Human NKT cells promote monocyte differentiation into suppressive myeloid antigen-presenting cells.
ABSTRACT: NKT cells have been shown to promote peripheral tolerance in a number of model systems, yet the processes by which they exert their regulatory effects remain poorly understood. Here, we show that soluble factors secreted by human NKT cells instruct human peripheral blood monocytes to differentiate into myeloid APCs that have suppressive properties. NKT instructed monocytes acquired a cell surface phenotype resembling myeloid DCs. However, whereas control DCs that were generated by culturing monocytes with recombinant GM-CSF and IL-4 had a proinflammatory phenotype characterized by the production of IL-12 with little IL-10, NKT-instructed APCs showed the opposite cytokine production profile of high IL-10 with little or no IL-12. The control DCs efficiently stimulated peripheral blood T cell IFN-gamma secretion and proliferation, whereas NKT-instructed APCs silenced these T cell responses. Exposure to NKT cell factors had a dominant effect on the functional properties of the DCs, since DCs differentiated by recombinant GM-CSF and IL-4 in the presence of NKT cell factors inhibited T cell responses. To confirm their noninflammatory effects, NKT-instructed APCs were tested in an in vivo assay that depends on the activation of antigen-specific human T cells. Control DCs promoted substantial tissue inflammation; however, despite a marked neutrophilic infiltrate, there was little edema in the presence of NKT-instructed APCs, suggesting the inflammatory cascade was held in check. These results point to a novel pathway initiated by NKT cells that can contribute to the regulation of human antigen-specific Th1 responses.
Project description:Comparison of the RNA expression profiles of CD14+ monocytes from human peripheral blood with derived dendritic cells (DCs) and macrophages (MACs) obtained by exposure with GM-CSF/IL-4 and GM-CSF, respectively, and with mature DCs and MACs after lipopolysaccharide (LPS) exposure The expression profiles of RNA of human CD14+ monocytes were compared with derived immature dendritic cells (iDCs) and macrophages (iMACs) following GM-CSF/IL-4 and GM-CSF incubation, and then activation/maturation with lypopolysaccharyde (LPS) using the Affymetrix PrimeView Human Gene Expression array (Affymetrix, Santa Clara, CA). This platform allows the interrogation of >36,000 transcrits and variants per sample. The samples were hybridized in the array following the manufacturerâ??s instructions. Total RNA isolated by standard procedures from CD14+ cells (total monocytes, MOs) corresponding to three sets of samples of monocytes (MOs), derived immature DCs and MACs (iDCS and iMACS) and activated/mature DCs and MACs following incubation with LPS (mDCS and mMACs)
Project description:Both classical DCs (cDCs) and monocyte-derived DCs (Mo-DCs) are capable of cross-priming CD8(+) T cells in response to cell-associated antigens. We found that Ly-6C(hi)TREML4(-) monocytes can differentiate into Zbtb46(+) Mo-DCs in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) but that Ly-6C(hi)TREML4(+) monocytes were committed to differentiate into Ly-6C(lo)TREML4(+) monocytes. Differentiation of Zbtb46(+) Mo-DCs capable of efficient cross-priming required both GM-CSF and IL-4 and was accompanied by the induction of Batf3 and Irf4. However, monocytes require IRF4, but not BATF3, to differentiate into Zbtb46(+) Mo-DCs capable of cross-priming CD8(+) T cells. Instead, Irf4(-/-) monocytes differentiate into macrophages in response to GM-CSF and IL-4. Thus, cDCs and Mo-DCs require distinct transcriptional programs of differentiation in acquiring the capacity to prime CD8(+) T cells. These differences may be of consideration in the use of therapeutic DC vaccines based on Mo-DCs.
Project description:NKT cells are innate lymphocytes that can recognize self or foreign lipids presented by CD1d molecules. NKT cells have been shown to inhibit the development of autoimmunity in murine model systems, however, the pathways by which they foster immune tolerance remain poorly understood. Here we show that autoreactive human NKT cells stimulate monocytes to differentiate into myeloid APCs that have a regulatory phenotype characterized by poor conjugate formation with T cells. The NKT cell instructed myeloid APCs show elevated expression of the inhibitory ligand PD-L2, and blocking PD-L1 and PD-L2 during interactions of the APCs with T cells results in improved cluster formation and significantly increased T cell proliferative responses. The elevated expression of PD-L molecules on NKT-instructed APCs appears to result from exposure to extracellular ATP that is produced during NKT-monocyte interactions, and blocking purinergic signaling during monocyte differentiation results in APCs that form clusters with T cells and stimulate their proliferation. Finally, we show that human monocytes and NKT cells that are injected into immunodeficient mice co-localize together in spleen and liver, and after 3 days in vivo in the presence of NKT cells a fraction of the myeloid cells have upregulated markers associated with differentiation into professional APCs. These results suggest that autoreactive human NKT cells may promote tolerance by inducing the differentiation of regulatory myeloid APCs that limit T cell proliferation through expression of PD-L molecules.
