Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

Summary

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

Abstract

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 naïve 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.

Introduction

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. Myeloi....

Protocol

All studies were performed in compliance with procedures approved by the Case Western Reserve University School of Medicine's Institutional Animal Care and Use Committee.

1. Prepare Bone Marrow-derived Dendritic Cells (BMDCs)

1. Sterilize all the surgical instruments via autoclaving and perform the experiment in class II biological safety cabinet with appropriated safety procedures.
2. Euthanize C57BL/6 mice 8 - 10 weeks of age by using CO₂ chamber. Place the mouse on a dissecting board and rinse with 70% ethanol. Excise tibia-fibula and femur bones by using a surgical scissor and place them with 70% ethanol i....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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