An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Summary

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Abstract

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.

Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5⁺) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5⁺ fraction are subsequently stained with αGalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 10⁸ iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Introduction

Murine invariant natural killer T (iNKT) cells are a distinct population of innate T lymphocytes selected in the thymus by CD1d-expressing cortical thymocytes ^1,2. iNKT cells express a T cell receptor (TCR) comprised of an invariant Vα14-Jα18 TCR chain paired with either Vβ8, Vβ7 or Vβ2 TCRs ³, which is capable of recognizing endogenous as well as foreign lipid antigens in the context of CD1d. For example, murine iNKT cells recognize and are activated by an endogenous lipid antigen called isoglobotrihexosylceramide (iGb3) ⁴, as well as α-galactosylceramide (αGalCer) ^5,6, a glycolipid isolat....

Protocol

In this study, adult (6-8 weeks) female C57Bl/6 mice were used. Mice were housed and bred according to the guidelines of the Ghent University vivarium. All animal procedures were approved by the Institutional Animal Care and Ethics Committee.

1. Preparation of Mononuclear Cells (MNCs) from Mouse Spleen

1. Sacrifice the mouse by cervical dislocation.
2. Place the mouse back down on the dissecting board and fix the hind and forelegs with pins.
3. Sterilize the skin-surface with 70% (vol/vol) ethanol/H₂O.
4. Cut the skin along the abdominal midline to the thorax and expose the spleen using sterile surgi....

Results

Isolation of splenic mononuclear cells using a density gradient takes approximately 1 hr and eliminates the use of reagents required to lyse red blood cells (RBCs). A high yield of viable cells is obtained using this method and debris generated during straining of the organ is removed. Typically, the frequency of iNKT cells within the splenic lymphocyte pool ranges between 1 and 5% of total T lymphocytes however, this can vary depending on the age, sex and health status of the animals used. Approximately 10⁶ i.......

Discussion

Critical steps in the current protocol include the isolation and subsequent enrichment of CD5⁺ lymphocytes (Section 1 & 2), FACS sorting (Section 3) and the initial plating of iNKT cells (Section 4). Of the steps performed in Section 1, remember to carefully layer the splenic cellular suspension over the density gradient medium such that a distinct cellular interphase is generated following centrifugation. The subsequent enrichment for CD5⁺ lymphocytes by magnetic cell separation (Section 2) min.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material
recombinant murine IL-2	eBiosciences	14-8021
recombinant murine IL-12	eBiosciences	39-8122-65
recombinant murine IL-7	eBiosciences	14-8071
purified anti-mouse CD3e (145-2C11)	eBiosciences	16-0032-86
purified anti-mouse CD28 (37.51)	eBiosciences	16-0281-85
e450-conjugated anti-mouse CD19 (eBio1D3)	eBiosciences	48-0193-82
e450-conjugated anti-mouse CD8α (53-6.7)	eBiosciences	48-0081-82
FITC-conjugated anti-mouse CD5 (53-7.3)	eBiosciences	11-0051-81
V500-conjugated anti-mouse CD3ε (500A2)	BD biosciences	560771
anti-mouse CD5 (Ly-1) microbeads	Macs Miltenyi Biotec	130-049-301
purified anti-mouse CD16/32 (2.4G2)	Macs Miltenyi Biotec	130-092-575
MACS MS Columns	Macs Miltenyi Biotec	130-042-201
Trypan Blue, 0.4% (wt/vol)	Gibco	15250-061
RPMI 1640 medium	Gibco	12633-020
1x PBS	Gibco	10010-056	[Ca2+/Mg2+ - free]
Fetal calf serum	Gibco	10270
L-Glutamine	Gibco	25030-123
Penicillin-streptomycin	Sigma-Aldrich	P4458-100ML
Bovine serum albumin	Sigma-Aldrich	A7906-100MG
β-Mercaptoethanol	Sigma-Aldrich	M3148
Ethylenediaminetetraacetic acid	Sigma-Aldrich	EDS-1KG
Ficoll-Paque plus	GE Healthcare	71-7167-00 AF
Equipment
96-well plate F-bottom	Greiner-bio one	657-160
24-well plate	Greiner-bio one	665-180
6-well plate	Greiner-bio one	655-180
15 ml falcon tube	Greiner-bio one	188-271
50 ml falcon tube	Greiner-bio one	227-261
70 µm filter	Greiner-bio one	542-070
30 µm filter	Millipore	SVGP01050
MiniMACS separator	Macs Miltenyi Biotec	130-042-102
MS Columns	Macs Miltenyi Biotec	130-042-201
Water-jacketed CO2 incubator	VWR
Hemocytometer	VWR
Dissection Kit	VWR
BD FACSAria III	BD Biosciences