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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 stages3,4, 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^.......

Representative Results

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.......

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.......

Acknowledgements

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.).

....

Materials

NameCompanyCatalog NumberComments
Ammonium-Chloride-Potassium (ACK) solutionin house0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA, pH 7.2-7.4
anti-FITC MicrobeadsMiltenyi Biotec130-048-701
anti-PE MicrobeadsMiltenyi Biotec130-048-801
Brefeldin ASigmaB6542
CD19 -FITCBiolegend115506clone 6D5
CD1d-tetramer -PENIH tetramer core facilitymouse PBS57-Cd1d-tetramers
CD4 -PeCy7Biolegend100528clone RM4-5
Fc blockerBD Bioscience553142
Fetal Bovine Serum (FBS)EurocloneECS0186Lheat-inactivated and filtered .22 before use
FOXP3 Transcription factor staining buffereBioscience00-5523-00
H2 (IAb) -FITCBiolegend114406clone AF6-120.1
hrIL-2Chiron Corp
IonomycinSigmaI0634
LD ColumnsMiltenyi Biotec130-042-901
LS ColumnsMiltenyi Biotec130-042-401
MACS buffer (MB)in house0.5% Bovine Serum Albumin (BSA; Sigma-Aldrich) and 2Mm EDTA
MS ColumnsMiltenyi Biotec130-042-201
Non-essential amino acidsGibco11140-035
Penicillin and streptomycin (Pen-Strep)Lonza15140-122
PermWashBD Bioscience51-2091KZ
PFASigmaP6148
Phosphate buffered saline (PBS)EuroCloneECB4004L
PMASigmaP1585
Pre-Separation Filters (30 µm)Miltenyi Biotec130-041-407
Recombinat Mouse IL-7R&D System407-ML-025
RPMI 1640 with glutamaxGibco61870-010
sodium pyruvateGibco11360-039
TCRβ -APCBiolegend109212clone H57-597
αCD3CD28 mouse T activator DynabeadsGibco11452D
β-mercaptoethanolGibco31350010

References

  1. Bendelac, A., Savage, P. B., Teyton, L. The biology of NKT cells. Annual Review of Immunology. 25, 297-336 (2007).
  2. Brennan, P. J., Brigl, M., Brenner, M. B. Invariant natural killer T cells: an innate activation scheme linked to ....

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