Project description:Interleukin-21 (IL-21) has broad actions on T and B cells, but its actions in innate immunity are poorly understood. Here we show that IL-21 induced apoptosis of conventional dendritic cells (cDCs) via STAT3 and Bim, and this was inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). ChIP-Seq analysis revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3. Expression of IL-21 in vivo decreased cDC numbers, and this was prevented by GM-CSF. Moreover, repetitive ?-galactosylceramide injection of mice induced IL-21 but decreased GM-CSF production by natural killer T (NKT) cells, correlating with decreased cDC numbers. Furthermore, adoptive transfer of wild-type CD4+ T cells caused more severe colitis with increased DCs and interferon-? (IFN-?)-producing CD4+ T cells in Il21r(-/-)Rag2(-/-) mice (which lack T cells and have IL-21-unresponsive DCs) than in Rag2(-/-) mice. Thus, IL-21 and GM-CSF exhibit cross-regulatory actions on gene regulation and apoptosis, regulating cDC numbers and thereby the magnitude of the immune response.
Project description:Immunotherapy using dendritic cells (DCs) is a promising treatment modality for cancer. However, the limited number of functional DCs from peripheral blood has been linked to the unsatisfactory clinical efficacies of current DC-based cancer immunotherapies. We previously generated proliferating antigen-presenting cells (APCs) by genetically engineering myeloid cells derived from induced pluripotent stem cells (iPSC-pMCs), which offer infinite functional APCs for broad applications in cancer therapy. Herein, we aimed to further enhance the antitumor effect of these cells by genetic modification. GM-CSF gene transfer did not affect the morphology, or surface phenotype of the original iPSC-pMCs, however, it did impart good viability to iPSC-pMCs. The resultant cells induced GM-CSF-dependent CD8<sup>+</sup> T cell homeostatic proliferation, thereby enhancing antigen-specific T cell priming <i>in vitro</i>. Administration of the tumor antigen-loaded GM-CSF-producing iPSC-pMCs (GM-pMCs) efficiently stimulated antigen-specific T cells and promoted effector cell infiltration of the tumor tissues, leading to an augmented antitumor effect. To address the potential tumorigenicity of iPSC-derived products, irradiation was applied and found to restrict the proliferation of GM-pMCs, while retaining their T cell-stimulatory capacity. Furthermore, the irradiated cells exerted an antitumor effect equivalent to that of bone marrow-derived DCs obtained from immunocompetent mice. Additionally, combination with immune checkpoint inhibitors increased the infiltration of CD8<sup>+</sup> or NK1.1<sup>+</sup> effector cells and decreased CD11b<sup>+</sup>/Gr-1<sup>+</sup> cells without causing adverse effects. Hence, although GM-pMCs have certain characteristics that differ from endogenous DCs, our findings suggest the applicability of these cells for broad clinical use and will provide an unlimited source of APCs with uniform quality.
Project description:Dendritic cells (DCs) are professional antigen-presenting cells with the ability to induce primary T-cell responses. They are commonly produced by culturing monocytes in the presence of IL-4 and GM-CSF (cells produced in this manner are called conventional DCs). Here we report the generation of two functionally distinct subsets of DCs derived from programmable cells of monocytic origin (PCMOs) in the presence of IL-3 or tumor necrosis factor alpha (TNF-α). Monocytes were treated with macrophage colony-stimulating factor (M-CSF) and IL-3 for 6 days and then incubated with IL-4 and IL-3 (for IL-3 DCs) or with IL-4, GM-CSF and TNF-α (for TNF-α DCs) for 7 days. Monocytes were then loaded with tumor lysate (used as antigen), and poly (I∶C) was added. The maturation factors TNF-α and monocyte conditioned medium (MCM) were added on days 4 and 5, respectively. The phenotypes of the DCs generated were characterized by flow cytometry, and the cells' phagocytic activities were measured using FITC-conjugated latex bead uptake. T-cell proliferation and cytokine release were assayed using MTT and commercially available ELISA kits, respectively. We found that either IL-3DCs or TNF-α DCs induce T-cell proliferation and cytokine secretion; the cytokine release pattern showed reduced IL-12/IL-10 and IFN-γ/IL-4 ratios in both types of DCs and in DC-primed T-cell supernatant, respectively, which confirmed that the primed T cells were polarized toward aTh2-type immune response. We concluded that PCMOs are a new cell source that can develop into two functionally distinct DCs that both induce a Th2-type response in vitro. This modality can be used as a DC-based immunotherapy for autoimmune diseases.
Project description:Dendritic cells (DCs) play a key role in the initiation of an immune response and are known as "professional" APCs because of their ability to activate naive T cells. A widely used method to generate DCs in vitro is to culture bone marrow (BM) cells or blood monocytes in the presence of GM-CSF and IL-4. In this study, we show that a small population of NK cells residing in the BM of RAG-/-, but not RAG-/- ?c chain-/- mice, remain in the DC culture and is the source of IFN-? produced after stimulation with LPS. These cells, which may represent early promoters of LPS-induced responses, have to be taken into account when interpreting experiments using BM-derived DCs.
