Immunology and Infection
Published: February 15th, 2021
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....
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
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.......
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.......
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.).....
|Ammonium-Chloride-Potassium (ACK) solution
|0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA, pH 7.2-7.4
|NIH tetramer core facility
|Fetal Bovine Serum (FBS)
|heat-inactivated and filtered .22 before use
|FOXP3 Transcription factor staining buffer
|H2 (IAb) -FITC
|MACS buffer (MB)
|0.5% Bovine Serum Albumin (BSA; Sigma-Aldrich) and 2Mm EDTA
|Non-essential amino acids
|Penicillin and streptomycin (Pen-Strep)
|Phosphate buffered saline (PBS)
|Pre-Separation Filters (30 µm)
|Recombinat Mouse IL-7
|RPMI 1640 with glutamax
|αCD3CD28 mouse T activator Dynabeads
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