This work was supported by Agency for Science, Technology and Reasearch Core Funding (to J.E.C).

<ol><li>Jolles, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paul+Langerhans%5BTitle%5D)+AND+%22J+Clin+Pathol%22%5BJournal%5D)">Paul Langerhans.</a> J Clin Pathol. 55 (4), 243-24 (2002).</li>
<li>Steinman, R. M., Cohn, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+a+novel+cell+type+in+peripheral+lymphoid+organs+of+mice.+I.+Morphology%2C+quantitation%2C+tissue+distribution%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution.</a> J Exp Med. 137 (5), 1142-1162 (1973).</li>
<li>Steinman, R. M., Swanson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+endocytic+activity+of+dendritic+cells%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">The endocytic activity of dendritic cells.</a> J Exp Med. 182 (2), 283-288 (1995).</li>
<li>Hemmi, H., Akira, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TLR+signalling+and+the+function+of+dendritic+cells%5BTitle%5D)+AND+%22Chem+Immunol+Allergy%22%5BJournal%5D)">TLR signalling and the function of dendritic cells.</a> Chem Immunol Allergy. 86, 120-135 (2005).</li>
<li>Mueller, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+maintaining+peripheral+tolerance%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Mechanisms maintaining peripheral tolerance.</a> Nat Immunol. 11 (1), 21-27 (2010).</li>
<li>Inaba, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+presentation+of+phagocytosed+cellular+fragments+on+the+major+histocompatibility+complex+class+II+products+of+dendritic+cells%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells.</a> J Exp Med. 188 (11), 2163-2173 (1998).</li>
<li>Caux, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((B70%2FB7-2+is+identical+to+CD86+and+is+the+major+functional+ligand+for+CD28+expressed+on+human+dendritic+cells%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">B70/B7-2 is identical to CD86 and is the major functional ligand for CD28 expressed on human dendritic cells.</a> J Exp Med. 180 (5), 1841-1847 (1994).</li>
<li>Paavonen, J., Lehtinen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interactions+between+human+papillomavirus+and+other+sexually+transmitted+agents+in+the+etiology+of+cervical+cancer%5BTitle%5D)+AND+%22Curr+Opin+Infect+Dis%22%5BJournal%5D)">Interactions between human papillomavirus and other sexually transmitted agents in the etiology of cervical cancer.</a> Curr Opin Infect Dis. 12 (1), 67-71 (1999).</li>
<li>Ohtani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mammalian+target+of+rapamycin+and+glycogen+synthase+kinase+3+differentially+regulate+lipopolysaccharide-induced+interleukin-12+production+in+dendritic+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Mammalian target of rapamycin and glycogen synthase kinase 3 differentially regulate lipopolysaccharide-induced interleukin-12 production in dendritic cells.</a> Blood. 112 (3), 635-643 (2008).</li>
<li>Wilde, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dendritic+cells+in+renal+biopsies+of+patients+with+ANCA-associated+vasculitis%5BTitle%5D)+AND+%22Nephrol+Dial+Transplant%22%5BJournal%5D)">Dendritic cells in renal biopsies of patients with ANCA-associated vasculitis.</a> Nephrol Dial Transplant. 24 (7), 2151-2156 (2009).</li>
<li>Al-Hello, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+enterovirus+strain+isolated+from+diabetic+child+belongs+to+a+genetic+subcluster+of+echovirus+11%2C+but+is+also+neutralised+with+monotypic+antisera+to+coxsackievirus+A9%5BTitle%5D)+AND+%22J+Gen+Virol%22%5BJournal%5D)">An enterovirus strain isolated from diabetic child belongs to a genetic subcluster of echovirus 11, but is also neutralised with monotypic antisera to coxsackievirus A9.</a> J Gen Virol. 89, 1949-1959 (2008).</li>
<li>Ziegler-Heitbrock, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nomenclature+of+monocytes+and+dendritic+cells+in+blood%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Nomenclature of monocytes and dendritic cells in blood.</a> Blood. 116 (16), 74-80 (2010).</li>
<li>Osugi, Y., Vuckovic, S., Hart, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myeloid+blood+CD11c%28%2B%29+dendritic+cells+and+monocyte-derived+dendritic+cells+differ+in+their+ability+to+stimulate+T+lymphocytes%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Myeloid blood CD11c(+) dendritic cells and monocyte-derived dendritic cells differ in their ability to stimulate T lymphocytes.</a> Blood. 100 (8), 2858-2866 (2002).</li>
<li>Reynolds, G., Haniffa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+and+Mouse+Mononuclear+Phagocyte+Networks%3A+A+Tale+of+Two+Species%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Human and Mouse Mononuclear Phagocyte Networks: A Tale of Two Species.</a> Front Immunol. 6, (2015).</li>
<li>Jefford, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+comparison+of+DCs+generated+in+vivo+with+Flt3+ligand+or+in+vitro+from+blood+monocytes%3A+differential+regulation+of+function+by+specific+classes+of+physiologic+stimuli%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Functional comparison of DCs generated in vivo with Flt3 ligand or in vitro from blood monocytes: differential regulation of function by specific classes of physiologic stimuli.</a> Blood. 102 (5), 1753-1763 (2003).</li>
<li>Menck, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+human+monocytes+by+double+gradient+centrifugation+and+their+differentiation+to+macrophages+in+teflon-coated+cell+culture+bags%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation of human monocytes by double gradient centrifugation and their differentiation to macrophages in teflon-coated cell culture bags.</a> J Vis Exp. (91), (2014).</li>
<li>Zhou, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+human+granulocyte+and+monocyte+isolation+procedures+on+functional+studies%5BTitle%5D)+AND+%22Clin+Vaccine+Immunol%22%5BJournal%5D)">Impact of human granulocyte and monocyte isolation procedures on functional studies.</a> Clin Vaccine Immunol. 19 (7), 1065-1074 (2012).</li>
<li>Menon, V., Thomas, R., Ghale, A. R., Reinhard, C., Pruszak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometry+protocols+for+surface+and+intracellular+antigen+analyses+of+neural+cell+types%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Flow cytometry protocols for surface and intracellular antigen analyses of neural cell types.</a> J Vis Exp. (94), (2014).</li>
<li>Faresjo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+useful+guide+for+analysis+of+immune+markers+by+fluorochrome+%28Luminex%29+technique%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">A useful guide for analysis of immune markers by fluorochrome (Luminex) technique.</a> Methods Mol Biol. 1172, 87-96 (2014).</li>
<li>Baker, H. N., Murphy, R., Lopez, E., Garcia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conversion+of+a+capture+ELISA+to+a+Luminex+xMAP+assay+using+a+multiplex+antibody+screening+method%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Conversion of a capture ELISA to a Luminex xMAP assay using a multiplex antibody screening method.</a> J Vis Exp. (65), (2012).</li>
<li>Defawe, O. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+and+qualification+of+a+multiplex+bead+array+to+assess+cytokine+and+chemokine+production+by+vaccine-specific+cells%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Optimization and qualification of a multiplex bead array to assess cytokine and chemokine production by vaccine-specific cells.</a> J Immunol Methods. 382 (1-2), 117-128 (2012).</li>
<li>Nicholls, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioenergetic+profile+experiment+using+C2C12+myoblast+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Bioenergetic profile experiment using C2C12 myoblast cells.</a> J Vis Exp. (46), (2010).</li>
<li>Chunharas, A., Pabunruang, W., Hongeng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Congenital+self-healing+Langerhans+cell+histiocytosis+with+pulmonary+involvement%3A+spontaneous+regression%5BTitle%5D)+AND+%22J+Med+Assoc+Thai%22%5BJournal%5D)">Congenital self-healing Langerhans cell histiocytosis with pulmonary involvement: spontaneous regression.</a> J Med Assoc Thai. 85, 1309-1313 (2002).</li>
