Isolation of Murine Lymph Node Stromal Cells

Summary

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Abstract

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host.

Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Introduction

Lymph nodes are specialized compartments where adaptive immune responses against foreign and self-antigens are initiated and coordinated. The procedure presented here describes a short enzymatic digestion combined with automated mechanical pipetting to obtain lymph node single cell suspension and gain access to viable lymph node stromal cells that maintain the surface expression of several molecules.

Lymph node stromal cell form the scaffold of the lymph node and fulfill three major functions: first they filter body fluids to sample antigens, pathogens and their pathogen associated molecular pattern (PAMPs), as well as cytokines and danger associated molecular pattern (DAMPs) present in the body. Second, they attract and instruct antigen presenting cells (APC) and lymphocytes to interact and initiate adaptive immune responses; and third, they provide a structural environment for the homeostasis and differentiation of lymphocytes^1-3. During inflammation lymph node stromal cells produce growth factors, cytokines and chemokines, adapt to swelling thereby organizing the interaction between dendritic cells (DCs), T-, and B- cells. The orchestration of immune responses is only possible due to the complex structural architecture formed by different stromal cell populations.

Lymph node stromal cells are CD45 negative cells and can be distinguished by the expression of CD31 or gp38 in fibroblastic and endothelial cells^1-6. Gp38⁺CD31^- defines T zone reticular cells (TRC, also known as FRC: fibroblastic reticular cells), gp38⁺CD31⁺ defines lymphatic endothelial cells (LEC), gp38^-CD31⁺ defines blood endothelial cells (BEC). Further, characterization of the subpopulations revealed the existence of other lymph node stromal cells. Indeed, a small pericyte-like cell population was characterized within the gp38^-CD31^- population⁷. Therefore, adaptation of the isolation procedure is advantageous for identification and characterization of the functional properties of different lymph node stromal cells.

Before the development of lymph node stromal cells digestion protocols the study of lymph node stromal cells was limited to in situ observations using tissue section and microscopy. Nevertheless, structural and functional studies showed important characteristics of lymph node stromal cells. Lymph node stromal cells are associated with podoplanin, collagen and extracellular matrix (ECM) proteins to form a complex 3 dimensional structure called conduit system, which transports lymph and associated-low molecular mass proteins from the subcapsular sinus of the lymph node to the high endothelial venules in the T cells zone⁸. DCs are in close contact with stroma cells and may be observed protruding into the tubular conduit structure to sample fluid and detect antigens⁸. The interaction of lymph node stromal cells (TRCs and LECs) with DCs is mediated by the release and presentation of chemokines CCL21 and CCL19^9,10. CCL19 and CCL21 are recognized by the CCR7 receptor facilitating DCs and T cells to migrate to the lymph node T cell zone^4,11. Despite using similar chemokines, DCs and T cells have different migration routes into the lymph nodes¹². Later, using enzymatic digestion of the lymph node and isolation of pure lymph node stromal cells, functional studies were performed on the role of the different lymph node stromal cells and their ability to interact with DCs and T/B cells^6,13. First, the crosstalk between IFN-γ producing effector T cells and lymph node stromal cells induces the production of the metabolite nitric oxide shown to dampen T cell responses and proliferation in the secondary lymphoid organs^14-16. Second, lymph node stromal cells have been reported to support the differentiation of regulatory DC subsets via the production of IL-10¹⁷, and to modulate naïve T cell homeostasis via the production of IL-7^6,18. Third, TLR expression in lymph node stromal cells suggests that stromal cells are susceptible to signal derived from an infection or self-molecules released during tissue injury. Indeed, the treatment of lymph node stromal cells with the ligand of TLR3 poly(I:C) induces a modest upregulation of major histocompatibility complex class I expression and upregulation of co-inhibitory molecule PD-L1, but not of costimulatory molecules, resulting in dramatic changes in peripheral tissue antigens expression¹⁹. Several groups have shown lymph node stromal cells express peripheral tissue antigens and induce tolerance of self-reactive T cells^19,21-27. Therefore, understanding the interactions between lymph node stromal cells and the other migratory and resident lymph node cells will help to find new target molecules to allow activation or suppression of immune responses during inflammation. Therefore, the implementation of the published enzymatic separation of the lymph node is needed.

