Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Summary

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Abstract

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4⁺ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4⁺ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17^- and CCL22-induced movement of CCR4⁺ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 µm pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

Introduction

This original transmigration method was presented by Stephen Boyden in 1962 in the Journal of Experimental Medicine¹. Much of what we know about chemotaxis and chemokinetics would not be possible without the development of the Boyden chamber. Prior to the discovery of the first chemokine in 1977, ex vivo transmigration systems were used to learn about serum-factors that could arrest cellular movement in macrophages while amplifying cellular motility in neutrophils^1,2. A massive wealth of knowledge has been developed regarding immune cell migration, and to date, 47 chemokines ha....

Protocol

All methods described here were reviewed and approved by the Institutional Animal Care and Use Committees at the University of Nebraska Medical Center (UNMC) and the University of Utah.

1. Setup and Preparation of Reagents

1. Prepare complete RPMI (Roswell Park Memorial Institute) media.
   1. Add 10 mL of heat-inactivated fetal bovine serum (FBS) to 90 mL of RPMI.
   2. Add 1 mL of 100x Penicillin-Streptomycin-Glutamine to 100 mL of 10% FBS RPMI.
2. Prepare ILC2 Expansion Media.
   1. Add IL-2 and IL-33 (20 ng/mL each cytokine) to 10 mL of complete RPMI.
   ....

Results

CCR4 expression on CD4+ T cells and ILC2.

For the success of the ex vivo transmigration experiment, it is imperative to determine whether the lymphocytes are responsive to CCL17 and CCL22 through CCR4; therefore, we determined CCR4 expression on both CD4⁺ T cells and ILC2 by flow cytometry. While it is well known that OVA-specific CD4⁺ helper T cells express CCR4, less is known of the expression of CCR4 on ILC2. Figure 1 show.......

Discussion

Herein, we present a well-established method for assessing chemokine-induced migration of lymphocytes in an ex vivo transmigration system. There are several critical steps in the protocol, the first of which is verifying the expression of the correct chemokine receptor on the immune cells in the experiment. In our hands, we chose CCR4 because of the body of literature that highlights the importance of CCR4 on Th2 helper T cells in allergic inflammation. Ovalbumin-induced inflammation was shown previously to be limited by.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.4% Trypan Blue	Sigma-Aldrich	15250061
1 mL syringe	BD Bioscience	329424	U-100 Syringes Micro-Fine 28 G 1/2" 1cc
100x Penicillin-Streptomycin, L-Glutamine	Gibco	10378-016	Dilute to 1x in RPMI media
15 mL conical tubes	Olympus Plastics	28-101	polypropylene tubes
3 μm transwell inserts	Genesee Scientific	25-288	24-well plate containing 12 transwell inserts
3x stabilizing fixative	BD Pharmigen	338036	Prepare 1x solution according to manufacturers protocol
5 mL polystyrene tubes	STEM Cell Technologies	38007
50 mL conical tubes	Olympus Plastics	28-106	polypropylene tubes
8-chamber easy separation magnet	STEM Cell Technologies	18103
ACK Lysing Buffer	Life Technologies Corporation	A1049201
Advanced cell strainer, 40 μm	Genesee Scientific	25-375	nylon mesh, 40 μm strainers
Aluminum Hydroxide, Reagent Grade	Sigma-Aldrich	239186-25G	20 mg/mL
anti-mouse CCR4; APC-conjugated	Biolegend	131211	0.5 μg/test
anti-mouse CD11b	BD Pharmigen	557396	0.5 μg/test
anti-mouse CD11c; PE eFluor 610	Thermo-Fischer Scientific	61-0114-82	0.25 μg/test
anti-mouse CD16/32, Fc block	BD Pharmigen	553141	0.5 μg/test
anti-mouse CD19; APC-eFluor 780 conjugated	Thermo-Fischer Scientific	47-0193-82	0.5 μg/test
anti-mouse CD3; PE Cy 7-conjugated	BD Pharmigen	552774	0.25 μg/test
anti-mouse CD45; PE conjugated	BD Pharmigen	56087	0.5 μg/test
anti-mouse ICOS (CD278)	BD Pharmigen	564070	0.5 μg/test
anti-mouse NK1.1 (CD161); FITC-conjugated	BD Pharmigen	553164	0.25 μg/test
anti-mouse ST2 (IL-33R); PerCP Cy5.5 conjugated	Biolegend	145311	0.5 μg/test
Automated Cell Counter	BIORAD	1450102
Automated Dissociator	MACS Miltenyi Biotec	130-093-235
Bovine Serum Albumin, Lyophilized Powder	Sigma-Aldrich	A2153-10G	0.5% in serum-free RPMI
Cell Counter Clides	BIORAD	1450015
Chicken Egg Ovalbumin, Grade V	Sigma-Aldrich	A5503-10G	500 μg/mL
Collagenase, Type 1, Filtered	Worthington Biochemical Corporation	CLSS-1, purchase as 5 X 50 mg vials (LS004216)	25 U/mL in RPMI
Compensation beads	Affymetrix	01-1111-41	1 drop per contol tube
Dissociation Tubes	MACS Miltenyi Biotec	130-096-335
FACS Buffer	BD Pharmigen	554657	1x PBS + 2% FBS, w/ sodium azide; stored at 4 °C
Heat Inactivated-FBS	Genesee Scientific	25-525H	10% in complete RPMI & ILC2 Expansion Media
Mouse CCL17	GenScript	Z02954-20	50 ng/mL
Mouse CCL22	GenScript	Z02856-20	50 ng/mL
Mouse CD4+ T cell enrichment kit	STEM Cell Technologies	19852
Mouse IL-2	GenScript	Z02764-20	20 ng/mL
Mouse ILC2 enrichment kit	STEM Cell Technologies	19842
Mouse recombinant IL-33	STEM Cell Technologies	78044	20 ng/mL
RPMI	Life Technologies Corporation	22400071
Separation Buffer	STEM Cell Technologies	20144	1x PBS + 2% FBS; stored at 4 °C
Small animal nebulizer and chamber	Data Sciences International
Sterile saline	Baxter	2F7124; NDC 0338-0048-04	0.9% Sodium Chloride