<li>Bai, L., Feuerer, M., Beckhove, P., Umansky, V., Schirrmacher, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+dendritic+cells+from+human+bone+marrow+mononuclear+cells%3A+advantages+for+clinical+application+in+comparison+to+peripheral+blood+monocyte+derived+cells%5BTitle%5D)+AND+%22Int+J+Oncol%22%5BJournal%5D)">Generation of dendritic cells from human bone marrow mononuclear cells: advantages for clinical application in comparison to peripheral blood monocyte derived cells.</a> Int J Oncol. 20 (2), 247-253 (2002).</li>
<li>Felzmann, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monocyte+enrichment+from+leukapharesis+products+for+the+generation+of+DCs+by+plastic+adherence%2C+or+by+positive+or+negative+selection%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">Monocyte enrichment from leukapharesis products for the generation of DCs by plastic adherence, or by positive or negative selection.</a> Cytotherapy. 5 (5), 391-398 (2003).</li>
<li>Berger, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+elutriation+of+monocytes+within+a+closed+system+%28Elutra%29+for+clinical-scale+generation+of+dendritic+cells%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Efficient elutriation of monocytes within a closed system (Elutra) for clinical-scale generation of dendritic cells.</a> J Immunol Methods. 298 (1-2), 1-2 (2005).</li>
<li>Sallusto, F., Lanzavecchia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+presentation+of+soluble+antigen+by+cultured+human+dendritic+cells+is+maintained+by+granulocyte%2Fmacrophage+colony-stimulating+factor+plus+interleukin+4+and+downregulated+by+tumor+necrosis+factor+alpha%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha.</a> J Exp Med. 179 (4), 1109-1118 (1994).</li>
<li>Pace, S. T., Gelman, B. B., Wong, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+Langerhans+cell+histiocytosis+of+the+lacrimal+gland+in+an+adult%5BTitle%5D)+AND+%22Can+J+Ophthalmol%22%5BJournal%5D)">Primary Langerhans cell histiocytosis of the lacrimal gland in an adult.</a> Can J Ophthalmol. 50 (3), 40-43 (2015).</li>
<li>Ravanfar, P., Wallace, J. S., Pace, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diaper+dermatitis%3A+a+review+and+update%5BTitle%5D)+AND+%22Curr+Opin+Pediatr%22%5BJournal%5D)">Diaper dermatitis: a review and update.</a> Curr Opin Pediatr. 24 (4), 472-479 (2012).</li>
<li>Gebhardt, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+case+of+cutaneous+Rosai-Dorfman+disease+refractory+to+imatinib+therapy%5BTitle%5D)+AND+%22Arch+Dermatol%22%5BJournal%5D)">A case of cutaneous Rosai-Dorfman disease refractory to imatinib therapy.</a> Arch Dermatol. 145 (5), 571-574 (2009).</li>
<li>Vazquez, P., Robles, A. M., de Pablo, F., Hernandez-Sanchez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-neural+tyrosine+hydroxylase%2C+via+modulation+of+endocrine+pancreatic+precursors%2C+is+required+for+normal+development+of+beta+cells+in+the+mouse+pancreas%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">Non-neural tyrosine hydroxylase, via modulation of endocrine pancreatic precursors, is required for normal development of beta cells in the mouse pancreas.</a> Diabetologia. 57 (11), 2339-2347 (2014).</li>
<li>Pellacani, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+melanoma+types+based+on+reflectance+confocal+microscopy%5BTitle%5D)+AND+%22Exp+Dermatol%22%5BJournal%5D)">Distinct melanoma types based on reflectance confocal microscopy.</a> Exp Dermatol. 23 (6), 414-418 (2014).</li>
<li>Shi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatic+involvement+of+Langerhans+cell+histiocytosis+in+children--imaging+findings+of+computed+tomography%2C+magnetic+resonance+imaging+and+magnetic+resonance+cholangiopancreatography%5BTitle%5D)+AND+%22Pediatr+Radiol%22%5BJournal%5D)">Hepatic involvement of Langerhans cell histiocytosis in children--imaging findings of computed tomography, magnetic resonance imaging and magnetic resonance cholangiopancreatography.</a> Pediatr Radiol. 44 (6), 713-718 (2014).</li>
<li>Haustein, M., Terai, N., Pablik, J., Pillunat, L. E., Sommer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapy-resistant+swelling+of+the+upper+eyelid+in+childhood%5BTitle%5D)+AND+%22Ophthalmologe%22%5BJournal%5D)">Therapy-resistant swelling of the upper eyelid in childhood.</a> Ophthalmologe. 111 (1), 53-57 (2014).</li>
<li>Haidinger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+versatile+role+of+mammalian+target+of+rapamycin+in+human+dendritic+cell+function+and+differentiation%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">A versatile role of mammalian target of rapamycin in human dendritic cell function and differentiation.</a> J Immunol. 185 (7), 3919-3931 (2010).</li>
<li>Macedo, C., Turquist, H., Metes, D., Thomson, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunoregulatory+properties+of+rapamycin-conditioned+monocyte-derived+dendritic+cells+and+their+role+in+transplantation%5BTitle%5D)+AND+%22Transplant+Res%22%5BJournal%5D)">Immunoregulatory properties of rapamycin-conditioned monocyte-derived dendritic cells and their role in transplantation.</a> Transplant Res. 1 (1), 16(2012).</li>
<li>Fischer, R., Turnquist, H. R., Taner, T., Thomson, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+rapamycin+in+the+induction+of+tolerogenic+dendritic+cells%5BTitle%5D)+AND+%22Handb+Exp+Pharmacol%22%5BJournal%5D)">Use of rapamycin in the induction of tolerogenic dendritic cells.</a> Handb Exp Pharmacol. 188 (188), 215-232 (2009).</li>
<li>Nicolette, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dendritic+cells+for+active+immunotherapy%3A+optimizing+design+and+manufacture+in+order+to+develop+commercially+and+clinically+viable+products%5BTitle%5D)+AND+%22Vaccine%22%5BJournal%5D)">Dendritic cells for active immunotherapy: optimizing design and manufacture in order to develop commercially and clinically viable products.</a> Vaccine. 25, 47-60 (2007).</li>
<li>Han, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+3+clinical+dendritic+cell+maturation+protocols+containing+lipopolysaccharide+and+interferon-gamma%5BTitle%5D)+AND+%22J+Immunother%22%5BJournal%5D)">Evaluation of 3 clinical dendritic cell maturation protocols containing lipopolysaccharide and interferon-gamma.</a> J Immunother. 32 (4), 399-407 (2009).</li>
<li>Paananen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+and+biological+analysis+of+echovirus+9+strain+isolated+from+a+diabetic+child%5BTitle%5D)+AND+%22J+Med+Virol%22%5BJournal%5D)">Molecular and biological analysis of echovirus 9 strain isolated from a diabetic child.</a> J Med Virol. 69 (4), 529-537 (2003).</li>
<li>HC, O. N., Wilson, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Limitations+with+in+vitro+production+of+dendritic+cells+using+cytokines%5BTitle%5D)+AND+%22J+Leukoc+Biol%22%5BJournal%5D)">Limitations with in vitro production of dendritic cells using cytokines.</a> J Leukoc Biol. 75 (4), 600-603 (2004).</li>
<li>Mest, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose-induced+insulin+secretion+is+potentiated+by+a+new+imidazoline+compound%5BTitle%5D)+AND+%22Naunyn+Schmiedebergs+Arch+Pharmacol%22%5BJournal%5D)">Glucose-induced insulin secretion is potentiated by a new imidazoline compound.</a> Naunyn Schmiedebergs Arch Pharmacol. 364 (1), 47-52 (2001).</li>
<li>Puc, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+activity+after+cold+preservation+of+pancreatic+islet+cells+treated+with+pefloxacin+%28PFX%29%5BTitle%5D)+AND+%22Ann+Transplant%22%5BJournal%5D)">Mitochondrial activity after cold preservation of pancreatic islet cells treated with pefloxacin (PFX).</a> Ann Transplant. 3 (1), 38-41 (1998).</li>
<li>Alarcon, C., Serna, J., Perez-Villamil, B., de Pablo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthesis+and+differentially+regulated+processing+of+proinsulin+in+developing+chick+pancreas%2C+liver+and+neuroretina%5BTitle%5D)+AND+%22FEBS+Letters%22%5BJournal%5D)">Synthesis and differentially regulated processing of proinsulin in developing chick pancreas, liver and neuroretina.</a> FEBS Letters. 436 (3), 361-366 (1998).</li>
<li>Jekunen, A. P., Kairemo, K. J., Paavonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+of+Hand-Schuller-Christian+syndrome+by+a+monoclonal+antibody%5BTitle%5D)+AND+%22Clin+Nucl+Med%22%5BJournal%5D)">Imaging of Hand-Schuller-Christian syndrome by a monoclonal antibody.</a> Clin Nucl Med. 22 (11), 771-774 (1997).</li>
<li>Pearce, E. J., Everts, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dendritic+cell+metabolism%5BTitle%5D)+AND+%22Nat+Rev+Immunol%22%5BJournal%5D)">Dendritic cell metabolism.</a> Nat Rev Immunol. 15 (1), 18-29 (2015).</li>
</ol>