Previously published protocols use different combinations of collagenase-based enzymatic digestion with low mechanical stress^6,19,20. However, long incubations with digestion enzymes or the different combination of digestion enzyme might degrade various surface molecules required to analyze the activation status and to identify new lymph node stromal cells. Depending on the type of the stromal cell analysis, the Link Protocol or Fletcher Protocol might be more suited. In the described procedure, a slightly shorter enzymatic digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells. This procedure enables highly reproducible isolation and distinction of lymph node stromal cell populations with low variability and more than 95% viability. The freshly isolated lymph node stromal cells can be directly used for surface marker expression, protein analysis, and transcriptional studies, as well as establishment of stromal cells lines to perform functional assays in vitro.

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Protocol

In this video publication and protocol, all animal procedures were conducted in accordance to the animal protocol approved by the Cantonal Authority Basel-Stadt, Switzerland.

1. Lymph Nodes Preparation and Digestion

1. Pre-heat water in a beaker to 37 °C on a magnetic stirrer with heating plate.
2. Prepare Basic Medium as following: DMEM medium (without pyruvate) supplemented with 2% FCS, 1.2 mM CaCl₂ and Pen/Strep (100 units of penicillin, 100 µg of streptomycin).
3. Sterilize all dissection instruments before use.
4. Euthanize lymph node donor mice per CO₂ asphyxiation and aseptically dissect the lymph nodes. Do not dissect the surrounding fat. NOTE: This protocol is optimized for peripheral skin-draining lymph node (inguinal, brachial, axillary).
5. Place lymph nodes in a sterile Petri dish containing 2 ml ice cold basic medium.
6. Disrupt the lymph node capsule using two 25 G needles fixed on 1 ml syringe.
7. Transfer the disrupted lymph node tissue in a 5 ml polypropylene round-bottom tube containing 750 µl basic medium supplemented with 1 mg/ml Collagenase IV and 40 µg/ml DNAse I.
8. Add one sterile magnetic stirrer in each tube.
9. Place tube in the beaker with 37 °C preheated water and stir the tubes at a slow rate (1 round/sec) for 30 min.
10. Remove the tube from the magnetic stirrer with heating plate and let lymph node fragments settle.
11. Carefully remove the supernatant enriched in "non-stromal cell". NOTE: If the analysis of T, B, dendritic cells and CD45^-gp38^-CD31^- is foreseen, save the "non-stromal cells" fraction.
12. Wash remaining lymph node tissue once with 750 µl basic medium. NOTE: This step is necessary only if working with immune-competent mice.
13. Let lymph node fragments settle.
14. Remove the non-stromal cell-floating fraction.
15. Add to the lymph node fragments 750 µl basic medium supplemented with 3.5 mg/ml Collagenase D and 40 µg/ml DNase I.
16. Place tube back in the beaker containing the 37 °C preheated water.
17. Digest lymph node tissue for 5 min while slowly stirring.
18. Disaggregate lymph node tissue fragments by pipetting and mixing 700 µl for 10 cycles at maximal speed using an automated multichannel pipette. NOTE: This disrupts lymph node tissue to improve digestion.
19. Place the tube back in the beaker with 37 °C preheated water.
20. Digest lymph node tissue fragments for another 10 min while slowly stirring.
21. Disaggregate lymph node tissue fragments by pipetting and mixing for 99 cycles at maximal speed using an automated multichannel pipette.
22. Add 7.5 µl of 0.5 M EDTA to ensure maintenance of single cell suspension.
23. Disaggregate lymph node tissue fragments by pipetting and mixing for 99 cycles at maximal speed using an automated multichannel pipette.
24. Add 750 µl basic medium and pass cells through a 70 µm nylon mesh.
25. Centrifuge cell suspension 5 min at 1,500 x g, 4 °C.
26. Stromal cells can now be used for further analysis.

