Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

Summary

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

Abstract

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (Bénézech et al.¹⁴). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP⁺ embryonic adipose tissue and make a LN/ EYFP⁺ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP⁺ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Introduction

Lymph nodes (LNs) are key organs of the immune system situated at strategic sites in the body, along the lymphatic vasculature network. They enable filtration of antigens and pathogens and provide a site for antigen presentation to lymphocytes and induction of adaptive immune responses. The stroma, which forms the basic structure of the LN and orchestrates the movement of the different hematopoietic participants of the adaptive immune response, is central to the function of these organs. Different populations of stromal cells supply essential and specific cues for the movements, localization, survival, proliferation and maturation of the hematopoietic component of the immune system^1-3. Adult LN stromal cells fall in three categories: the blood endothelial cells, the lymphatic endothelial cells and fibroblasts. These three categories encompass heterogeneous populations. The fibroblastic populations contain fibroblastic reticular cells (FRC), follicular dendritic cells (FDC), marginal reticular cells (MRC), while the fibroblasts forming the capsule, the medulla and other cells which are not yet identified^1-5. The origins and the mechanisms governing the maturation of the different LN stromal cell populations are unclear and the absence of specific markers allowing the fate mapping of specific LN stromal cell populations rends their study particularly difficult. However, a full understanding of the ontogeny of LN stromal cells is necessary to the comprehension of adaptive immune responses, the mechanisms contributing to tolerance and is at the basis of the development of artificial LNs.

So far, the study of LN stromal cell origin and development has been mostly limited to the direct assessment of LN development in embryos and newborns in wild type mice and different knockout mouse strains^6-9. These approaches are limited by the embryonic and perinatal lethality of some of the mouse strains carrying deletions in genes important for LN development. Moreover, some of the genes essential for lymphoid tissue development are also involved in a wide range of biological process as it is the case for RANK^10-11 or NF-κB2¹². To address these issues, isolation and transplantation of embryonic LNs under the kidney capsule of a host mouse have been performed^12-13. This technique allows, for example, the transfer of genetically modified embryonic LNs in a wild type environment to assess organ development and the recruitment and organization of host cells. However, the growth of embryonic LN grafted under the kidney capsule of an adult host is impaired, thus limiting the use of this technique.

LNs and fat deposits are anatomically closely associated and they develop simultaneously during embryogenesis. We recently demonstrated that the association LN/adipose tissue plays a crucial role in the provision of stromal cell progenitors for the LNs. In particular, signaling through the LTβR controls the fate of adipocyte precursor cells by blocking adipogenesis and instead promoting maturation towards a LN stromal cell phenotype¹⁴. Here we describe a method based on generation of LN-fat pad chimeras allowing the tracing of adipose tissue derived cells in the developing LN. This method will be useful to determine the contribution of adipose tissues to different LN stromal cell populations and combined with the use of tissues from genetically modified mouse strains, will allow a better understanding of the mechanisms controlling the differentiation of the different LN stromal cell subsets.

Protocol

Mice were bred and maintained under SPF conditions in the Biomedical Service Unit at the University of Birmingham according to UK Home Office and local ethics committee regulations. All procedures described in this protocol are covered under a Project License approved by both local ethics committee and the Home Office. 1. Isolation of Newborn LNs Sacrifice the newborn mice by cervical dislocation. Section the head, and open the body with scissors from the top of the thora…

Representative Results

Three weeks after transplantation, the LN/fat pad chimera is recovered from the kidney. The chimera is now very similar to a normal LN in its own fat pad, and the LN is visible in the center of the adipose tissue (Figure 1). If the LN can't be identified, it is possible that it was lost during the transplantation on the kidney. When assessing the role of genes potentially important in LN development, the LNs recovered may remain very small and more difficult to find. This is the case when adipose tis…

Discussion

In this article we presented a method to assay and quantify the contribution of adipose tissue progenitor cells to the developing LNs and two techniques that allow the analysis of their progeny. Dissection of embryonic fat pads and newborns LNs are delicate and require manual skills gained by a lot of practice prior to the generation of the actual LN-fat pad chimera. To control the quality of the dissections, flow-cytometric analysis can be performed on the fat pads and LNs. Embryonic fat pad preparation should be…

Materials

2-Mercaptoethanol	Sigma	M3148
50 mm Sterilin Petri Dish	Appleton Woods	SC265
90 mm Sterilin Petri Dish	Appleton Woods	SC260
Adhesive slides	Surgipath	00202
Anti-mouse CD31 eFluor 450	eBioscience	48-0311
Anti-mouse CD4 Alexa 647	eBioscience	51-0041
Anti-mouse CD45 PerCP-Cy5.5	eBioscience	45-0451
Anti-mouse IgM Rhodamine Red	Stratech	715-296-020-JIR
Anti-mouse Podoplanin PE/Cy7	Biolegend	127411
Anti-mouse Podoplanin purified	eBioscience	14-5381
Collagenase D	Roche	11088858001
DMEM Medium	Sigma	D5671
DNase I	Sigma	DN25-1G
Dumont #5 Forceps Inox Biologie	FST	11252-20	Extra fine forceps for embryonic and newborn dissections
EDTA solution 0.5 M	Sigma	E7889
ERTR-7	Biogenesis
Fetal bovine serum	Sigma	F9665
Hardened fine iris scissors straight 11 cm	FST	14090-11	Small scissors for dissection
HEPES solution	Sigma	H0887
Isopore Membrane Filters 0.8 μm ATTP	Millipore	ATTPO 1300
L-Glutamine solution	Sigma	G7513
MEM Nonessential Amino Acid Solution (100x)	Sigma	M7145
O.C.T. Compound	Tissue-Tek	4583
Penicillin-Streptomycin	Sigma	P4458
Plastic box with lid	Watkins and Doncaster	E6052	Sandwich box for in vitro organ culture. Drill two holes in the lid to allow gas exchange in the CO2 incubator.
RPMI 1640 Medium	Sigma	R0883
Sponges Vulkan Underwrap	Patterson Medical	004383
Stereomicroscope	Leica	LEICA MZ95	Dissecting microscope with zoom
Thermomixer	Eppendorf	5436
Vectashield Mounting Medium with DAPI	Vector Laboratories	H-1200