Open access fees for this article were provided by, Agilent Technologies.

This paper describes a method to generate from monocytes immature moDCs, tolerogenic moDCs and mature moDCs. The important steps in this protocol are discussed in detail in the following paragraphs. It is important to note that human peripheral blood is used as a starting material in this protocol and universal precautions for handling human blood should be practiced. Although it is technically feasible to derive DCs from bone marrow in humans 24, the in vitro DC differentiation from cells found in peripheral blood is preferred due to the availability of peripheral blood compared to bone marrow. Among the cells found in peripheral blood, hematopoietic CD34+ stem cells and monocytes are commonly used for the in vitro generation of DCs. Hematopoietic CD34+ stem cells are cultured with GM-CSF and TNF-&#945; to derive CD1a+ and CD14+ subsets which are then further differentiated into Langerhans like cells and dendritic cells. Conversely, monocytes are cultured in GM-CSF and IL-4 to generate immature moDCs. Several protocols are used for the enrichment of monocytes from peripheral blood; for example, by adherence to plastic dishes, elutriation and isolation kits 25, 26. The advantages of the adherence protocol are minimum damage to cells and relatively cost effective however cell purity might be compromised; and an extra step is required to detach the cells for further experiments. Elutriation is a technique that separates cells based on their size and density. The advantages of elutriation are cell viability and monocytes can be readily used for further experiments; however this technique is limited by the availability of an elutriator and the inability to separate different populations of cells (T cells and monocytes) with similar sedimentation parameters. Commercially available isolation kits utilize magnetic microbeads to either positively select or negatively select the monocytic population. Some protocols are biased towards monocyte isolation using the negative selection as the isolated monocytes remain &#34;untouched&#34; (not bound by markers or microbeads). In this protocol, CD14 beads were used to positive select human monocytes from PBMCs. CD14 lacks a cytoplasmic domain and binding of antibody to CD14 does not trigger signal transduction. Moreover, the microbeads will detach from monocytes after culture and hence does not hinder the differentiation process. In addition, CD14 is strongly expressed on most monocytes and weakly on neutrophils and some myeloid dendritic cells, hence this method of isolation results in higher cell purity than the other methods17.
Blood monocytes can be differentiated into DCs or macrophages and the fate of monocytes greatly depends on the cytokine environment. In this paper, moDCs are generated by adding granulocyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin 4 (IL-4) to human peripheral blood monocytes. GM-CSF is required for monocyte survival and IL-4 exerts an inhibitory activity on macrophage differentiation; and the combinatorial addition of GM-SCSF and IL-4 to monocytes yield a higher percentage of immature moDCs compared to individual cytokine27. There are other protocols that generate moDCs by adding tumor necrosis factor alpha (TNF-&#945;), interferon alpha (IFN-&#945;) and Interleukin 13 (IL-13) to peripheral blood monocytes 28,29,30. The combination of GM-CSF and IL-4 was optimized in the 1990s and now an accepted protocol that generates plastic immature DCs differentiated into immunogenic or tolerogenic moDCs and polarized into Th1, Th2 or Th17 promoting moDCs.
Immature moDCs are differentiated into tolerogenic moDCs by the addition of vitamin D3 and dexamethasone. There are several protocols to generate tolerogenic DCs for example, via nuclear factor-kappa B (NF-kB) inhibition, &#946;-catenin activation, Vitamin D3, Dexamethasone and Rapamycin 31,32,33,34,35,9,36,37. Although both vitamin D3 and dexamethasone alone have been reported to induce a tolerogenic effect on DCs, the combination of vitamin D3 and dexamethasone result in a greater suppression of alloproliferation than when individual drugs are used. Therefore, the existing protocol for generation of tolerogenic DCs was modified to a combination of vitamin D3 and dexamethasone. This method is currently being accepted as a model for human tolerogenic DCs with a therapeutic utility. It is also important to note that the reconstituted Vitamin D3 and dexamethasone have a short shelf life.
In this protocol, Lipopolysaccharides (LPS) was added as a DC maturation inducer. Immature moDCs can also be induced to maturation using pro-inflammatory cocktail: (TNF-&#945;), Interleukin 1 beta (IL-1&#946;), Interleukin 6 (IL-6) and prostaglandin E2) or pro-inflammatory cytokines (TNF-&#945; and interferon gamma (IFN-&#915;)). Pro-inflammatory cocktail generates mature moDCs with high co-stimulatory and migratory functions but they produce relatively low levels of IL-1238. TNF-&#945; or IFN-&#915; alone is not able to induce a stable dendritic phenotype 39. LPS stimulates Toll-like receptor 4 (TLR4), mediates the activation of NF-kB and mitogen activated protein kinases (MAPKs) to induce DC maturation. DC maturation induced by LPS shows an up-regulation of DC maturation markers (CD83, CD86, HLA-DR) and also led to the production of IL-12p70. In addition, this step can be further modified to pair LPS with TLR3 agonists to produce mature DCs for clinical cancer vaccines. In this paper, tolerogenic DCs are shown to be resistant to maturation upon LPS treatment. These semi-mature like DCs are not immunogenic and do not release pro-inflammatory cytokines 40.
