Name: Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies (Video) | JoVE Journal
Uploaded: 2021-02-15T13:30:00.000+00:00
Duration: 8 min 37 s
Description: Learn to purify and expand mouse invariant natural killer T cells from spleen for immunology research. This protocol achieves 30-fold expansion in two weeks for in vitro and in vivo studies.

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5 minutes

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.).

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
<ol>
	<li>Euthanize iV&#945;14-J&#945;18 mice by inhalation of CO2 according to the institutional policy. 
	NOTE: iV&#945;14-J&#945;18 mice must be 8 weeks old or older. To avoid rejection of the cells from the in vivo transfer of the cells, consider that cells isolated from female mice can be adoptively transferred both in male and female recipients, whereas cells isolated from male mice can be transferred only in male recipients. For in vitro experiments, no gender bias must be considered. More mice can be pooled in order to obtain more cells.</li>
	<li>Dissect the mouse spleen and smash it through a 70 nm cell strainer to obtain a single-cell suspension in 10 mL of phosphate buffered saline (PBS) with 2% fetal bovine serum (FBS).</li>
	<li>Centrifuge at 300 x g for 5 min.</li>
	<li>Remove the supernatant by inversion and process with erythrocyte lysis buffer. Resuspend the cell pellet with 1 mL of sterile ACK (Ammonium-Chloride-Potassium) Lysing Buffer, incubate for 3 min at room temperature, and block with 5 mL of PBS with 2% FBS. Centrifuge at 300 x g for 5 min. 
	NOTE: ACK is commercially available; it consists of a solution of 0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA (ethylenediaminetetraacetic acid) dissolved in bidistilled H2O, pH 7.2-7.4. If homemade, sterilize by filtration with a 0.22 &#181;m filter.</li>
	<li>Remove the supernatant by inversion. Resuspend the cell pellet in 3 mL of PBS with 2% FBS and remove fat residues by pipetting. If needed, pool the cells coming from different mice.</li>
	<li>Count the cells and keep 50 &#181;L for FACS analysis.</li>
</ol>
2. T cell enrichment
NOTE: For the enrichment steps, work fast, keep the cells cold and use solutions pre-cooled at 4 &#176;C overnight and then kept on ice
<ol>
	<li>Centrifuge at 300 x g for 5 min.</li>
	<li>Resuspend all the cells in the appropriate amount of PBS with 2% FBS (500 &#181;L for 107 cells) and Fc blocker (2.5 &#181;L x 107 cells); incubate for 15 min at room temperature (RT).</li>
	<li>Wash with 1-2 mL of MACS separation buffer (MB) per 107 total cells and centrifuge at 300 x g for 10 min. 
	NOTE: MACS buffer is commercially available. It consists of PBS pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA. If homemade, sterilize by filtration with a 0.22 &#181;m filter.</li>
	<li>Remove the supernatant by inversion and stain the cells with CD19-FITC and H2(IAb)-FITC (use 5 &#181;L x 107 cells in 100 &#181;L of MB). Mix well and incubate for 15 min in the dark at 4-8 &#176;C.</li>
	<li>Wash cells by adding 1&#8722;2 mL of MB per 107 cells and centrifuge at 300 x g for 10 min.</li>
	<li>Pipette off the supernatant completely and resuspend the cell pellet in 90 &#181;L of MB per 107 total cells. Add 10 &#181;L of Anti-FITC MicroBeads per 107 total cells. Mix well and incubate for 15 min in the dark at 4-8 &#176;C.</li>
	<li>Wash the cells by adding 1&#8722;2 mL of MB per 107 cells and centrifuge at 300 x g for 10 min.</li>
	<li>Pipette off the supernatant completely and resuspend up to 1.25 x 108 cell in 500 &#181;L of MB.</li>
	<li>Place a LD column in the magnetic field of the MACS separator to proceed to the depletion. To avoid clogging, apply a pre-separation filter on the LD column and rinse with 2 mL of MB.</li>
	<li>When the column reservoir is empty, apply the cell suspension onto the filter. Collect the unlabeled cells that pass through the column.</li>
	<li>Wash 3 times with 1 mL of MB, only when the column reservoir is empty. Collect the total effluent, which will be enriched in T cells, and count the cells. Always keep 50 &#181;L for FACS analysis.</li>
</ol>
3. iNKT cell enrichment
<ol>
