Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Summary

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Abstract

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Introduction

Since their discovery, dendritic cells (DC) have been a focus of extensive research due to their unique ability to skew T cell differentiation¹. Over the past several decades, an extensive research effort has sought to define the various DC subsets and their function during tumor progression and immunity ². DCs are composed of heterogeneous cell populations that differ from each other in their pattern-recognition receptors, tissue distribution, and migratory and antigen presentation capabilities^3,4,5. Compared to other DC subset....

Protocol

The protocols below refer to the isolation of mouse MoDC, yet the overall principles may apply to other DC subsets cells, as well. 12 - 16-week-old C57Bl/6j mice were maintained in an American Association for the Accreditation of Laboratory Animal Care–accredited animal facility. All protocols were approved by Stanford University and Tel-Aviv University Institutional Animal Care and Use Committee.

1. Isolation of Tumor Associated Monocyte-Derived DC

1. In a laminar flow hood, remove tumors from CO₂ euthanized mice and place tumors in RPMI medium without fetal bovine serum (FBS).
   NOTE: It is highly recommende....

Results

We initially compared the capacity of antibodies from naïve syngeneic and allogeneic mice to bind to tumor cells. To this end, B16F10 and LMP tumor cell lines were fixed in paraformaldehyde and washed extensively. B16F10 is a melanoma cell line, which was originally isolated from lung metastases in C57Bl/6 mice. LMP is a pancreatic tumor cell that was isolated from KrasG12D/+, LSL-Trp53R172H/+, and Pdx-1-Cre mice, and grows steadily in 129F1 mice. To obtain IC, tumor cells were incubated .......

Discussion

Given the large number of DC required for vaccinating mice (approximately 2-4 x 10⁶ DC per one mouse), most of the vaccination strategies in mice are based on isolation of DC from BM and spleen followed by their ex vivo activation. However, attempts to activate tumor DC in vivo, using the same conditions for activating spleen and BM DC, have often been unsuccessful in producing effective immunity. In two subsequent publications, Carmi et al. have found that blood and tumor MoDC diffe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ficoll-Paque PREMIUM	GE-Healthcare	17-5442-02
OptiPrep	StemCell Technologies	07820
CD45 MicroBeads	Miltenyi	130-052-301
EasySep Monocyte Isolation Kit	StemCell Technologies	19861
Collagenase IV	Sigma	C9697-50MG	Test each lot for endotoxin
DNase I	Sigma	DN25-10MG
HBSS	ThermoFisher	14025092
FBS	ThermoFisher	16140071	Test each lot for endotoxin
PE-CD11c	Biolegend	117307
APC-CD11b	Biolegend	101211
Brilliant Violet 650 MHCII	Biolegend	107641
AF48- CD86	Biolegend	105017
APC/Cy7-Ly-C6	Biolegend	108423
PE/Cy7-CD15	Biolegend	135523