Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

Summary

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer’s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer’s patch.

Abstract

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer's patch (PP). Common dendritic cell progenitors (CDPs, lin^- c-kit^lo CD115⁺ Flt3⁺) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar^-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1⁺ and recipients being CD45.2⁺. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar^-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Introduction

Here, we demonstrate a protocol to assess whether common dendritic cell progenitors (CDPs) are capable of giving rise to the plasmacytoid dendritic cell (pDC) population in Peyer's patch (PP). The overall goal of using this method was to evaluate the developmental regulation of pDCs in Peyer's patch (PP pDCs). The reason this is important is that PP pDCs differ from pDCs found in other tissues, including bone marrow, blood and spleen, and therefore it is unclear whether PP pDCs and other pDC populations are developmentally and/or functionally related. Specifically, pDCs are widely known for being the principal type I interferon (IFN) producers within the hemat....

Protocol

Institutional approval must be obtained in advance for all experimental manipulations described herein involving mice. These include the use of C57BL6 mice for isolation of bone marrow progenitor cells, Ifnar^-/- mice as recipients for adoptive transfer of hematopoietic progenitors and use of HGT for cytokine overexpression in vivo. Appropriate housing and animal care must also be provided by the investigator or institution. Furthermore, institutional approval may be required for the plasmids used in hydrodynamic gene transfer (i.e. recombinant DNA approval). The studies described here were approved by the Institutional Animal....

Results

Our results show the gating strategy for the isolation of CDPs from mouse bone marrow cells (Figure 1), as detailed in Protocols 2 and 3. CDPs comprise approximately 0.1% of total bone marrow cells, and roughly 4-6 x 10⁴ CDPs can be isolated from one mouse. Upon adoptive transfer, CDPs differentiate into pDCs and cDCs¹⁰.

Discussion

The adoptive transfer technique described herein assessed the contribution of CDPs to the PP pDC population in recipient mice that are deficient in PP pDCs (e.g. Ifnar^-/- mice). In future experiments, it will be important to evaluate the potential of other progenitor subsets in generating PP pDCs, in particular whether PP pDCs derive from Flt3⁺ CLPs. This question is significant since it remains unclear why PP pDCs are uniquely sensitive to IFNAR-STAT1 signals for PP accrual

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6J	JAX	664
B6.SJL	JAX	2014
RPMI	Invitrogen	11875-093
15 ml Conical tubes	BD Biosciences	352095
50 ml Conical tubes	BD Biosciences	352070
Sterile surgical tweezers
Sterile small pair scissors
Sterile large pair scissors
40 μm cell strainer	BD Biosciences	352340
35 μm cell strainer cap tubes	BD Biosciences	352235
RBC lysing buffer	Sigma	R7757
FACS buffer			PBS, 2 mM EDTA, 1% FCS, filter sterilized
Percoll	GE Healthcare	17089102
10x HBSS	Sigma	H4641
Collagenase IV	Worthington	LS004188
Goat anti-rat IgG microbeads	Milteyni Biotec	130-048-501
LD column	Milteyni Biotec	130-042-901
Rat anti-D3	BD Biosciences	555273
Rat anti-CD19	BD Biosciences	553783
Rat anti-CD11b	BD Biosciences	553308
Rat anti-CD11c	BD Biosciences	553799
Rat anti-Ter119	BD Biosciences	553671
Anti-CD3 (PerCP)	eBiosciences	45-0031
Anti-CD19 (PerCP)	eBiosciences	45-0193
Anti-CD11b (PerCP)	eBiosciences	45-0112
Anti-CD11c (PerCP)	eBiosciences	45-0114
Anti-F4/80 (PerCP)	eBiosciences	45-4801
Anti-Ter119 (PerCP)	eBiosciences	45-5921
Anti-Sca-1 (PE.Cy7)	eBiosciences	25-5981
Anti-CD115 (APC)	eBiosciences	17-1152
Anti-c-kit (APC.Cy7)	eBiosciences	47-1171
Anti-IL-7R (Pacific Blue)	eBiosciences	48-1271
Anti-Flt3 (PE)	eBiosciences	12-1351
Anti-CD45.1 (APC.Cy7)	BD Biosciences	560579
Anti-CD45.2 (PE.Cy7)	Biolegend	109830
Anti-CD11c (Pacific Blue)	eBiosciences	48-0114
Anti-B220 (APC)	eBiosciences	25-0452
Anti-Siglec-H (PE)	eBiosciences	12-0333
Anti-PDCA-1 (FITC)	eBiosciences	Nov-72
Cell sorter	BD Biosciences		e.g. BD Fortessa
Heat lamp
Mouse restrainer
1 ml Syringes	Becton Dickinson	309602
27½ G needles (sterile)	Becton Dickinson	305109