	<li>Centrifuge at 300 x g for 5 min and remove the supernatant by inversion.</li>
	<li>Stain the cells with CD1d-tetramer-PE (mouse PBS57-CD1d-tetramer), according to the antibody titration in 50 &#181;L of MB per 106 cells. Mix well and incubate for 30 min in the dark on ice. 
	NOTE: The procedure can also be performed with APC-labelled mCD1d tetramers and anti-APC beads; adjusting the fluorochromes used in the following staining accordingly. In the present protocol we used mouse PBS-57-CD1d-tetramers provided by NIH. &#945;galactosylceramide (&#945;Gal-Cer) is the prototypical antigen recognized by iNKT cells; PBS-57 is an analogue of &#945;Gal-Cer with improved solubility9; the NIH Tetramer Facility provides PBS-57 ligand complexed to CD1d tetramers. However, other CD1d dimers/tetramers/dextramers are commercially available and can be loaded with a lipidic antigen as &#945;Gal-Cer. We envisage the possibility to adjust the protocol for their use.</li>
	<li>Wash the cells by adding 1&#8722;2 mL of MB per 107 cells and centrifuge at 300 x g for 10 min.</li>
	<li>Pipette off the supernatant completely and resuspend the cell pellet in 80 &#181;L of MB per 107 total cells. Add 20 &#181;L of Anti-PE MicroBeads per 107 total cells. Mix well and incubate for 15 min in the dark at 4-8 &#176;C.</li>
	<li>Wash cells by adding 1&#8722;2 mL of MB per 107 cells and centrifuge at 300 x g for 10 min.</li>
	<li>Pipette off the supernatant completely and resuspend up to 108 cells in 500 &#181;L of MB. Otherwise if cells exceed 108, adjust the volume accordingly.</li>
	<li>According to the cell count, place a LS (up to 108) or MS (up to 107) column in the magnetic field of the MACS separator. Rinse the column with MB (3 mL for LS, 500 &#181;L for MS).</li>
	<li>Apply the cell suspension onto the column.</li>
	<li>Collect unlabeled cells that pass through. Wash the column 3 times by adding the appropriate amount of MB (3 x 3 mL for LS column, 3 x 500 &#181;L for MS column) only when the column reservoir is empty. The total effluent is the negative fraction.</li>
	<li>Remove the column from the magnetic field and place it on a new collection tube.</li>
	<li>Pipette MB onto the column (5 mL for LS column or 1 mL for MS column); push the provided plunger into the column and flush out the positive fraction (enriched in iNKT cells).</li>
	<li>To further increase the iNKT cell recovery, centrifuge the negative fraction at 300 x g for 10 min and repeat steps 3.6-3.7 with a new LS or MS column. Pool the positive fractions and determine cell count. Keep 50 &#181;L of both positive and negative fractions for FACS analysis.</li>
	<li>Check the Purification steps by FACS analysis. Samples include: spleen ex-vivo, T cell enriched fraction, iNKT positive fraction and iNKT negative fraction. Stain the cells with: CD19-FITC, IAb-FITC, CD1d-tetramer-PE, TCR&#946;-APC and DAPI. 
	NOTE: The expected recovery from one iV&#945;14-J&#945;18 mouse is 2x106 iNKT cells .</li>
</ol>
4. iNKT cell activation and expansion
<ol>
	<li>Activate purified iNKT cells with mouse T activator anti-CD3/CD28 magnetic beads in a 1:1 ratio.
	<ol>
		<li>Centrifuge the iNKT cell positive fraction at 300 x g for 5 min.</li>
		<li>Meanwhile transfer the appropriate volume of anti-CD3/CD28 magnetic beads to a tube and add an equal volume of PBS, vortex for 5 seconds. Place the tube on a magnet for 1 minute and discard the supernatant.</li>
		<li>Remove the tube from the magnet and resuspend the washed magnetic beads in the proper volume of complete RPMI (RPMI 1640 medium, 10% heat-inactivated FCS, 1 mM sodium pyruvate, 1% Non-essential amino acids, 10 U/ml penicyllin and streptomycin, 50 &#956;M &#946;-Mercaptoethanol) to have 5 x 105 iNKT cells in 1 mL. Use this suspension to resuspend the centrifuged iNKT positive fraction.</li>
	</ol>
	</li>
	<li>Plate 1 mL of the cell suspension (5 x 105 iNKT cells) and anti-CD3/CD28 magnetic beads in a 48 well plate with 20 U/mL IL-2 and incubate at 37 &#176;C.</li>
	<li>After 5 days, add 10 ng/mL IL-7.</li>
	<li>Split the cells 1:2 when they reach 80-90% confluence, always add 20U/mL IL-2 and 10 ng/mL IL-7. In these conditions, iNKT cells can be expanded for up to 15 days.</li>
</ol>