2. Staining of Lymph Node Stromal Cells

1. Use a combination of anti-CD45, anti-podoplanin (gp38), anti-CD31 antibodies to successfully recognize TRC, LEC, BEC and DN cells.
2. Incubate the digested lymph node tissue with Live/Dead tracker, anti-CD45-FITC (1:200), anti-gp38-PE (1:200), anti-CD31-APC (1:200) in 100 μl HBSS containing 2% FCS for at least 20 min, 4 °C, in the dark.
3. Wash the cells adding 500 l of HBSS containing 2% FCS
4. Centrifuge cell suspension 3 min at 1500 x g, 4°C.
5. Re-suspend in 100 μl of HBSS containing 2% FCS.
6. Run the stained cells in a flow cytometer equipped with the following optics: excitation source with up to three lasers: blue (488 nm, air-cooled, 20 mW solid state), red (633 nm, 17 mW HeNe), and violet (405 nm, 30 mW solid state).
7. Gate CD45^- cells to exclude hematopoietic cells.
8. Gate singlets (FSC-W) and live cells (LIVE/DEAD tracker) to exclude doublets and dead cells.
9. Plot gp38 vs. CD31 to visualize TRC (gp38⁺CD31^-), LEC (gp38⁺CD31⁺), BEC (gp38^-CD31⁺) and DN (gp38^-CD31^-) cells (see Figure 1).

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Results

The present protocol is a modified digestion protocol published by Link et al., 2007⁶ with a shorter digestion time (45 min maximum) due to mechanical disaggregation with an automated multichannel pipette. In addition, the procedure is more standardized, minimizes degradation of surface markers on different lymph node stromal cells and allows the handling of more than one sample at the same time.

Collagenase IV and Collagenase D in Links protocol⁶ and current pro...

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Discussion

The study of lymph node stromal cells recently became a research focus due to the development of two published digestion protocols^6,13. Both protocols are adequate to gain single lymph node stromal cells but differ in the use of digestion enzymes and the time of digestion. Since stromal cells and their surface markers are sensitive to enzymatic digestion and mechanical stress, an optimized protocol is required.

The isolation of viable lymph node stromal cells from freshly dissected ...

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Materials

Name	Company	Catalog Number	Comments
DMEM	LifeTechnologies-Gibco	41965-039
FCS			Final concentration 2%
CaCl2	Sigma	499609	Final concentration 1.2 mM
Collagenase IV	Worthington Biochemical Corporation		>160 units per mg dry weight, use at final concentration of 1 mg/ml
Collagenase D	Roche	11088882001	use at 3.5 mg/ml
DNAse I	Roche	11284932001	use at 40 µg/ml
Stiring magnets	FAUST		5 mm long-2 mm ø
Polystyrene round bottom tubes 5 ml	Falcon-BD Bioscience
Magnetic stirrer with heating function	IKA-RCT-standard	9720250
Petri dishes 100 mm, sterile	TPP	6223201
25 G needles	Terumo
anti mouse CD45 Ab	Biolegend		Clone 30-F11
anti mouse CD11c Ab	Biolegend		Clone N418
anti mouse Podoplanin Ab	Biolegend		Clone 8.1.1
anti mouse CD31 Ab	Biolegend		Clone MEC13.3
Eppendorf Xplorer plus, Multichannel	Eppendorf	4861 000.821/830	1.250 µl max. volume
anti mouse CD140a	Biolegend		Clone APA5
anti mouse CD80	Biolegend		Clone 16-10A1
anti mouse CD40	Biolegend		Clone 1C10
anti mouse I-Ab	Biolegend		Clone AF6-120.1
anti mouse CD274 (PD-L1)	Biolegend		Clone 10F.9G2
LIVE/DEAD Fixable Near-IR Dead Cell stain kit	Invitrogen	L10119