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 ...

This protocol enables the purification of INK T-cells, which are very rare, and allows a 30-fold expansion in their number. The purification can be performed in one day and produce a large number of ready-to-use INK T-cells for in vivo and in vitro studies in two weeks. Demonstrating the procedure will be Gloria Delfanti, a post-doc in the lab.After dissecting the mouse spleen, smash it through a 70 nanometer cell strainer to obtain a single cell suspension in 10 milliliters of PBS with 2%FBS. Centrifuge at 300 times G for five minutes. Remove the supernatant and resuspend the cell pellet with one milliliter of sterile ammonium chloride potassium lysine buffer.Incubate the cells for three minutes at room temperature, then block with five milliliters of PBS with 2%FBS. Centrifuge at 300 times G for five minutes. After removing the supernatant, resuspend the cell pellet in three milliliters of PBS with 2%FBS and remove fat residues by pipetting.For the enrichment steps, keep the cells cold and use solutions pre-cooled at four degrees Celsius and then kept on ice. Centrifuge at 300 times G for five minutes. Resuspend all the cells in the appropriate amount of PBS with 2%FPS and Fc blocker and incubate for 15 minutes at room temperature.Wash with one to two milliliters of MACS separation buffer per 10 million cells and centrifuge at 300 times G for 10 minutes. After removing the supernatant, stain the cells with CD19-FITC and H2-IAb-FITC and incubate for 15 minutes in the dark at four to eight degrees Celsius. Wash cells by adding one to two milliliters of MACS buffer per 10 million cells and centrifuge at 300 times G for 10 minutes.Remove supernatant and resuspend the cell pellet in 90 microliters of MACS buffer per 10 million cells. Add 10 microliters of anti-FITC microbeads per 10 million cells. Mix well and incubate for 15 minutes in the dark at four to eight degrees Celsius.Wash the cells by adding one to two milliliters of MACS buffer per 10 million cells and centrifuge at 300 times G for 10 minutes. Remove the supernatant and resuspend up to 125 million cells in 500 microliters of MACS buffer. Place an LD column in the magnetic field of the MACS separator.To avoid clogging, apply a pre-separation filter on the LD column and rinse it with two milliliters of MACS buffer. Apply the cell suspension onto the filter and collect the unlabeled cells that pass through the column. Wash the empty column reservoir three times with one milliliter of MACS buffer.Collect the T-cell enriched effluent and count the cells, keeping 50 microliters for FACS analysis. After centrifuging at 300 times G for five minutes, remove the supernatant and stain the cells with CD1d tetramer-PE. Mix well and incubate for 30 minutes in the dark on ice.Wash the cells by adding one to two milliliters of MACS buffer per 10 million cells. After centrifugation at 300 times G for 10 minutes, remove the supernatant and resuspend the cell pellet in 80 microliters of MACS buffer per 10 million cells. Add 20 microliters of anti-PE microbeads per 10 million cells.Mix well and incubate for 15 minutes in the dark at four to eight degrees Celsius. Wash cells by adding one to two milliliters of MACS buffer per 10 million cells and centrifuge at 300 times G for 10 minutes. Remove the supernatant and resuspend up to 100 million cells in 500 microliters of MACS buffer.According to the cell count, place the LS or MS column in the magnetic field of the MACS separator and rinse the column with MACS buffer. Apply the cell suspension onto the column, collect unlabeled cells that pass through and wash the column three times as described previously. This is the negative fraction.Remove the column from the magnetic field and place it on a new collection tube. Pipette MACS buffer onto the column and push the provided plunger into the column to flush out the positive fraction enriched in INK T-cells. To further increase INK T-cell recovery, centrifuge the negative fraction at 300 times G for 10 minutes and repeat previous steps with a new LS or MS column.Pull the positive fractions and determine cell count. Keep 50 microliters of both positive and negative fractions for FACS analysis to check the purity. To activate INK T cells at a one-to-one ratio, transfer the appropriate volume of anti-CD3 and CD28 magnetic beads to a tube and at an equal volume of PBS, then vortex for five seconds.Place the tube on a magnet for one minute and discard the supernatant. Remove the tube from the magnet and resuspend the washed magnetic beads in the proper volume of RPMI. Centrifuge purified INK T-cells at 300 times G for five minutes and mix them with mouse T-activator anti-CD3 or CD28 magnetic beads.Plate one milliliter of the cell suspension, anti-CD23 and CD28 magnetic beads in a 48-well plate with 20 units per milliliter IL-2, then incubate at 37 degrees Celsius. After five days, add 10 nanograms per milliliter IL-7. Split the cells in half when they reach 80 to 90%confluence, always adding 20 units per milliliter IL-2 and 10 nanograms per milliliter IL-7.Under these conditions, INK T-cells can be expanded for up to 15 days. Using this protocol, INK T-cells are enriched from the spleen of transgenic mice through an immunomagnetic separation process. After enrichment, the cells can be expanded with anti-CD3 and CD28 beads, resulting in a 30-fold expansion on average by day 14 of the culture.The strong activation with anti-CD3 and anti-CD beads induced the downregulation of the IMK T-cell TCR expression on the cell surface and a double negative population appeared. The majority of expanded INK T-cells are CD4 negative. Characterization of the lineage-specific transcription factors PLZF and ROR gamma T made it possible to identify the NKT1, NKT2, and NKT17 phenotypes on enriched INK T-cells at day zero and 14.The enriched INK T-cells show a T80-like effector phenotype, which is conserved after 14 days of expansion as confirmed by the secretion of both interferon gamma and IL-4 after PMA and ionomycin stimulation. During the purification steps, remember to work fast with pre-cooled solutions and get rid of any fat residues that may be seen while pipetting. Expanded INK T-cells can be exploited for in vitro assays and in vivo transfers into mice, even after functional modifications via gene transfer or editing.In vitro expansion of INK T-cells overcomes the limitation given by the paucity, making them exploitable in preclinical studies in the fields of tumor immune surveillance, infectious diseases, and auto-immunity.

purification-expansion-mouse-invariant-natural-killer-t-cells-for

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

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.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...