<ol><li>Bendelac, A., Savage, P. B., Teyton, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biology+of+NKT+cells%5BTitle%5D)+AND+%22Annual+Review+of+Immunology%22%5BJournal%5D)">The biology of NKT cells.</a> Annual Review of Immunology. 25, 297-336 (2007).</li>
<li>Brennan, P. J., Brigl, M., Brenner, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Invariant+natural+killer+T+cells%3A+an+innate+activation+scheme+linked+to+diverse+effector+functions%5BTitle%5D)+AND+%22Nature+Reviews%3A+Immunology%22%5BJournal%5D)">Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions.</a> Nature Reviews: Immunology. 13 (2), 101-117 (2013).</li>
<li>Pellicci, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+natural+killer+T+%28NKT%29+cell+developmental+pathway+iInvolving+a+thymus-dependent+NK1.1%28-%29CD4%28%2B%29+CD1d-dependent+precursor+stage%5BTitle%5D)+AND+%22Journal+of+Experimental+Medicine%22%5BJournal%5D)">A natural killer T (NKT) cell developmental pathway iInvolving a thymus-dependent NK1.1(-)CD4(+) CD1d-dependent precursor stage.</a> Journal of Experimental Medicine. 195 (7), 835-844 (2002).</li>
<li>Benlagha, K., Kyin, T., Beavis, A., Teyton, L., Bendelac, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+thymic+precursor+to+the+NK+T+cell+lineage%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A thymic precursor to the NK T cell lineage.</a> Science. 296 (5567), 553-555 (2002).</li>
<li>Lee, Y. J., Holzapfel, K. L., Zhu, J., Jameson, S. C., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Steady-state+production+of+IL-4+modulates+immunity+in+mouse+strains+and+is+determined+by+lineage+diversity+of+iNKT+cells%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells.</a> Nature Immunology. 14 (11), 1146-1154 (2013).</li>
<li>Griewank, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Homotypic+interactions+mediated+by+Slamf1+and+Slamf6+receptors+control+NKT+cell+lineage+development%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Homotypic interactions mediated by Slamf1 and Slamf6 receptors control NKT cell lineage development.</a> Immunity. 27 (5), 751-762 (2007).</li>
<li>Cortesi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bimodal+CD40%2FFas-Dependent+Crosstalk+between+iNKT+Cells+and+Tumor-Associated+Macrophages+Impairs+Prostate+Cancer+Progression%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Bimodal CD40/Fas-Dependent Crosstalk between iNKT Cells and Tumor-Associated Macrophages Impairs Prostate Cancer Progression.</a> Cell Reports. 22 (11), 3006-3020 (2018).</li>
<li>Heczey, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Invariant+NKT+cells+with+chimeric+antigen+receptor+provide+a+novel+platform+for+safe+and+effective+cancer+immunotherapy%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy.</a> Blood. 124 (18), 2824-2833 (2014).</li>
<li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modified+alpha-galactosyl+ceramide+for+staining+and+stimulating+natural+killer+T+cells%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">A modified alpha-galactosyl ceramide for staining and stimulating natural killer T cells.</a> Journal of Immunological Methods. 312 (1-2), 34-39 (2006).</li>
<li>Chiba, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+reliable+generation+of+invariant+natural+killer+T-cell+lines+in+vitro%5BTitle%5D)+AND+%22Immunology%22%5BJournal%5D)">Rapid and reliable generation of invariant natural killer T-cell lines in vitro.</a> Immunology. 128 (3), 324-333 (2009).</li>
<li>Crowe, N. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+antitumor+immunity+mediated+by+NKT+cell+subsets+in+vivo%5BTitle%5D)+AND+%22Journal+of+Experimental+Medicine%22%5BJournal%5D)">Differential antitumor immunity mediated by NKT cell subsets in vivo.</a> Journal of Experimental Medicine. 202 (9), 1279-1288 (2005).</li>
<li>de Lalla, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+profibrotic+cytokines+by+invariant+NKT+cells+characterizes+cirrhosis+progression+in+chronic+viral+hepatitis%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Production of profibrotic cytokines by invariant NKT cells characterizes cirrhosis progression in chronic viral hepatitis.</a> Journal of Immunology. 173 (2), 1417-1425 (2004).</li>
<li>Tian, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD62L%2B+NKT+cells+have+prolonged+persistence+and+antitumor+activity+in+vivo%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">CD62L+ NKT cells have prolonged persistence and antitumor activity in vivo.</a> Journal of Clinical Investigation. 126 (6), 2341-2355 (2016).</li>
<li>Gaya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Initiation+of+Antiviral+B+Cell+Immunity+Relies+on+Innate+Signals+from+Spatially+Positioned+NKT+Cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.</a> Cell. 172 (3), 517-533 (2018).</li>
<li>Rotolo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+Anti-lymphoma+Activity+of+CAR19-iNKT+Cells+Underpinned+by+Dual+CD19+and+CD1d+Targeting%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting.</a> Cancer Cell. 34 (4), 596-610 (2018).</li>
<li>Schneidawind, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Third-party+CD4%2B+invariant+natural+killer+T+cells+protect+from+murine+GVHD+lethality%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Third-party CD4+ invariant natural killer T cells protect from murine GVHD lethality.</a> Blood. 125 (22), 3491-3500 (2015).</li>
<li>Schneidawind, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD4%2B+invariant+natural+killer+T+cells+protect+from+murine+GVHD+lethality+through+expansion+of+donor+CD4%2BCD25%2BFoxP3%2B+regulatory+T+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">CD4+ invariant natural killer T cells protect from murine GVHD lethality through expansion of donor CD4+CD25+FoxP3+ regulatory T cells.</a> Blood. 124 (22), 3320-3328 (2014).</li>
<li>Schneidawind, D., Pierini, A., Negrin, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulatory+T+cells+and+natural+killer+T+cells+for+modulation+of+GVHD+following+allogeneic+hematopoietic+cell+transplantation%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Regulatory T cells and natural killer T cells for modulation of GVHD following allogeneic hematopoietic cell transplantation.</a> Blood. 122 (18), 3116-3121 (2013).</li>
<li>Leveson-Gower, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low+doses+of+natural+killer+T+cells+provide+protection+from+acute+graft-versus-host+disease+via+an+IL-4-dependent+mechanism%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Low doses of natural killer T cells provide protection from acute graft-versus-host disease via an IL-4-dependent mechanism.</a> Blood. 117 (11), 3220-3229 (2011).</li>
<li>Coman, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+CD4-+invariant+NKT+lymphocytes+regulate+graft+versus+host+disease%5BTitle%5D)+AND+%22Oncoimmunology%22%5BJournal%5D)">Human CD4- invariant NKT lymphocytes regulate graft versus host disease.</a> Oncoimmunology. 7 (11), 1470735(2018).</li>
<li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NKT+Cells+Coexpressing+a+GD2-Specific+Chimeric+Antigen+Receptor+and+IL15+Show+Enhanced+In+vivo+Persistence+and+Antitumor+Activity+against+Neuroblastoma%5BTitle%5D)+AND+%22Clinical+Cancer+Research%22%5BJournal%5D)">NKT Cells Coexpressing a GD2-Specific Chimeric Antigen Receptor and IL15 Show Enhanced In vivo Persistence and Antitumor Activity against Neuroblastoma.</a> Clinical Cancer Research. 25 (23), 7126-7138 (2019).</li>
<li>Heczey, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-GD2+CAR-NKT+cells+in+patients+with+relapsed+or+refractory+neuroblastoma%3A+an+interim+analysis%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Anti-GD2 CAR-NKT cells in patients with relapsed or refractory neuroblastoma: an interim analysis.</a> Nature Medicine. 26 (11), 1686-1690 (2020).</li>
<li>Exley, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adoptive+Transfer+of+Invariant+NKT+Cells+as+Immunotherapy+for+Advanced+Melanoma%3A+A+Phase+I+Clinical+Trial%5BTitle%5D)+AND+%22Clinical+Cancer+Research%22%5BJournal%5D)">Adoptive Transfer of Invariant NKT Cells as Immunotherapy for Advanced Melanoma: A Phase I Clinical Trial.</a> Clinical Cancer Research. 23 (14), 3510-3519 (2017).</li>
<li>Wolf, B. J., Choi, J. E., Exley, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+Approaches+to+Exploiting+Invariant+NKT+Cells+in+Cancer+Immunotherapy%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">Novel Approaches to Exploiting Invariant NKT Cells in Cancer Immunotherapy.</a> Frontiers in Immunology. 9, 384(2018).</li>
</ol>

The authors have nothing to disclose.

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 iV&#945;14-J&#945;18 transgenic mice on purpose to reduce the number of mice needed for the procedure.
Another successful protocol for iNKT cell expansion from iV&#945;14-J&#945;18 transgenic mice is avail...

Invariant Natural killer T cells (iNKT cells) are innate-like T lymphocytes that express a semi-invariant &#945;&#946; T cell receptor (TCR), formed in mice by an invariant V&#945;14-J&#945;18 chain paired with a limited set of diverse V&#946; 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 CD4- subset. Through this program, iNKT cells acquire distinct T helper (TH) effector phenotypes, namely TH1 (iNKT1), TH2 (iNKT2) and TH17 (iNKT17), identifiable by the expression of the transcription factors T-bet, GATA3, PLZF, and ROR&#947;t, respectively5. iNKT cells recognize a range of microbial lipids but are also self-reactive against endogenous lipids that are upregulated in the context of pathological situations of cell stress and tissue damage, such as cancer and autoimmunity2. Upon activation, iNKT cells modulate the functions of other innate and adaptive immune effector cells via direct contact and cytokine production2.
The investigations of iNKT cells have been facilitated by mouse models, including CD1d-deficient or J&#945;18-deficient mice, and by the production of antigen-loaded CD1d tetramers plus the generation of monoclonal antibodies (mAbs) specific for the human semi-invariant TCR. However, the generation of primary mouse iNKT cell line has proved difficult. To better characterize the antitumor functions of iNKT cells and to utilize them for adoptive cell therapy, we set up a protocol to purify and expand splenic iNKT cells of iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg)6, in which iNKT cells are 30 times more frequent than in wild type mice.
Expanded iNKT cells can be exploited for in vitro assays, and in vivo upon transfer back into mice. In this setting, for example, we have shown their potent anti-tumor effects7. Moreover, in vitro expanded iNKT cells are amenable to functional modification via gene transfer or editing prior to their injection in vivo8, allowing insightful functional analysis of molecular pathways, as well as paving the way for advanced cell therapies.

<table><tbody><tr><td>Ammonium-Chloride-Potassium (ACK) solution</td><td>in house</td><td></td><td>0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA, pH 7.2-7.4</td></tr><tr><td>anti-FITC Microbeads</td><td>Miltenyi Biotec</td><td>130-048-701</td><td></td></tr><tr><td>anti-PE Microbeads</td><td>Miltenyi Biotec</td><td>130-048-801</td><td></td></tr><tr><td>Brefeldin A</td><td>Sigma</td><td>B6542</td><td></td></tr><tr><td>CD19 -FITC</td><td>Biolegend</td><td>115506</td><td>clone 6D5</td></tr><tr><td>CD1d-tetramer -PE</td><td>NIH tetramer core facility</td><td></td><td>mouse PBS57-Cd1d-tetramers</td></tr><tr><td>CD4 -PeCy7</td><td>Biolegend</td><td>100528</td><td>clone RM4-5</td></tr><tr><td>Fc blocker</td><td>BD Bioscience</td><td>553142</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Euroclone</td><td>ECS0186L</td><td>heat-inactivated and filtered .22 before use</td></tr><tr><td>FOXP3 Transcription factor staining buffer</td><td>eBioscience</td><td>00-5523-00</td><td></td></tr><tr><td>H2 (IAb) -FITC</td><td>Biolegend</td><td>114406</td><td>clone AF6-120.1</td></tr><tr><td>hrIL-2</td><td>Chiron Corp</td><td></td><td></td></tr><tr><td>Ionomycin</td><td>Sigma</td><td>I0634</td><td></td></tr><tr><td>LD Columns</td><td>Miltenyi Biotec</td><td>130-042-901</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>MACS buffer (MB)</td><td>in house</td><td></td><td>0.5% Bovine Serum Albumin (BSA; Sigma-Aldrich) and 2Mm EDTA</td></tr><tr><td>MS Columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Gibco</td><td>11140-035</td><td></td></tr><tr><td>Penicillin and streptomycin (Pen-Strep)</td><td>Lonza</td><td>15140-122</td><td></td></tr><tr><td>PermWash</td><td>BD Bioscience</td><td>51-2091KZ</td><td></td></tr><tr><td>PFA</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>EuroClone</td><td>ECB4004L</td><td></td></tr><tr><td>PMA</td><td>Sigma</td><td>P1585</td><td></td></tr><tr><td>Pre-Separation Filters (30 &amp;micro;m)</td><td>Miltenyi Biotec</td><td>130-041-407</td><td></td></tr><tr><td>Recombinat Mouse IL-7</td><td>R&amp;amp;D System</td><td>407-ML-025</td><td></td></tr><tr><td>RPMI 1640 with glutamax</td><td>Gibco</td><td>61870-010</td><td></td></tr><tr><td>sodium pyruvate</td><td>Gibco</td><td>11360-039</td><td></td></tr><tr><td>TCR&amp;beta; -APC</td><td>Biolegend</td><td>109212</td><td>clone H57-597</td></tr><tr><td>&amp;alpha;CD3CD28 mouse T activator Dynabeads</td><td>Gibco</td><td>11452D</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Gibco</td><td>31350010</td><td></td></tr></tbody></table>

purification and expansion of mouse invariant natural killer t cells for in vitro and in vivo studies

Purification and Expansion of Mouse Invariant Natural Killer T Cells 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 iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg 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.

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...

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 ...

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Research

JoVE Journal

Immunology and Infection

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Naïve CD4+ T cells polarize to various subsets depending on the environment at the time of activation.  The differentiation of naïve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

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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.

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Learn to purify and expand mouse invariant natural killer T cells from spleen for immunology research. This protocol achieves 30-fold expansion in two weeks for in vitro and in vivo studies.

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

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