Name: development and functional characterization of murine tolerogenic dendritic cells
Uploaded: 2018-05-18T14:30:00
Description: Watch this Scientific Journal Video about Development and Functional Characterization of Murine Tolerogenic Dendritic Cells at JoVE.com

We thank Reata Pharmaceuticals for providing CDDO-DFPA. We also acknowledge the support of the Jane and Lee Seidman Chair in Pediatric Cancer Innovation (John Letterio). This work was supported by Department of Defense [W81XWH-12-1-0452]; the Angie Fowler Adolescent and Young Adult Cancer Research Initiative at the Case Comprehensive Cancer Center; and the Callahan Graduate Scholar Award for Hsi-Ju Wei from F.J. Callahan Foundation.

All studies were performed in compliance with procedures approved by the Case Western Reserve University School of Medicine&#39;s Institutional Animal Care and Use Committee.
1. Prepare Bone Marrow-derived Dendritic Cells (BMDCs)
<ol>
	<li>Sterilize all the surgical instruments via autoclaving and perform the experiment in class II biological safety cabinet with appropriated safety procedures.</li>
	<li>Euthanize C57BL/6 mice 8 - 10 weeks of age by using CO2 chamber. Place the mou...

This paper describes an efficient protocol that may be used to reproducibly to generate iDCs and to subsequently differentiate them into TolDCs, and we propose that this may be applied to evaluate the capacity of new molecular target agents to induce the TolDC phenotype. As described in this report, we followed a sequence in which we first analyzed TolDC expression of surface ligands by flow cytometry, followed by an assessment of the DC cytokine profile as measured by qRT-PCR and ELISA. Finally, the immunoregulatory fun...

