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

Summary

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

Abstract

Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence naïve 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 naïve 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.

Introduction

Dendritic cells (DCs) are critical mediators between innate and adaptive immunity. DCs, which mainly reside in mucosal membranes, skin and lymphoid tissue, are the primary antigen presenting cells (APCs)¹. DCs uptake foreign proteins and process and present them on major histocompatibility (MHC) proteins to naïve T cells. DCs specifically express MHC class II proteins, such as human leukocyte antigen-DR (HLA-DR) in humans. The activation state of the DCs upon antigen exposure is critical for the downstream T cell response². Immature DCs express various pattern recognition receptors (PRRs) that recognize classes of m....

Protocol

All human peripheral blood mononuclear cell (PBMC) samples were obtained from the University of Pennsylvania's Human Immunology Core from deidentified donors with prior approval from the University of Pennsylvania's Institutional Review Board (IRB) with patient consent.

Optional: While in this method, we used freshly isolated PBMCs obtained from an academic laboratory, PBMCs can be isolated from either whole blood or leukapheresis-enriched blood products. We recommend using the density gradient centrifugation method, as this is a well-established and reliable method that is described elsewhere²³

Results

We have described a protocol for human PBMCs, isolate both CD3⁺ T cells and CD14⁺ monocytes using commercially available magnetic separation kits, differentiate monocytes into CD14^-, HLA-DR⁺, CD141⁺, CD1c⁺ moDCs using GM-CSF and IL-4, treat them for 24 h, and co-culture with autologous T cells with anti-CD3/CD28 stimulation for 72 h. An experimental schematic is shown in Figure 1.

Isolation .......