The limitations of this protocol lie in the differentiation process. The process takes 8 days from Day 0 to Day 7 which poses a difficulty to be adapted into high throughput analyses. A modification in the protocol is required to shorten the differentiation process yet yield high numbers of viable DCs in the different states. Secondly, the DCs are generated by addition of cytokines in this protocol and these cytokines do not sustain DC population for long period of time. Moreover, cytokines are used in concentrations much higher than in vivo and could result in biased development of pathways that are not physiologically identical to in vivo DCs. For example in vitro cultures of DC precursors have been shown to respond to GM-CSF, which is not an essential cytokine for normal DC differentiation in vivo41. Nevertheless, cytokine stimulation can be a useful method to generate high numbers of DC in vitro for experimentation. The ability to subject these cells generated from this protocol to other analyses such as immunofluorescence staining, flow cytometry, allo reaction studies and metabolic studies increases the usefulness of this method. These in vitro DCs serve as a good model to improve the knowledge of DC development, maturation and antigen presentation which is previously difficult to do with the rare numbers of in vivo DCs.
DCs&#39; ability to regulate immunological immunity versus tolerance makes them attractive candidates in therapeutics against cancer and autoimmune diseases 42,43,44,45. Immunogenic DCs generated in this protocol can be used to improve vaccination efficacy against infectious diseases and tumors; while tolerogenic DCs can be used to control unwanted T cell responses and prevent rejections following transplantation. The intricate balance between immunity and tolerance depends immensely on DC differentiation status. DC differentiation is a coordinated cellular program that is governed by multiple signaling pathways and metabolic fate. Different differentiation states of DC differ in bioenergetic and biosynthesis needs; for example, activated DCs require more energetic metabolic adaptations important for survival and migration as compared to DCs at resting state. It is important to note that vitamin D3, dexamethasone and rapamycin are known for their ability to induce tolerogenic DCs, have been described to influence DC metabolism. In this paper, the energetic metabolism of moDCs from different differentiation states were characterized using extracellular flux analyzers and tolerogenic moDCs exhibited the highest metabolic plasticity and LPS-induced maturation decreased this plasticity. Anabolic metabolism supports DCs maturation while catabolic metabolism influences tolerogenic DC functions46. The DCs generated from this protocol can be used to assess whether altering the metabolic state of DCs hold the key to modifying immunity and tolerance in therapeutics. In conclusion, we presented a protocol for the generation of immature, tolerogenic and mature moDCs crucial for studying DCs&#39; immunoregulatory functions.

DC was first described by Paul Langerhans (Langerhans cells) in the late nineteenth century as referenced by Jolles 1 and characterized by Ralph Steinman and Zanvil Cohn in 1973 who recognized them as professional antigen presenting cells 2. DCs are found in peripheral blood and in most tissues of the body, especially abundant in tissues that are exposed to the external environment such as the skin (present as Langerhans cells) and in the linings of the nose, lungs, stomach and intestines which enable them to encounter extrinsic antigens. Immature DCs have endocytic capability but relatively low capacity to stimulate T cells 3. Immature DCs express various pattern recognition receptors (PRRs) that capture pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) 4. Activating danger signals drive maturation towards immunogenic DCs while self-molecules result in T cell unresponsiveness and apoptosis 5. Immunogenic DCs are characterized by the upregulation of MHC molecules and co-stimulatory surface molecules and their ability to prime naive T cells 6,7.
Immature DCs can also be matured towards a Treg-inducing or tolerogenic state in response to Vitamin D3 metabolite 1a,25(O)2D3 and certain immunosuppressive agents like Interleukin-10 (IL-10), dexamethasone and rapamycin 8-9. Tolerogenic DCs are characterized by their expression of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) containing surface receptors and ligands. Signal transduction of ITIMs containing ILT family members, ILT3 and ILT4 in tolerogenic DCs inhibit alloproliferation and drive Foxp3+ Treg expansion 10,11. These unique properties of tolerogenic DCs lead to their profound potency in vivo, namely the ability to induce durable tolerance to transplanted allogeneic grafts and suppress the development of autoimmune diseases. Tolerogenic DCs may be viewed therefore as a subtype of mature-polarized DCs which function in the inhibition of immune activation.
Currently, there are two general subsets of dendritic cells in human peripheral blood: plasmacytoid DCs and myeloid DCs 12. Circulating DCs are rare constituting to less than 2% of leukocytes in human blood and this poses a difficulty to the isolation of an appropriate number of DCs to study their immunoregulatory functions. To overcome this problem, monocyte differentiated DCs are used as an in vitro model for the study of dendritic cell function. These in vitro DCs have similar receptors and functions compared to in vivo DCs. Detailed comparison of in vivo DCs and in vitro generated monocyte derived DCs (moDCs) are investigated by other laboratories 13,14,15. It is also reported that moDCs and CD1c+ DCs were equivalent at antigen presenting and inducing T cell function15.
In this paper, we describe a method of generating immature moDCs from peripheral blood monocytes and then differentiating them into immunogenic and tolerogenic DCs. These monocyte derived dendritic cells (moDCs) are characterized by their surface markers, cytokine profile, immunoregulatory functions and metabolic states. Immunogenic and tolerogenic dendritic cells produce different cytokines which result in expansion of either allogenic T cells or regulatory T cells. In this paper, cytokine profiling is performed with systems using multiplex technology. Growth medium of cells are incubated with antibody immobilized color coded beads and read in a compact analyzer. Metabolic states of DCs are analyzed using extracellular flux analyzers that measure oxygen consumption rate, an indicator of cellular respiration, and extracellular acidification rate which reflects glycolytic flux in dendritic cells. Measurement of these bioenergetics rates provides a means to track the changes in cellular metabolism which are vital in dendritic cell development and function.