Dendritic cells (DCs) are an integral part of the innate immune system and were first discovered and characterized by Ralph Steinman and Zanvil Cohn in 1973 as primary professional antigen presenting cells1. DCs have been shown to play an important role in immune activation by presenting processed antigens to T cells and B cells via major histocompatibility complexes (MHC) in secondary lymphoid organs to link the innate and adaptive immune systems2. In the mammalian immune system, there are at least two categories of DCs which have been described as myeloid DCs and plasmacytoid DCs (pDCs)3. Myeloid DCs, also known as conventional DCs (cDCs), are characterized by the expression of CD11c and can be differentiated as immature DCs (iDCs) in vitro from bone marrow progenitor cells or peripheral blood monocytes using granulocyte-macrophage-colony-stimulating factor (GM-CSF) and IL-4 in murine or human species, respectively4.
Activating &#39;danger&#39; signals, such as pathogen-associated molecular patterns (PAMP) or damage-associated molecular patterns (DAMP), will drive iDCs maturation toward immunogenic DCs as mature DCs (mDCs) via binding various pattern recognition receptors on the DC surface5. Immunogenic DCs further prime na&#239;ve T cell proliferation and differentiation through upregulation of MHCII2, costimulatory ligands (CD80, CD86, and CD40)6, cytokines, and other soluble mediators7. A cascade of pro-inflammatory mediator production from Immunogenic DCs is essential for cytokine-mediated T cell differentiation. For example, both IFN-&#947; and IL-12 are necessary for Th1 differentiation8 and IL-1, IL-6, and IL-23 are critical for na&#239;ve T cell polarization towards Th17 cells9. Although mature DCs react to foreign antigens, uncontrolled DC activation by self-antigens may cause tolerance ablation and foster the development of autoimmune diseases by generating autoreactive T cells whose activation leads to tissue destruction10.
Recent reports have provided clear evidence of DC plasticity, exemplified by their ability to interact with different cues within their tissue microenvironment and to differentiate into distinct effector/suppressor DC subsets. The anti-inflammatory mediators, such as IL-1011, TGF-&#946;12, and HO-113 have been shown to play an important role in immune suppression by inducing tolerogenic DCs (TolDCs). These TolDCs acquire regulatory functions and suppress T cell proliferation14. Moreover, the lack of co-stimulation by DCs and the production of anti-inflammatory mediators from TolDCs both contribute to the induction of regulatory T cells (Tregs) and also effectively inhibit both Th1 and Th17 differentiation and expansion15. In past two decades, the therapeutic potential of TolDCs has been reported by several investigators. In these studies, the administration of ex-vivo generated TolDCs not only ameliorated pathological symptoms in different preclinical models of autoimmune diseases16 but also led to the development of immune tolerance in patients17,18. Interestingly, today the TolDCs therapy has been considered as an alternative or adjunctive approach for autoimmune diseases in several clinical trials, including type 1 diabetes mellitus19, rheumatoid arthritis20,21, multiple sclerosis (MS)22,23,24, and Crohn&#39;s disease25.
There are a variety of protocols that have been employed to develop TolDCs and several laboratories have reported methods for generation and phenotypic characterization of TolDCs. These methods can be used to reproducibly generate TolDCs in vitro from hematopoietic progenitors and to stably maintain them in a tolerogenic state in vivo26,27,28,29. The iDCs can be converted into TolDCs by exposure to various immunomodulatory pharmacological agents or anti-inflammatory cytokines. For example, Vitamin D3 is a well-known pharmacological agent known to augment IL-10 production and suppress IL-12 secretion from DCs and to thereby boost their immunosuppressive function30. Moreover, when DCs are exposed to potent inflammatory stimuli, such as lipopolysaccharides (LPS), several pharmacological agents such as dexamethasone31, rapamycin32, and corticosteroids33 have been shown to induce the TolDC phenotype by reducing DC surface expression of CD40, CD80, CD86, and MHCII34. IL-10 and TGF-&#946; are the most-studied anti-inflammatory cytokines to induce DC tolerance35 and the concomitant exposure to both of these cytokines have been shown to induce a tolerogenic phenotype in DCs36.
Since the tolerogenic DC is defined by functional characteristics rather than by phenotypic markers, there is a great need to develop a consistent method for cellular and functional characterization of TolDCs. Moreover, a rigorous and consistent protocol must be established for the consistent evaluation and characterization of the tolerogenic DC phenotype if we are to effectively and reproducibly compare the ability of new agents to induce the TolDC phenotype in the laboratory. Here we provide a detailed protocol with step-by-step methods to isolate iDCs from hematopoietic progenitors of mice and to subsequently analyze the efficacy of new agents under evaluation for their capacity to convert iDCs into TolDCs, providing a robust functional and phenotypic characterization of TolDCs both in vitro and in vivo. This description includes an elaborate method to characterize the TolDCs by their surface ligands, cytokine profile, and immunosuppressive functions in vitro. We also provide an example of a method to explore the potential therapeutic application of these TolDCs in a pre-clinical model of MS, experimental autoimmune encephalomyelitis (EAE). This established protocol will help investigators to evaluate the capacity of new agents to promote the induction of TolDCs and will facilitate the effort to broaden the scope of TolDC therapeutic development.

