4 on functional DC maturation. To further understand IL-4's effects on DC maturation and function in vitro, we choose the most commonly used maturation factor tumor necrosis factor (TNF)-α. Methods: Human monocyte-derived iDC were treated for 48 h with GM-CSF and TNF-α in the presence (IL-4 +-DC) or absence (IL-4 −-DC) of IL-4 and functions of both DC populations were compared. Results: On mixed lymphocyte reaction assay, IL-4 +-DC were less potent than IL-4 −-DC at inducing the proliferation of allogeneic CD4 + T cells and the proportion of activated T cells expressing CD69 and/or CD25 was smaller. Interleukin-4 reduced the cell-surface expression of TNF-α-induced DC maturation markers CD83, CD86, HLA-DR and CD25 and generated a heterogeneous population of DCs. IL-4 +-DC secreted less IL-12 and more IL-10 than IL-4 −-DC following activation by soluble CD40L, and IL-4 +-DC-activated T cells secreted lesser amounts of T helper (Th) 1 cytokines (IL-2 and interferon-γ). Importantly, IL-4 impaired the in vitro migratory capacity of DCs in response to CCL21 and CCL19 chemokines. This effect was related to reduced expression of CCR7 at both mRNA and protein levels. Conclusion: Interleukin-4 used with GM-CSF and TNF-α during the maturation of DCs in vitro impaired DC functions and disturbed the maturation effect of TNF-α. Finally, our study reinforces the view that the quality of the DC maturation stimulus, which regulates DC migration and cytokine production, may be a decisive feature of the immunogenicity of DCs.
Experimental Hematology, 2012
Plasmacytoid dendritic cells (pDC), a subset of dendritic cells characterized by a rapid and massive type-I interferon secretion through the Toll-like receptor pathway in response to viral infection, play important roles in the pathogenesis of several diseases, such as chronic viral infections (e.g., hepatitis C virus, human immunodeficiency virus), autoimmunity (e.g., psoriasis, systemic lupus erythematosus), and cancer. As pDC represent a rare cell type in the peripheral blood, the goal of this study was to develop a new method to efficiently generate large numbers of cells from a limited number of CD34(+) cord blood progenitors to provide a tool to resolve important questions about how pDC mediate tolerance, autoimmunity, and cancer. Human CD34(+) hematopoietic progenitor cells isolated from cord blood were cultured with a combination of Flt3-ligand (Flt3L), thrombopoietin (TPO), and one of the following cytokine: interleukin (IL)-3, interferon-β(IFN-β), or prostaglandin E2(PGE(2)). Cells obtained in the different culture conditions were analyzed for their phenotype and functional characteristics. The addition of IL-3 cooperates with Flt3L and TPO in the induction of pDC from CD34(+) hematopoietic progenitor cells. Indeed, Flt3L/TPO alone or supplemented with prostaglandin E2 or interferon-β produced smaller amounts of pDC from hematopoietic progenitor cells. In addition, pDC generated in Flt3L/TPO/IL-3 cultures exhibited morphological, immunohistochemical, and functional features of peripheral blood pDC. We showed that IL-3, in association with Flt3L and TPO, provides an advantageous tool for large-scale generation of pDC. This culture condition generated, starting from 2 × 10(5) CD34(+) cells, up to 2.6 × 10(6) pDC presenting features of blood pDC.
International Immunology, 2005
Dendritic cells (DCs) are the most potent professional antigen-presenting cells which can activate T cells to induce the primary immune response. For clinical studies, DCs are often differentiated in vitro from peripheral blood mononuclear cells (PBMCs) through treatment with granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4. However, IL-13, a cytokine closely related to IL-4, has also been reported to induce differentiation equally or more efficiently when used with GM-CSF. For the present study, we compared the DC characteristics exhibited by iDCs and LPS-matured DCs differentiated from PBMCs using GM-CSF and IL-4 or IL-13. Physical characteristics examined include cellular morphology and surface phenotype. Functional traits investigated include FITC-dextran uptake, IL-10 and IL-12 production, allostimulation and cytokine production by stimulated T cells and antigen-specific T cell stimulation. Compared with IL-13-derived DCs, IL-4 treatment yielded more differentiated DCs, with extensive dendrites and higher expression of DC-SIGN, DEC-205, CD86 and HLA-DR. In addition, IL-4 DCs were more efficient at inducing allogeneic T cell proliferation and immature IL-4 DCs had higher endocytic activity at low FITC-dextran concentrations (1 lg ml ÿ1). Although IL-13 was capable of generating DCs from PBMCs, it was not as effective as IL-4 in generating DC phenotype and functionality. Thus, the use of GM-CSF and IL-4 is the more efficient treatment for inducing DC differentiation from PBMCs.