<table><tbody><tr><td>Ficoll</td><td>GE Healthcare</td><td>17-1440-03</td><td>PBMC isolation</td></tr><tr><td>Syringe</td><td>Becton, Dickinson</td><td>302832</td><td>PBMC isolation</td></tr><tr><td>1.5 ml centrifuge tube</td><td>Axygen</td><td>MCT-150-C</td><td>PBMC isolation</td></tr><tr><td>15 ml falcon tube</td><td>Falcon</td><td>352096</td><td>PBMC isolation</td></tr><tr><td>50 ml falcon tube</td><td>Falcon</td><td>352070</td><td>PBMC isolation</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>PBMC isolation</td></tr><tr><td>0.2 &amp;micro;m filter</td><td>Sartorius stedim biotech</td><td>17597</td><td>PBMC isolation</td></tr><tr><td>MACs kit</td><td>Miltenyi biotec</td><td>130-042-201</td><td>Monocyte enrichment</td></tr><tr><td>MiniMACS Separator</td><td>Miltenyi biotec</td><td>130-042-102</td><td>Monocyte enrichment</td></tr><tr><td>Cell culture grade water</td><td>Invitrogen, Life Technologies</td><td></td><td>Cell culture</td></tr><tr><td>RPMI</td><td>Gibco, Life Technologies</td><td>11875-093</td><td>Cell culture</td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30070103</td><td>Cell culture</td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco, Life Technologies</td><td>15140</td><td>Cell culture</td></tr><tr><td>Phosphate&lt;br/&gt; Buffered Saline</td><td>Gibco, Life Technologies</td><td>10010-031</td><td>Cell culture</td></tr><tr><td>NEAA</td><td>Gibco, Life Technologies</td><td>11140-040</td><td>Cell culture</td></tr><tr><td>EDTA</td><td>Gibco, Life Technologies</td><td>15575</td><td>Cell culture</td></tr><tr><td>HEPES</td><td>Gibco, Life Technologies</td><td>15630-080</td><td>Cell culture</td></tr><tr><td>Sodium Pyruvate</td><td>Gibco, Life Technologies</td><td>11360-070</td><td>Cell culture</td></tr><tr><td>GM-CSF</td><td>Miltenyi biotec</td><td>130-093-868</td><td>Cell culture</td></tr><tr><td>IL-4</td><td>Miltenyi biotec</td><td>130-093-924</td><td>Cell culture</td></tr><tr><td>Vitamin D3</td><td>Sigma</td><td>D1530</td><td>Cell culture</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D2915</td><td>Cell culture</td></tr><tr><td>LPS</td><td>Sigma</td><td>l2755</td><td>Cell culture</td></tr><tr><td>trypan blue</td><td>Gibco, Life Technologies</td><td>15250-061</td><td>Cell culture</td></tr><tr><td>PerCP-conjugated HLADR&amp;nbsp;</td><td>BioLegend</td><td>307628</td><td>Cytometry</td></tr><tr><td>PE-conjugated CD80</td><td>BD Biosciences</td><td>557227</td><td>Cytometry</td></tr><tr><td>PE-conjugated CD83&amp;nbsp;</td><td>BD Biosciences</td><td>556855</td><td>Cytometry</td></tr><tr><td>PE-conjugated CD86&amp;nbsp;</td><td>BD Biosciences</td><td>555665</td><td>Cytometry</td></tr><tr><td>APC-conjugated CD11c</td><td>BD Biosciences</td><td>340544</td><td>Cytometry</td></tr><tr><td>PE-conjugated CD14&amp;nbsp;</td><td>Miltenyi biotec</td><td>130-091-242</td><td>Cytometry</td></tr><tr><td>PE-conjugated BDCA3&amp;nbsp;</td><td>Miltenyi biotec</td><td>130-090-514</td><td>Cytometry</td></tr><tr><td>APC-conjugated ILT3&amp;nbsp;</td><td>eBioscience</td><td>12-5139-73</td><td>Cytometry</td></tr><tr><td>Isotype matched&lt;br/&gt; PerCP- conjugated Mab</td><td>BioLegend</td><td>400250</td><td>Cytometry</td></tr><tr><td>Isotype matched&lt;br/&gt; PE- conjugated Mab</td><td>Miltenyi biotec</td><td>130-091-835</td><td>Cytometry</td></tr><tr><td>Isotype matched&lt;br/&gt; APC- conjugated Mab</td><td>Miltenyi biotec</td><td>130-091-836</td><td>Cytometry</td></tr><tr><td>BD LSR II Flow Cytometer</td><td>BD Pharmingen</td><td>BD LSR II&amp;nbsp;</td><td>Cytometry</td></tr><tr><td>cytofix/cytoperm</td><td>BD Biosciences</td><td>554714</td><td>Cytometry</td></tr><tr><td>APC/CY7-conjugated CD25</td><td>BD pharmingen</td><td>557753</td><td>Cytometry</td></tr><tr><td>PE/CY7-conjugated CD4</td><td>Biolegend</td><td>300512</td><td>Cytometry</td></tr><tr><td>PerCP-conjugated CD3</td><td>Biolegend</td><td>300428</td><td>Cytometry</td></tr><tr><td>EasySep Human CD4+ T cell enrichment kit</td><td>STEMCELL Technologies</td><td>19052</td><td>Alloreaction study</td></tr><tr><td>EasySep magnet&amp;nbsp;</td><td>STEMCELL Technologies</td><td>18000</td><td>Alloreaction study</td></tr><tr><td>Cell Trace CFSE cell proliferation kit</td><td>Molecular probes</td><td>C34554</td><td>Alloreaction study</td></tr><tr><td>HBSS</td><td>Gibco, Life Technologies</td><td>14025092</td><td>Alloreaction study</td></tr><tr><td>Alexa Fluor 647-conjugated FoxP3</td><td>BD Biosciences</td><td>560889</td><td></td></tr><tr><td>Milliplex MAP Human&lt;br/&gt; Cytokine/Chemokine&lt;br/&gt; magnetic bead panel</td><td>Millipore</td><td>HCYTOMAG-60K</td><td>Cytokine analysis</td></tr><tr><td>5 ml Polystyrene tube</td><td>Falcon</td><td>352058</td><td>Cytokine analysis</td></tr><tr><td>Luminex Sheath Fluid&amp;nbsp;</td><td>Millipore</td><td>SHEATHFLUID</td><td>Cytokine analysis</td></tr><tr><td>FLEXMAP 3D system with xPONENT software</td><td>Luminex Corporation</td><td>FLEXMAP 3D</td><td>Cytokine analysis</td></tr><tr><td>MitoTracker Red CMXRos</td><td>Cell Signalling</td><td>9082</td><td>Mitochondrial activity</td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>D2650</td><td>Mitochondrial activity</td></tr><tr><td>XF Assay Medium (OCR)</td><td>Seahorse Bioscience</td><td>102352-000</td><td>Metabolic adaptation</td></tr><tr><td>Glucose&amp;nbsp;</td><td>Sigma Aldrich</td><td>G8769</td><td>Metabolic adaptation</td></tr><tr><td>XF Base Medium (ECAR)</td><td>Seahorse Bioscience</td><td>102353-100</td><td>Metabolic adaptation</td></tr><tr><td>L-glutamine</td><td>Gibco, Life Technologies</td><td>25030-081</td><td>Metabolic adaptation</td></tr><tr><td>Calibrant</td><td>Seahorse Bioscience</td><td>100840-000&amp;nbsp;</td><td>Metabolic adaptation</td></tr><tr><td>XF Cell Mito Stress kit</td><td>Seahorse Bioscience</td><td>103015-100</td><td>Metabolic adaptation</td></tr><tr><td>XF Glycolysis Stress kit</td><td>Seahorse Bioscience</td><td>103020-100</td><td>Metabolic adaptation</td></tr><tr><td>Seahorse&amp;nbsp;</td><td>Seahorse Bioscience</td><td>XFe96</td><td>Metabolic adaptation</td></tr></tbody></table>

