Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Summary

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Abstract

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iVα14-Jα18 transgenic mice (iVα14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iVα14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

Introduction

Invariant Natural killer T cells (iNKT cells) are innate-like T lymphocytes that express a semi-invariant αβ T cell receptor (TCR), formed in mice by an invariant Vα14-Jα18 chain paired with a limited set of diverse Vβ chains¹, which is specific for lipid antigens presented by the MHC class I-related molecule CD1d². iNKT cells undergo an agonist selection program resulting in the acquisition of an activated/innate effector phenotype already in the thymus, which occurs through several maturation stages^3,4, producing a CD4⁺ and a....

Protocol

Procedures described here were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) (no. 1048) at the San Raffaele Scientific Institute.

NOTE: All the procedures must be performed under sterile conditions. All the reagents used are listed in the Table of Materials.

1. Spleen processing

1. Euthanize iVα14-Jα18 mice by inhalation of CO₂ according to the institutional policy.
   NOTE: iV^.......

Discussion

Here we show a reproducible and feasible protocol to obtain millions of ready-to-use iNKT cells. Due to the paucity of these cells in vivo, a method to expand them was highly needed. The protocol we propose requires neither a particular instrumentation nor a high number of mice. We exploited iVα14-Jα18 transgenic mice on purpose to reduce the number of mice needed for the procedure.

Another successful protocol for iNKT cell expansion from iVα14-Jα18 transgenic mice is avail.......

Materials

Name	Company	Catalog Number	Comments
Ammonium-Chloride-Potassium (ACK) solution	in house		0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA, pH 7.2-7.4
anti-FITC Microbeads	Miltenyi Biotec	130-048-701
anti-PE Microbeads	Miltenyi Biotec	130-048-801
Brefeldin A	Sigma	B6542
CD19 -FITC	Biolegend	115506	clone 6D5
CD1d-tetramer -PE	NIH tetramer core facility		mouse PBS57-Cd1d-tetramers
CD4 -PeCy7	Biolegend	100528	clone RM4-5
Fc blocker	BD Bioscience	553142
Fetal Bovine Serum (FBS)	Euroclone	ECS0186L	heat-inactivated and filtered .22 before use
FOXP3 Transcription factor staining buffer	eBioscience	00-5523-00
H2 (IAb) -FITC	Biolegend	114406	clone AF6-120.1
hrIL-2	Chiron Corp
Ionomycin	Sigma	I0634
LD Columns	Miltenyi Biotec	130-042-901
LS Columns	Miltenyi Biotec	130-042-401
MACS buffer (MB)	in house		0.5% Bovine Serum Albumin (BSA; Sigma-Aldrich) and 2Mm EDTA
MS Columns	Miltenyi Biotec	130-042-201
Non-essential amino acids	Gibco	11140-035
Penicillin and streptomycin (Pen-Strep)	Lonza	15140-122
PermWash	BD Bioscience	51-2091KZ
PFA	Sigma	P6148
Phosphate buffered saline (PBS)	EuroClone	ECB4004L
PMA	Sigma	P1585
Pre-Separation Filters (30 µm)	Miltenyi Biotec	130-041-407
Recombinat Mouse IL-7	R&D System	407-ML-025
RPMI 1640 with glutamax	Gibco	61870-010
sodium pyruvate	Gibco	11360-039
TCRβ -APC	Biolegend	109212	clone H57-597
αCD3CD28 mouse T activator Dynabeads	Gibco	11452D
β-mercaptoethanol	Gibco	31350010