Discussion

Here we describe a reliable and versatile method to assess the functionality of immunomodulatory agents to induce tolDCs from moDCs and validate their functionality to generate Tregs from autogenic T cells ex vivo. There are several critical steps in this protocol. First, monocytes are notoriously sensitive cells and must be obtained from fresh, not previously frozen PBMCs for the best results. Monocytes should be isolated as soon as possible and placed in the differentiation cocktail. Typically, poor monocyte y.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.1-10 µL Filtered Pipet tips	VWR	76322-158	General Cell Culture
1.5 mL Centrifuge Tube	VWR	77508-358	General Cell Culture
10 mL Serological Pipets	VWR	414004-267	General Cell Culture
100-1000 µL Filtered Pipet tips	VWR	76322-164	General Cell Culture
15 mL Conical Tube	VWR	77508-212	General Cell Culture
20-200 µL Filtered Pipet tips	VWR	76322-160	General Cell Culture
2-Mercaptoethanol	MP Biomedical	194834	T Cell Culture
50 mL Conical Tube	VWR	21008-736	General Cell Culture
60 x 15 mm Dish, Nunclon Delta	Thermo Fischer	150326	General Cell Culture
96 Well Conical (V) Bottom Plate, Non-Treated Surface	Thermo Fischer	277143	General Cell Culture
96 well Flat Bottom Plate	Thermo Fischer	161093	General Cell Culture
APC/Cyanine7 anti-human CD272 (BTLA) Antibody	Biolegend	344518	Flow Cytometry
Attune NxT (Red/Blue Laser, 7 Channel)	Thermo Fischer	A24863	Flow Cytometry
BSA	Thermo Fischer	15260-037	General Cell Culture
CD14 Monoclonal Antibody (61D3), PE	Thermo Fischer	12-0149-42	Flow Cytometry
CD1c Monoclonal Antibody (L161), PE-Cyanine7	Thermo Fischer	25-0015-42	Flow Cytometry
CD209 (DC-SIGN) Monoclonal Antibody (eB-h209), PerCP-Cyanine5.5	Thermo Fischer	45-2099-42	Flow Cytometry
CD25 Monoclonal Antibody (CD25-4E3), APC	Thermo Fischer	17-0257-42	Flow Cytometry
CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH1), PE	Thermo Fischer	12-5983-42	Flow Cytometry
CD4 Monoclonal Antibody (RPA-T4), Alexa Fluor 488	Thermo Fischer	53-0049-42	Flow Cytometry
CD40 Monoclonal Antibody (5C3), APC	Thermo Fischer	17-0409-42	Flow Cytometry
CD40 Monoclonal Antibody (5C3), APC-eFluor 780	Thermo Fischer	47-0409-42	Flow Cytometry
CD69 Monoclonal Antibody (FN50), PE	Thermo Fischer	MA1-10276	Flow Cytometry
CD86 Monoclonal Antibody (BU63), FITC	Thermo Fischer	MHCD8601	Flow Cytometry
CD8a Monoclonal Antibody (RPA-T8), PE-Cyanine7	Thermo Fischer	25-0088-42	Flow Cytometry
Conical Bottom (V-well) 96 Well Plate	Thermo Fischer	2605	Flow Cytometry
Cryogenic Vials, 2 mL	Thermo Fischer	430488	T Cell Culture
Dimethylsulfoxide (DMSO), Sequencing Grade	Thermo Fischer	20688	General Cell Culture
DPBS	Thermo Fischer	14200166	General Cell Culture
EasySep Human Monocyte Isolation Kit	Stem Cell Technologies	19359	Cell Separation
EasySep Human T Cell Isolation Kit	Stem Cell Technologies	17951	Cell Separation
EasySep Magnet	Stem Cell Technologies	18000	Cell Separation
EDTA	Thermo Fischer	AIM9260G	General Cell Culture
Falcon Round-Bottom Polystyrene Tubes, 5 mL	Stem Cell Technologies	38025	Cell Separation
Fc Receptor Binding Inhibitor Polyclonal Antibody	Thermo Fischer	14-9161-73	Flow Cytometry
Fetal Bovine Serum	Thermo Fischer	A5670701	General Cell Culture
Fixable Viability Dye eFluor 780	Thermo Fischer	65-0865-18	Flow Cytometry
Foxp3 / Transcription Factor Staining Buffer Set	Thermo Fischer	00-5523-00	Flow Cytometry
FOXP3 Monoclonal Antibody (PCH101), PE-Cyanine5.5	Thermo Fischer	35-4776-42	Flow Cytometry
HBSS	Thermo Fischer	14170-112	General Cell Culture
Heat Inactivated Fetal Bovine Serum	Thermo Fischer	A5670801	General Cell Culture
HEPES (1 M)	Thermo Fischer	15630106	moDC Cell Culture
HLA-DR Monoclonal Antibody (L243), Alexa Fluor 488	Thermo Fischer	A51009	Flow Cytometry
Human CD3/CD28/CD2 T Cell Activator	StemCell Technologies	10970	T Cell Culture
Human GM-CSF Recombinant Protein	Thermo Fischer	300-03	moDC Cell Culture
Human IL-10 ELISA Kit, High Sensitivity	Thermo Fischer	BMS215-2HS	ELISA
Human IL-4, Animal-Free Recombinant Protein	Thermo Fischer	AF-200-04	moDC Cell Culture
Human PBMC (Freshly Isolated)	UPenn HIC	N/A	Cells
Human TNF alpha ELISA Kit	Thermo Fischer	BMS223-4	ELISA
Light Microscope (DMi1)	Lucia	391240	General Cell Culture
Lipopolysaccaride (LPS)	Invivogen	tlrl-eblps	moDC Cell Culture
LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit	Thermo Fischer	L34975	Flow Cytometry
MEM Non-Essential Amino Acids Solution (100x)	Thermo Fischer	11140050	T Cell Culture
Penicillin-Streptomycin (100x)	Thermo Fischer	15140122	General Cell Culture
Pipette Controller	VWR	77575-370	General Cell Culture
Rapamycin, 98+%	Thermo Fischer	J62473.MF	moDC Cell Culture
RPMI 1640 with Glutamax	Thermo Fischer	61870-036	General Cell Culture
Separation Buffer	Stem Cell Technologies	20144	Cell Separation
T Cell Stimulation Cocktail (500x)	Thermo Fischer	00-4970-93	T Cell Culture
UltraComp eBead Plus Compensation Beads	Thermo Fischer	01-3333-41	Flow Cytometry
Variable Pipette Set	Fischer Scientific	05-403-152	General Cell Culture