generation of immature, mature and tolerogenic dendritic cells with differing metabolic phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Monocyte Purification and Dendritic Cell Differentiation
Monocytes were purified from PBMCs by density centrifugation of peripheral blood (Figure 1A), followed by CD14+ positive selection magnetic separation (Figure 1B) and cultured in complete medium in the presence of GM-CSF and IL-4 to obtain immature dendritic cells (Figure 2A). Addition of vitamin D3 and dexamethasone post GM-CSF and IL-4 resulted in the differentiation of immature moDCs into tolerogenic moDCs (Figure 2B). LPS was added to induce maturation of immature moDCs to mature moDCs (Figure 2C) and tolerogenic moDCs were stimulated with LPS to verify resistance to maturation (Figure 2D). The blood samples used in this work were obtained from healthy donors with previous informed consent.
moDC Characterization
Surface Marker Characterization by Flow Cytometry
Analysis of DC surface markers showed that mature moDCs expressed the highest levels of maturation markers HLA-DR, CD83 and CD86 compared to LPS-treated tolerogenic moDCs, tolerogenic moDCs and immature moDCs (Figure 3). These results demonstrated that tolerogenic moDCs were resistant to maturation as compared to immature moDCs following LPS stimulation. In addition, LPS-treated tolerogenic moDCs and tolerogenic moDCs displayed increased expression of CD14, BDCA3 (CD141) and Immunoglobulin-like transcript (ILT)3 compared to immature and mature moDCs. The tolerogenic moDCs that we generated here is consistent with previous reports 23.
Functional characterization of moDCs
moDCs induced for maturation become immunogenic and release cytokines that promote the proliferation of CD4+ T-cells. We assessed the immunogenicity of the different moDC subtypes by measuring the proliferation of co-cultured T cells. Tolerogenic moDCs were poorly immunogenic compared with mature moDCs, as shown by low alloproliferation of CD4+ T-cells (Figure 4A). Tolerogenic moDCs are characterized by their low IFN-Γ, low IL-12p40, and high IL-10 cytokine production in alloreaction co-cultures with CD4+ T-cells (Figure 4B). Furthermore, increasing the number of tolerogenic moDCs in co-cultures of mature moDCs induced allospecific CD4+ T-cells increased the frequency of CD25high Foxp3+ regulatory T-cells (Figure 4C).
Analysis of Mitochondrial Activity
Red CMXRos is used to reflect the mitochondrial activity to analyze the mitochondrial membrane potential levels in moDCs. Tolerogenic moDCs were observed to have higher mitochondrial activity compared with the other moDC differentiated subtypes (Figure 5A). Next, the rate of mitochondrial oxygen consumption (OCR) is assessed for the different moDC subtypes using a bioanalyzer. OCR measurements allow high resolution insights to the metabolic profile, providing information including but not limited to basal respiration, spare respiratory capacity, proton leak and non-mitochondrial respiration. The measurement of OCR provides a means to assess the ability of cells to respond to stress. The cells are metabolically perturbed by the addition of three different compounds in succession. The first injection is Oligomycin (ATP Coupler) which inhibits complex V of the electron transport chain (ETC), inhibiting ATP synthesis. This step distinguishes the percentage of oxygen consumed for ATP synthesis and the percentage of oxygen consumed to overcome proton leak across the inner mitochondrial membrane. The second injection is FCCP (ETC accelerator) that disrupts ATP synthesis by transporting hydrogen ions across the mitochondrial membrane instead of through the proton channel of Complex V. The collapse of the mitochondrial membrane potential leads to a rapid consumption of energy and oxygen, without the generation of ATP. FCCP treatment can be used to calculate the spare respiratory capacity of cells. Maintenance of spare respiratory capacity under stress conditions is critical to cell survival. This capacity is determined by several factors, including the availability of substrate and functional capacity of the enzymes involved in the ETC. The third injection is a combination of Rotenone, a Complex I inhibitor, and Antimycin A, a Complex III inhibitor. This combination shuts down mitochondrial respiration and OCR is observed to decrease as a result of impaired mitochondrial function (Figure 5C). Tolerogenic moDCs displayed higher basal OCR levels than mature moDCs (Figure 5D). In addition, tolerogenic, LPS-tolerogenic, and immature moDCs showed increased spare respiratory capacity compared with mature moDCs (Figure 5E).
Metabolic Characterization of moDCs
Because lactic acid and protons are released from cells during glycolysis, we analyzed the glycolytic activity of moDCs by performing a real-time analysis of the rate of extracellular acidification (ECAR) (Figure 6B). In the presence of glucose, the glycolytic rate of all moDCs increased compared with the basal stage, with mature moDCs exhibiting higher glycolytic rate than immature moDCs (Figure 6D). Tolerogenic and immature moDCs exhibited higher maximal glycolysis (induced by oligomycin in presence of glucose) compared with LPS-treated moDCs (Figure 6B). The glycolytic capacity of tolerogenic and immature moDCs was higher than mature moDCs (Figure 6E). In contrast to their high glycolytic rate, glycolytic reserve was the lowest in mature moDCs (Figure 6F).
<img alt="Blood separation diagram, centrifuge and magnetic cell sorting with CD14 microbeads, PBMC isolation." src="/files/ftp_upload/54128/54128fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Monocyte Purification from Peripheral Blood. (A) 25 ml of blood is carefully layered onto 15 ml of Ficoll per 50 ml tube before centrifugation. PBMCs are concentrated in a layer below plasma after density centrifugation. (B) PBMCs are incubated with microbeads that are conjugated to monoclonal human CD14 antibodies (isotype: mouse IgG2a) and then loaded onto a column which is placed in the magnetic field of a Separator to isolate CD14+ monocytes. <a href="https://www.jove.com/files/ftp_upload/54128/54128fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscope images of moDC types: immature, tolerogenic, mature, LPS-tolerogenic, cell morphology comparison." src="/files/ftp_upload/54128/54128fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Morphological Characterization of moDCs. (A) 200 ng/ml of GM-CSF and IL-4 is added to purified CD14+ monocytes on Day 0, 4 and 6 to generate immature moDCs; and (B) an additional step of stimulation with 100 nM Vitamin D3 and 10 nM dexamethasone on Day 5 generates tolerogenic moDCs. Immature moDCs and tolerogenic moDCs are stimulated with 1 µg/ml LPS on Day 6 to generate (C) mature moDCs and (D) LPS-tolerogenic moDCs. <a href="https://www.jove.com/files/ftp_upload/54128/54128fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Dendritic cell marker expression, flow cytometry histograms; isotype, maturation state analysis." src="/files/ftp_upload/54128/54128fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Surface Marker Characterization by Flow Cytometry. Expression levels of surface markers HLA-DR, CD80, CD83, CD86, CD11, CD14, BDCA3 and LT3 in tolerogenic (green), LPS-tolerogenic (black), immature (red), mature (blue) moDCs. Isotype controls are shown in grey. Individual histogram for each cell type is plotted with Y-axis cell count against X-axis log of fluorophore intensity and overlaid. All histograms are representative of four independent experiments. This figure has been modified from J Immunol 194 (11), 5174-5186, doi: 10.4049/jimmunol.1303316 (June 1, 2015). Reproduced and republished with copyright permission. Copyright 2015. The American Association of Immunologists, Inc. <a href="https://www.jove.com/files/ftp_upload/54128/54128fig3large.jpg" target="_blank">Please click here to view a larger version of this figure. </a>