<table>
	<tbody>
		<tr>
			<td>CDDO-DFPA (RTA-408)</td>
			<td>Reata Pharmaceuticals</td>
			<td>in house synthesis</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Mouse GM-CSF</td>
			<td>Peprotech Inc.</td>
			<td>315-03</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>Mouse IL-4</td>
			<td>Peprotech Inc.</td>
			<td>214-14</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides (LPS)</td>
			<td>Sigma Aldrich Inc.</td>
			<td>L2880</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma Aldrich Inc.</td>
			<td>516732</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Pertussis toxin (PTX)</td>
			<td>R&#38;D systems</td>
			<td>3097</td>
			<td>EAE induction</td>
		</tr>
		<tr>
			<td>MOG (35&#8211;55) peptide</td>
			<td>21stCentury Biochemicals</td>
			<td>in house synthesis</td>
			<td>EAE induction</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Gibco, Life Technologies</td>
			<td>15250-061</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>RPMI-1640 plus L-glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>11875-093</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Non-essential amino acid (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>11140050</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>15630080</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td>Cell isolation</td>
		</tr>
		<tr>
			<td>PE-conjugated CD80</td>
			<td>BD Biosciences</td>
			<td>557227</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>PE-conjugated CD86</td>
			<td>BD Biosciences</td>
			<td>555665</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>PE-conjugated PD-L1</td>
			<td>BioLegend</td>
			<td>124307</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>APC-conjugated MHCII</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-112-388</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>APC-conjugated CD11c</td>
			<td>BD Biosciences</td>
			<td>340544</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Isotype matched PE</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-091-835</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Isotype matched APC</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-091-836</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>BioLegend</td>
			<td>423801</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Pan dendritic cell isolation kit</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-100-875</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>FcR Blocking Reagent</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-100-875</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Pan Dendritic Cell Biotin-Antibody Cocktail</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-100-875</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-100-875</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>CD4+ T cell isolation kit</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-104-454</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>CD4+ T cell Biotin-Antibody Cocktail</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-104-454</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-104-454</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>ACK lysing buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>A1049201</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD Biosciences</td>
			<td>309626</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>3 ml syringe</td>
			<td>BD Biosciences</td>
			<td>309588</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>25G needle</td>
			<td>BD Biosciences</td>
			<td>309626</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>23G needle</td>
			<td>BD Biosciences</td>
			<td>309588</td>
			<td>BMDC differentiation</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich Inc.</td>
			<td>A2058</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>15575020</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>LS Column</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-042-401</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Pre-Separation Filter</td>
			<td>Miltenyi Biotec Inc.</td>
			<td>130-095-823</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>collagenase D</td>
			<td>Sigma Aldrich Inc.</td>
			<td>11088858001</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher Scientific</td>
			<td>14025076</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>ovalbumin (OVA) peptide 323&#8211;329</td>
			<td>Sigma Aldrich Inc.</td>
			<td>O1641</td>
			<td>T cell proliferation assay</td>
		</tr>
		<tr>
			<td>Mouse IFN-&#947; TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm01168134_m1</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse IL-12a TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm00434165</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse IL-12 p70 DuoSet ELISA</td>
			<td>R&#38;D systems</td>
			<td>DY419-05</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>Mouse EDN-1 ELISA</td>
			<td>RayBiotech</td>
			<td>ELM-EDN1-1</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>TNF-&#945; TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm00443258</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse TNF-&#945; Quantikine ELISA Kit</td>
			<td>R&#38;D systems</td>
			<td>MTA00B</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>IL-6 TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm00446190</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse IL-6 Quantikine ELISA Kit</td>
			<td>R&#38;D systems</td>
			<td>M6000B</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>IL-23a TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm01160011</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Mouse IL-23 DuoSet ELISA</td>
			<td>R&#38;D systems</td>
			<td>DY1887-05</td>
			<td>ELISA</td>
		</tr>
		<tr>
			<td>IL-4 TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm99999154_m1</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>IL-10 TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm01288386_m1</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>TGF-&#946; TaqMan probe</td>
			<td>ThermoFisher Scientific</td>
			<td>Mm01178820_m1</td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>Anti-Heme Oxygenase 1 antibody</td>
			<td>Abcam</td>
			<td>ab13248</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>Anti-&#946;-actin antibody</td>
			<td>Abcam</td>
			<td>ab8226</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detection System</td>
			<td>Bio-Rad Inc.</td>
			<td></td>
			<td>qRT-PCR</td>
		</tr>
		<tr>
			<td>BD FACSCalibur Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometry</td>
		</tr>
	</tbody>
</table>

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.

The differentiation and selection of BMDCs: 
Bone marrow progenitor cells were cultured in complete RPMI medium in the presence of GM-CSF and IL-4 to differentiate into iDCs for 7 days (Figure 1A). On day 1, cells were small in size and showed spherical morphology. Washing with PBS before the replacement of fresh medium on Day 3 helped cells to form clusters and also increased the p...

Watch this Scientific Journal Video about Development and Functional Characterization of Murine Tolerogenic Dendritic Cells at JoVE.com

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

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 ...

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