Name: generation of immature, mature and tolerogenic dendritic cells with differing metabolic phenotypes
Uploaded: 2016-06-22T14:00:00
Duration: 6 min 9 s
Description: Watch this Scientific Journal Video about Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes at JoVE.com

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

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>
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			<td>Ficoll</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td>PBMC isolation</td>
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			<td>50 mL falcon tube</td>
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			<td>0.2 &#181;m filter</td>
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			<td>MACs kit</td>
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			<td>MiniMACS Separator</td>
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			<td>Phosphate 
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			<td>NEAA</td>
			<td>Gibco, Life Technologies</td>
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			<td>EDTA</td>
			<td>Gibco, Life Technologies</td>
			<td>15575</td>
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			<td>HEPES</td>
			<td>Gibco, Life Technologies</td>
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			<td>Sodium Pyruvate</td>
			<td>Gibco, Life Technologies</td>
			<td>11360-070</td>
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			<td>GM-CSF</td>
			<td>Miltenyi biotec</td>
			<td>130-093-868</td>
			<td>Cell culture</td>
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			<td>IL-4</td>
			<td>Miltenyi biotec</td>
			<td>130-093-924</td>
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			<td>Dexamethasone</td>
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			<td>D2915</td>
			<td>Cell culture</td>
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			<td>LPS</td>
			<td>Sigma</td>
			<td>l2755</td>
			<td>Cell culture</td>
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			<td>trypan blue</td>
			<td>Gibco, Life Technologies</td>
			<td>15250-061</td>
			<td>Cell culture</td>
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		<tr>
			<td>PerCP-conjugated HLADR&#160;</td>
			<td>BioLegend</td>
			<td>307628</td>
			<td>Cytometry</td>
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			<td>PE-conjugated CD80</td>
			<td>BD Biosciences</td>
			<td>557227</td>
			<td>Cytometry</td>
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			<td>PE-conjugated CD83&#160;</td>
			<td>BD Biosciences</td>
			<td>556855</td>
			<td>Cytometry</td>
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			<td>PE-conjugated CD86&#160;</td>
			<td>BD Biosciences</td>
			<td>555665</td>
			<td>Cytometry</td>
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		<tr>
			<td>APC-conjugated CD11c</td>
			<td>BD Biosciences</td>
			<td>340544</td>
			<td>Cytometry</td>
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		<tr>
			<td>PE-conjugated CD14&#160;</td>
			<td>Miltenyi biotec</td>
			<td>130-091-242</td>
			<td>Cytometry</td>
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		<tr>
			<td>PE-conjugated BDCA3&#160;</td>
			<td>Miltenyi biotec</td>
			<td>130-090-514</td>
			<td>Cytometry</td>
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		<tr>
			<td>APC-conjugated ILT3&#160;</td>
			<td>eBioscience</td>
			<td>12-5139-73</td>
			<td>Cytometry</td>
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			<td>Isotype matched 
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			<td>BioLegend</td>
			<td>400250</td>
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			<td>Isotype matched 
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			<td>Miltenyi biotec</td>
			<td>130-091-835</td>
			<td>Cytometry</td>
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			<td>Isotype matched 
			APC- conjugated Mab</td>
			<td>Miltenyi biotec</td>
			<td>130-091-836</td>
			<td>Cytometry</td>
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			<td>BD LSR II Flow Cytometer</td>
			<td>BD Pharmingen</td>
			<td>BD LSR II&#160;</td>
			<td>Cytometry</td>
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		<tr>
			<td>cytofix/cytoperm</td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Cytometry</td>
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		<tr>
			<td>APC/CY7-conjugated CD25</td>
			<td>BD pharmingen</td>
			<td>557753</td>
			<td>Cytometry</td>
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		<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>
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		<tr>
			<td>EasySep magnet&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>18000</td>
			<td>Alloreaction study</td>
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		<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 
			Cytokine/Chemokine 
			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&#160;</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>
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		<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&#160;</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&#160;</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&#160;</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-&#915;, 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="Figure 1" src="/files/ftp_upload/54128/54128fig1.jpg" /> 
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="Figure 2" src="/files/ftp_upload/54128/54128fig2.jpg" /> 
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 &#181;g/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="Figure 3" src="/files/ftp_upload/54128/54128fig3.jpg" /> 
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.&#160;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="Figure 4" src="/files/ftp_upload/54128/54128fig4.jpg" /> 
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-&#915; (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 &#177; 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 &#177; 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 &#177; 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="Figure 5" src="/files/ftp_upload/54128/54128fig5.jpg" /> 
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 &#177; 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 &#177; S.E.M.&#160;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="Figure 6" src="/files/ftp_upload/54128/54128fig6.jpg" /> 
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.&#160;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.

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