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

1. Sacrifice the newborn mice by cervical dislocation.
2. Section the head, and open the body with scissors from the top of the thoracic region to the bottom of the abdominal region and remove carefully all the viscera (heart, lungs, liver, intestine, kidney, bladder) from the abdominal cavity.
3. Transfer the body into a 90 mm Petri dish containing RF10 media (RPMI1640 media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine). From this step onwards, the tissues will be kept under sterile conditions and handled in a tissue culture hood.
4. Transfer the body into a new 90 mm Petri dish containing RF10.
5. Under the dissecting microscope, detach carefully the peritoneum from the skin in the inguinal region. The inguinal LN is situated at the intersection of three blood vessels in the fat pad.
6. Remove carefully the LN. Make sure that all adipose tissue is removed. Transfer the LN into a 50 mm Petri dish containing RF10 media. Keep the tissues on ice while continuing with the procedure.

2. Isolation of E18.5 Fat Pads

1. To generate mouse embryos at day 18.5 of gestation (E18.5), set up timed pregnancies by placing a female and a male mouse per cage overnight. Separate the mice in the morning and check for vaginal plugs (gestational day E0.5).
   1. Euthanize the plugged females at day 18.5 of pregnancy by cervical dislocation.
   2. Remove the embryos and placed them in a 90 mm Petri Dish containing RF10.
2. From this step onwards, tissues should be handled using sterile technique in the tissue culture hood. Under the dissecting microscope, section the head, open the embryos with scissors from the thoracic region to the bottom of the abdominal region and remove carefully all the viscera (heart, lungs, liver, intestine, kidney, bladder) from the body cavity.
3. Clean the body from all remaining visceral tissues and wash it in PBS to eliminate all traces of blood that could make the dissection more difficult.
4. Transfer the clean bodies into a fresh 90 mm Petri dish containing RF10.
5. Carefully detach the peritoneum from the skin in the inguinal region. The inguinal LN is situated at the intersection of three blood vessels in the fat pad.
6. Carefully remove the LN and discard it. It is very important to completely remove the LN to ensure that the fat pad used for the chimeras is not contaminated by any lymphoid stromal cells.
7. Remove the inguinal fat pad and transfer it into a 50 mm Petri dish containing RF10 media. Keep the tissues on ice while continuing with the procedure.

3. Generation of the LN-fat Pad Chimera

1. Prepare the in vitro organ culture system¹⁵.
   1. Sponge and filters preparation: This can be done in advance and sponges and filters stored sterile in a Petri dish in the culture hood.
      1. Cut the Vulkan Underwrap in 1-1.5 cm² pieces
      2. Boil the sponges for 2 hr in distilled water.
      3. Let the sponges dry for a couple of hours in the cell culture hood.
      4. Boil the filters for 20 min.
      5. Let the filters dry for a couple of hours in the cell culture hood.
   2. Prepare DMEM media complemented with 10% fetal bovine serum, 10 mM HEPES, 1x MEM Nonessential Amino Acid Solution, 50 mM 2-Mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-Glutamine.
   3. Add 2 ml of media in a 50 mm Petri dish.
   4. Arrange one sponge at the bottom of the Petri dish. Wet both sides of the sponge by submerging them in the media.
   5. Place one filter on top of the sponge. The filter is positioned at the liquid-air interface.
2. Under the dissecting microscope, carefully reassociate one newborn LN with one embryonic fat pad.
3. Place the LN-fat pad chimera on top of the filter.
4. Transfer the Petri dish in a rectangular plastic box with a couple of holes on the lid and containing water at the bottom to ensure humidity.
5. Seal the lid to the box with tape. Leave the two holes in the lid open.
6. Transfer the box to a cell culture incubator at 37 °C with 5% CO₂. Allow the box to equilibrate for 2 hr, before sealing the holes in the lid with tape.
7. Incubate the tissues for at least 2 days to allow the LN to attach to the fat pad and be transferable under the kidney capsule.

4. Transfer of the LN-fat Pad Chimera Under the Kidney Capsule of Host Mice

1. Collect the LN-fat pad chimeras from the filter and transfer them under the kidney capsule of host mice. The kidney capsule transfer method has already been described ¹⁶.

5. Isolation of the Transferred LNs

1. After 3-4 weeks under the kidney capsule, the LN-fat pads chimeras are ready to be harvested. Euthanize the host mice using approved guidance.
2. Remove the kidney from the host mice.
3. Under the dissecting microscope, isolate the LN-fat pad chimera and dissect out the LN.

6. Enzymatic Digestion of the Transferred LNs for Flow-Cytometric Analysis

1. Make one incision in the LN with small scissors to allow the enzymes to penetrate the organ and facilitate the digestion.
2. Place one LN in 1.5 ml Eppendorf containing 600 μl of digestion buffer (RPMI 1% FCS, 2.5 mg/ml Collagenase D, 100 μg/ml DNase I).
3. Incubate for 30 min at 37 °C on an agitating thermal block. Pipette up and down to help dissociation of tissues every 10 min.
4. Add 6 μl EDTA 0.5 M to the tube and pipette up and down to finish the dissociation of the tissues.
5. Centrifuge for 5 min at 490 x g.
6. Resuspend the cell pellet in the staining solution (PBS, 0.1% BSA, 5% rat serum and 5% mouse serum) containing the combination of primary antibodies.
7. Incubate for 30 min on ice.
8. Wash twice in PBS
9. Incubate with secondary antibodies for 20 min if required.
10. Wash 2x in PBS.
11. Analyze on flow-cytometer.

7. Analysis of the Transferred LNs by Immunofluorescence

1. Fixation for EYFP conservation during the freezing and cryosections process: Place the LN in a solution of PBS, 4% PFA and 10% sucrose and leave for 3-4 hr.
2. Put a single small drop of cold O.C.T. compound on a small piece of aluminum foil. Avoid air bubbles. Carefully place the LN without any liquid in the middle of the drop.
3. Freeze the organ by placing the aluminum foil on dry ice.
4. Cut 8-10 μm sections onto extra adhesives glass slides using a cryostat. Let the slides dry for 2 hr before storing them at -20 °C.
5. Staining procedures can vary according to primary antibodies used and can be optimized from the following protocol. Stain the sections with staining solution (PBS, 1% BSA) containing the combination of primary antibodies.
6. Incubate for 1 hr at room temperature in a humidified chamber.
7. Wash twice in PBS for 5 min
8. Incubate with secondary antibodies for 1 hr.
9. Wash twice in PBS for 5 min.
10. Mount slides in mounting media containing DAPI.
11. Capture images using a fluorescence or confocal microscope.

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

Name	Company	Catalog Number	Comments
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