Effect of an Immunomodulatory Pharmacological Agent on the Interaction Between Dendritic Cells and CD4+ T Cells

Abstract

Source: Wei, H., et al. Development and Functional Characterization of Murine Tolerogenic Dendritic Cells. J. Vis. Exp. (135), (2018).

This video demonstrates a method of converting dendritic cells (DCs) to tolerogenic DCs (TolDCs) using an immunomodulatory pharmacological agent. This conversion is confirmed by evaluating the TolDC function through their immunosuppressive effect on CD4+ T cells.

Protocol

1. Evaluate the Function of TolDCs In Vitro and In Vivo

1. T-cell Syngeneic Proliferation Assay
   1. Sterilize all the surgical instruments via autoclaving and perform the experiment in a class II biological safety cabinet with appropriate safety procedures.
   2. Prepare MACS buffer using 0.5% bovine serum albumin (BSA) and 2 mM EDTA in 500 ml PBS. Sterilize the buffer by filtering through a 0.2 µm filter.
      NOTE: Keep the buffer on the ice during the following experiment.
   3. To obtain CD4+ T cells, euthanize OT-II T-cell receptor (TCR) transgenic mice 8-10 we....

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
Pan dendritic cell isolation kit	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
CD4+ T cell isolation kit	Miltenyi Biotec Inc	130-104-454	T cell proliferation assay
CFSE	BioLegend	423801	T cell proliferation assay
FcR Blocking Reagent	Miltenyi Biotec Inc.	130-100-875	T cell proliferation assay
Anti-Biotin MicroBeads	Miltenyi Biotec Inc.	130-104-454	T cell proliferation assay
1 ml syringe	BD Biosciences	309626	T cell proliferation assay
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