<img alt="Graphs showing CD4+ proliferation and cytokine levels (IFNγ, IL-12p40, IL-10) in moDC experiment." src="/files/ftp_upload/54128/54128fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Functional Characterization of moDCs. (A) Quantification of alloproliferation frequency of CD4+ T-cells induced by co-culture with increasing numbers of tolerogenic (TOL; green), LPS-tolerogenic (L-TOL; black), immature (IMM; red) and mature (MAT; blue) moDCs. The data was pooled from four independent experiments; mean + S.E.M. Statistical differences between all moDCs versus mature moDCs were analyzed by two-way ANOVA with Dunnett multiple comparison post-test. (B) Cytokine analysis of IFN-Γ (left panel), IL-12p40 (middle panel) and Il-10 (right panel) in supernatants from alloreactions between CD4+ T-cells co-cultured with increasing numbers of either tolerogenic (green), immature (red) or mature (blue) moDCs. Data were pooled from six independent experiments; mean ± S.E.M. (C) CD4+ T-cell alloproliferation and regulatory T-cells expansion induced by co-culture with mature moDCs in the presence of increasing numbers of tolerogenic moDCs. Left Y-axis, frequency of CD4+ T-cell proliferation. Right Y-axis, frequency of CD25high Foxp3+ cells gated on proliferative CD4+ T-cells. The data were pooled from three independent experiments; mean ± S.E.M. Statistical differences between presence versus absence of tolerogenic moDCs were analyzed by one-way ANOVA with Dunnett multiple comparison post-test. (D) CD4+ T-cell alloproliferation (Left) and frequency of CD25highFoxP3+ cells (Right) induced by co-culture with tolerogenic moDCs in the presence of increasing numbers of mature moDCs. The data were pooled from two to three independent experiments; mean ± S.E.M. Statistical differences between the presence versus absence of mature moDCs were analyzed by two-way ANOVA with Dunnett multiple comparison post-test. This figure has been modified from J Immunol 194 (11), 5174-5186, doi: 10.4049/jimmunol.1303316 (June 1, 2015). Reproduced and republished with copyright permission. Copyright 2015. The American Association of Immunologists, Inc. <a href="https://www.jove.com/files/ftp_upload/54128/54128fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cellular respiration analysis with bar graphs and a line chart illustrating OCR changes and treatments." src="/files/ftp_upload/54128/54128fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Analysis of Mitochondrial Activity of moDCs. (A) Levels of mitochondrial membrane potential (Red CMXRos) in moDCs were obtained by flow cytometric analysis. The data of four independent experiments were pooled. Mean ± S.E.M. (B) Schematic representation of a real-time mitochondrial respiration. OCR analysis starting from basal respiration and after the addition of oligomycin (complex V inhibition), FCCP (maximal respiration induction), and rotenone/antimycin A mixture (electron transport chain [ETC] inhibition). The mitochondrial SRC (maximal basal subtracted from maximal respiration) is derived from the OCR curve. (C) Representative kinetic study of mitochondria OCR (pmol/min) in tolerogenic (TOL, green), LPS-tolerogenic (L-TOL, black), immature (IMM, red) and mature (MAT, blue) moDCs by using sequential addition of oligomycin (Olig), FCCP, and rotenone/antimycin A (Rot-AA). (D) OCR quantification of basal respiration of moDCs and (E) spare respiratory capacity of moDCs. The data were pooled from five independent experiments. Mean ± S.E.M. This figure has been modified from J Immunol 194 (11), 5174-5186, doi: 10.4049/jimmunol.1303316 (June 1, 2015). Reproduced and republished with copyright permission. Copyright 2015. The American Association of Immunologists, Inc. <a href="https://www.jove.com/files/ftp_upload/54128/54128fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Static equilibrium and glycolysis, ECAR rates, bar graphs, glycolytic parameters, research analysis." src="/files/ftp_upload/54128/54128fig6.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: Metabolic Characterization of moDCs. (A) Schematic representation of real-time glycolysis. ECAR analysis starts from basal ECAR, in which the cells were incubated in glucose-free media followed by the addition of glucose (glycolysis induction), oligomycin (which induces maximal cell glycolysis and complex V inhibition), and finally 2-deoxy-D-glucose (glycolysis inhibition). Glycolytic rate (glycolysis induction subtracted for basal ECAR), glycolytic capacity (maximal glycolysis subtracted for basal ECAR), and glycolytic reserve (maximal glycolysis subtracted for glycolysis induction) are derived from the ECAR curve. (B) Representative kinetic study of glycolysis-dependent ECAR (mpH/min) in tolerogenic (TOL, green), LPS-tolerogenic (L-TOL, black), immature (IMM, red), and mature (MAT, blue) moDCs by using sequential addition of glucose (Gluc), oligomycin (Olig), and 2-DG. (C) Bars show basal ECAR levels (D) glycolytic rate (E) glycolytic capacity, and (F) glycolytic reserve of moDCs. The data were pooled from three independent experiments; mean 6 SEM. Statistical differences were analyzed by one-way ANOVA with a Tukey multiple comparison post-test. This figure has been modified from J Immunol 194 (11), 5174-5186, doi: 10.4049/jimmunol.1303316 (June 1, 2015). Reproduced and republished with copyright permission. Copyright 2015. The American Association of Immunologists, Inc. <a href="https://www.jove.com/files/ftp_upload/54128/54128fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes at JoVE.com