Project description:Comparison of the RNA expression profiles of CD14+ monocytes from human peripheral blood with derived dendritic cells (DCs) and macrophages (MACs) obtained by exposure with GM-CSF/IL-4 and GM-CSF, respectively, and with mature DCs and MACs after lipopolysaccharide (LPS) exposure The expression profiles of RNA of human CD14+ monocytes were compared with derived immature dendritic cells (iDCs) and macrophages (iMACs) following GM-CSF/IL-4 and GM-CSF incubation, and then activation/maturation with lypopolysaccharyde (LPS) using the Affymetrix PrimeView Human Gene Expression array (Affymetrix, Santa Clara, CA). This platform allows the interrogation of >36,000 transcrits and variants per sample. The samples were hybridized in the array following the manufacturer’s instructions. Total RNA isolated by standard procedures from CD14+ cells (total monocytes, MOs) corresponding to three sets of samples of monocytes (MOs), derived immature DCs and MACs (iDCS and iMACS) and activated/mature DCs and MACs following incubation with LPS (mDCS and mMACs)
Project description:Human monocytes have been grouped into classical (CD14++CD16-), non-classical (CD14dimCD16++), and intermediate (CD14++CD16+) subsets. Documentation of normal function and variation in this complement of subtypes, particularly their differentiation potential to dendritic cells (DC) or macrophages, remains incomplete. We therefore phenotyped monocytes from peripheral blood of healthy subjects and performed functional studies on high-speed sorted subsets. Subset frequencies were found to be tightly controlled over time and across individuals. Subsets were distinct in their secretion of TNF?, IL-6, and IL-1? in response to TLR agonists, with classical monocytes being the most producers and non-classical monocytes the least. Monocytes, particularly those of the non-classical subtype, secreted interferon-? (IFN-?) in response to intracellular TLR3 stimulation. After incubation with IL-4 and GM-CSF, classical monocytes acquired monocyte-derived DC (mo-DC) markers and morphology and stimulated allogeneic T cell proliferation in MLR; intermediate and non-classical monocytes did not. After incubation with IL-3 and Flt3 ligand, no subset differentiated to plasmacytoid DC. After incubation with GM-CSF (M1 induction) or macrophage colony-stimulating factor (M-CSF) (M2 induction), all subsets acquired macrophage morphology, secreted macrophage-associated cytokines, and displayed enhanced phagocytosis. From these studies we conclude that classical monocytes are the principal source of mo-DCs, but all subsets can differentiate to macrophages. We also found that monocytes, in particular the non-classical subset, represent an alternate source of type I IFN secretion in response to virus-associated TLR agonists.
Project description:Due to their ability to present foreign antigens and prime naïve T cells, macrophages, and dendritic cells (DCs) are referred to as professional antigen-presenting cells (APCs). Although activated macrophages may function as APCs, these cells are particularly effective at directly engaging pathogens through phagocytosis, and production of antimicrobial compounds. On the other hand, DCs possess superb antigen-presenting and costimulatory capacity and they are essential for commencement and regulation of adaptive immune responses. In in vitro models, development of mature mammalian DCs from monocytes requires sequential exposure to growth factors (including GM-CSF and IL-4) and proinflammatory stimuli such as toll-like receptor (TLR) ligands. Currently, except for IL-4/13, neither orthologs nor functional analogs of the growth factors which are essential for the differentiation of mammalian DCs (including GM-CSF and FLT3) have been identified in teleosts and data about differentiation of piscine APCs is scant. In the present study, primary salmon mononuclear phagocytes (MPs) stimulated in vitro for 5-7 days with a B-class CpG oligodeoxynucleotides (ODN 2006PS) underwent morphological differentiation and developed "dendritic" morphology, characterized by long, branching pseudopodia. Transcriptional profiling showed that these cells expressed high levels of proinflammatory mediators characteristic for M1 polarized MPs. However, the cells treated with CpGs for 7 days downregulated their surface MHCII molecules as well as their capacity to endocytose ovalbumin and exhibited attenuated allostimulatory activity. This concurred with transcriptional downregulation of costimulatory CD80/86 and upregulation of inhibitory CD274 (B7-H1) genes. Despite their exhausted allostimulatory activity, these cells were still responsive to re-stimulation with gardiquimod (a TLR7/8 ligand) and further upregulated a wide array of immune genes including proinflammatory mediators such as intereukin-1 beta and tumor necrosis factor. Overall, the presented data highlight the disparate effects TLR ligands may have on the proinflammatory status of APCs, on one side, and their antigen-presenting/costimulatory functions, on the other. These findings also indicate that despite the poor phylogenetic conservation of the growth factors involved in the differentiation of DCs, some of the processes that orchestrate the development and the differentiation of professional APCs are conserved between teleosts in mammals.