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Immunology and Infection

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

Published: February 15th, 2021



1Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, 2Vita-Salute San Raffaele University
* These authors contributed equally

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.

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.

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 stages3,4, producing a CD4+ and a....

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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^.......

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The protocol described in this manuscript enables to enrich iNKT cells from the spleen of iVa14-Ja18 transgenic mice through an immunomagnetic separation process summarized in Figure 1A. Total spleen T cells are first negatively selected by depleting B cells and monocytes, followed by iNKT cell positive immunomagnetic sorting with PBS-57 lipid antigen loaded CD1d tetramers, that enable to specifically stain only iNKT cells. This protocol yields about 2 x 106 of 95-98% pure iNKT ce.......

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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.......

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We thank Paolo Dellabona and Giulia Casorati for scientific support and critical reading of the manuscript. We also thank the NIH Tetramer Core Facility for mouse CD1d tetramer. The study was funded by Fondazione Cariplo Grant 2018-0366 (to M.F.) and Italian Association for Cancer Research (AIRC) fellowship 2019-22604 (to G.D.).


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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

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