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

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Universidad San Sebastian

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Immunology and Infection

23.0K Views.  Agency for Science, Technology and Research. Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Video: Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Biology

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies 2.
Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production.
DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 &deg;C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine 3. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4 - 20 x 106 cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

The most commonly used method for generating large numbers of autologous dendritic cells (DCs) for use in tumor immunotherapy is described. The method uses IL-4 and GM-CSF to differentiate DCs from monocytes. The immature DCs are stimulated to mature and then loaded with antigens before they are injected back into the patient.

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Medicine

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided into conventional DCs (cDCs) and plasmacytoid DCs (pDCs). cDCs specializes in presenting antigens to and activate na&#239;ve T cells. On the other hand, pDCs can produce large quantities of type I interferons (IFN-I) during viral infection. The specification of DCs occurs at an early stage of DC progenitors in the bone marrow (BM) and is defined by a network of transcription factors (TFs). For example, cDCs highly express ID2, while pDCs highly express E2-2. Since more and more subsets of DCs are being identified, there is a growing interest in understanding specific TFs controlling DC development. Here, we establish a method to screen TFs critical for DCs differentiation in vitro by delivering lentivirus carrying short hairpin RNA (shRNA) into an immortalized hematopoietic stem and progenitor cell (iHSPCs) line. After the selection and in vitro differentiation, cDC and pDC potential of the stable knockdown cell lines are analyzed by flow cytometry. This approach provides a platform to identify genes potentially governing DC fates from progenitors in vitro.

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Here we provide a protocol for screening potential transcription factors involved in the development of dendritic cell (DC) using lentiviral transduction of shRNA to obtain stable knockdown cell lines for in vitro DC differentiation.

Dendritic cells (DCs) are important antigen-presenting cells that connect innate and adaptive immune responses. DCs are heterogeneous and can be divided ...

De-Sialylated Monocyte-Derived-DCs from Isolated PBMCs Using Sialidase Treatment

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

Author Spotlight: Dendritic Cells Maturation Using Sialidases-Based Enzymatic Treatment of the Cell Surface 

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical ...

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell (Treg) phenotype. TolDCs are currently under investigation as therapies for autoimmunity and transplantation, both as a cell therapy and method to induce tolDCs from endogenous DCs. To date, however, the number of known agents to induce tolDCs from na&#239;ve DCs is relatively small and their potency to generate Tregs in vivo has been inconsistent, particularly therapies that induce tolDCs from endogenous DCs. This provides an opportunity to explore novel compounds to generate tolerance.
Here we describe a method to test novel immunomodulatory compounds on monocyte-derived DCs (moDCs) in vitro and validate their functionality to generate autologous Tregs. First, we obtain PBMCs and isolate CD14+ monocytes and CD3+ T cells using commercially available magnetic separation kits. Next, we differentiate monocytes into moDCs, treat them with an established immunomodulator, such as rapamycin, dexamethasone, IL-10, or vitamin D3, for 24 h and test their change in tolerogenic markers as a validation of the protocol. Finally, we co-culture the induced tolDCs with autologous T cells in the presence of anti-CD3/CD28 stimulation and observe changes in Treg populations and T cell proliferation. We envision this protocol being used to evaluate the efficacy of novel immunomodulatory agents to reprogram already differentiated DCs towards tolDC.

We describe a procedure to assess the capacity for pharmacologic agents to generate tolerogenic dendritic cells from na&#239;ve monocyte-derived dendritic cells in vitro and validate their potency by autologous regulatory T cell generation.

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence na&#239;ve T cells toward a regulatory T cell ...

Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes

Source: Sim, W. J. et al., <a href="https://app.jove.com/v/54128">Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes.</a>&#160;J. Vis. Exp.&#160;(2016).
This video demonstrates the generation of immature, mature, and tolerogenic dendritic cells, or DCs, from human monocytes. The incubation of monocytes with growth factors and cytokines leads to the production of immature DCs. In response to immune regulatory molecules, immature DCs differentiate into tolerogenic and mature DCs.

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Summary

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Culture of myeloid dendritic cells from bone marrow precursors

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

A Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14⁺ Monocytes

Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes