Name: purification and expansion of mouse invariant natural killer t cells for in vitro and in vivo studies
Uploaded: 2021-02-15T13:30:00
Description: Watch this Scientific Journal Video about Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies at JoVE.com

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&#94...

<ol>
	<li>Bendelac, A., Savage, P. B., Teyton, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+NKT+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invariant+natural+killer+T+cells:+an+innate+activation+scheme+linked+to+diverse+effector+functions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+natural+killer+T+(NKT)+cell+developmental+pathway+iInvolving+a+thymus-dependent+NK1.1(-)CD4(+)+CD1d-dependent+precursor+stage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thymic+precursor+to+the+NK+T+cell+lineage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steady-state+production+of+IL-4+modulates+immunity+in+mouse+strains+and+is+determined+by+lineage+diversity+of+iNKT+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homotypic+interactions+mediated+by+Slamf1+and+Slamf6+receptors+control+NKT+cell+lineage+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimodal+CD40/Fas-Dependent+Crosstalk+between+iNKT+Cells+and+Tumor-Associated+Macrophages+Impairs+Prostate+Cancer+Progression.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invariant+NKT+cells+with+chimeric+antigen+receptor+provide+a+novel+platform+for+safe+and+effective+cancer+immunotherapy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+alpha-galactosyl+ceramide+for+staining+and+stimulating+natural+killer+T+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+reliable+generation+of+invariant+natural+killer+T-cell+lines+in+vitro.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+antitumor+immunity+mediated+by+NKT+cell+subsets+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+profibrotic+cytokines+by+invariant+NKT+cells+characterizes+cirrhosis+progression+in+chronic+viral+hepatitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD62L++NKT+cells+have+prolonged+persistence+and+antitumor+activity+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+Antiviral+B+Cell+Immunity+Relies+on+Innate+Signals+from+Spatially+Positioned+NKT+Cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Anti-lymphoma+Activity+of+CAR19-iNKT+Cells+Underpinned+by+Dual+CD19+and+CD1d+Targeting.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Third-party+CD4++invariant+natural+killer+T+cells+protect+from+murine+GVHD+lethality.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++invariant+natural+killer+T+cells+protect+from+murine+GVHD+lethality+through+expansion+of+donor+CD4+CD25+FoxP3++regulatory+T+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+T+cells+and+natural+killer+T+cells+for+modulation+of+GVHD+following+allogeneic+hematopoietic+cell+transplantation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+doses+of+natural+killer+T+cells+provide+protection+from+acute+graft-versus-host+disease+via+an+IL-4-dependent+mechanism.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+CD4-+invariant+NKT+lymphocytes+regulate+graft+versus+host+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKT+Cells+Coexpressing+a+GD2-Specific+Chimeric+Antigen+Receptor+and+IL15+Show+Enhanced+In+vivo+Persistence+and+Antitumor+Activity+against+Neuroblastoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-GD2+CAR-NKT+cells+in+patients+with+relapsed+or+refractory+neuroblastoma:+an+interim+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+Transfer+of+Invariant+NKT+Cells+as+Immunotherapy+for+Advanced+Melanoma:+A+Phase+I+Clinical+Trial.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Approaches+to+Exploiting+Invariant+NKT+Cells+in+Cancer+Immunotherapy.">Novel Approaches to Exploiting Invariant NKT Cells in Cancer Immunotherapy.</a> Frontiers in Immunology. 9, 384 (2018).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinat Mouse IL-7</td>
			<td>R&#38;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&#946; -APC</td>
			<td>Biolegend</td>
			<td>109212</td>
			<td>clone H57-597</td>
		</tr>
		<tr>
			<td>&#945;CD3CD28 mouse T activator Dynabeads</td>
			<td>Gibco</td>
			<td>11452D</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-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

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...

Watch this Scientific Journal Video about Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies at JoVE.com

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

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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