Name: an optimized method for isolating and expanding invariant natural killer t cells from mouse spleen
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D.E. and M.B.D. are members of a multidisciplinary research platform (MRP) of Ghent University, and the Ghent Researchers on Unfolded Proteins in Inflammatory Disease (GROUP-ID) consortium.

In this study, adult (6-8 weeks) female C57Bl/6 mice were used. Mice were housed and bred according to the guidelines of the Ghent University vivarium. All animal procedures were approved by the Institutional Animal Care and Ethics Committee.
1. Preparation of Mononuclear Cells (MNCs) from Mouse Spleen
<ol>
	<li>Sacrifice the mouse by cervical dislocation.</li>
	<li>Place the mouse back down on the dissecting board and fix the hind and forelegs with pins.</li>
	<li>Sterilize the skin-surface with 70% (vol/vol) ethanol/H2O.</li>
	<li>Cut the skin along the abdominal midline to the thorax and expose the spleen using sterile surgical instruments.</li>
	<li>After trimming the surrounding fat tissues, place the spleen into a 24 well plate containing 1 ml 1x phosphate buffered saline (PBS) at RT.</li>
	<li>Dissect the spleen using a sterile scalpel and transfer the pieces into a 70 &#181;m nylon filter placed within a 50 ml tube.</li>
	<li>Using a 2 ml syringe plunger, gently mash the spleen pieces through the cell strainer and wash with 5 ml 1x PBS.</li>
	<li>Add 3 ml of Ficoll-Paque to a 15 ml tube and overlay the density gradient medium with the 5 ml cellular suspension.</li>
	<li>Centrifuge at 400 x g for 20 min at RT without brake.</li>
	<li>Transfer the interphase to a fresh 15 ml tube, add 10 ml cold 1x PBS and centrifuge at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Resuspend the cells in 2 ml 1x PBS containing 0.5% bovine serum albumin (BSA) and 1 mM ethylenediaminetetraacetic acid (EDTA) (FACS buffer). Transfer 10 &#181;l of the cell suspension to a small tube, mix with 10 &#181;l of&#160;0.4%</li>
</ol>
2. Enrichment, Detection and Purification of Mouse iNKT Cells
<ol>
	<li>Enrichment of mouse splenic CD5positive (CD5+) lymphocytes.
	<ol>
		<li>(Optional) Stain 105 cells with FITC-conjugated anti-CD5 (4 &#181;l of 1:30 dilution/106 cells) for 30 min at 4 &#176;C in the dark, wash with 200 &#181;l FACS buffer and resuspend in 500 &#181;l FACS buffer. Prepare a 20 &#181;M working solution of 4&#39;,6-diamidino-2-phenylindole (DAPI) in 1x PBS, add 1 &#181;l thereof to 105 cells in FACS buffer and incubate at RT for 5 min. Acquire 104 pre-enriched cells on the flow cytometer.
		<ol>
			<li>Using FCS-H and FCS-W, electronically gate on FSC-Wlo cells to exclude cell doublets and aggregates (Figure 1A). Then electronically gate out DAPI+ (dead) cells within the FSC-Wlo population (Figure 1B), and use SSC-A and FCS-A to electronically select the lymphocyte population by size (Figure 1C).</li>
			<li>Using cells within the lymphocyte gate, plot SSC-A versus CD5 and electronically gate CD5+ lymphocytes as shown in Figure 1D. Acquire the remainder of the pre-enriched sample on the flow cytometer.</li>
		</ol>
		</li>
		<li>Adjust cell counts to 107 cells per 90 &#181;l in FACS buffer and add 10 &#181;l anti-CD5 microbeads.</li>
		<li>Incubate at 4 &#186;C for 15 min, wash once with 4 ml FACS buffer and resuspend in 500 &#181;l cold FACS buffer. Enrich for CD5+ lymphocytes using magnetic cell separation (MACS)columns according to manufacturer&#8217;s recommendations.</li>
		<li>Optional) Stain 105 cells with FITC-conjugated anti-CD5 (4 &#181;l of 1:30 dilution/106 cells) and DAPI as in 2.1.1. Acquire 104 enriched lymphocytes on the flow cytometer and verify that the electronic gates created in 2.1.1. select CD5+ lymphocytes in the enriched cellular fraction. Acquire 105 cells on the flow cytometer and analyze the percentage CD5+ lymphocytes present in the enriched fraction as shown in Figure 1E-H.</li>
	</ol>
	</li>
	<li>Detection and isolation of mouse iNKT cells by FACS.
	<ol>
		<li>Resuspend enriched CD5+ lymphocytes at a concentration of approximately 106 cells per 20 &#181;l in FACS buffer containing anti-CD16/CD32 (4 &#181;l of 1:50 dilution/106 cells) and PE-conjugated &#945;GalCer/CD1d tetramer (1 &#181;g/106 cells).</li>
		<li>Incubate for 30 min at RT in the dark.</li>
		<li>Dilute mouse antibodies (mAbs) anti-CD3&#949; V500 (4 &#181;l of 1:20 dilution/106 cells), anti-CD19 e450 (4 &#181;l of 1:50 dilution/106 cells) and anti-CD8&#945; e450 (4 &#181;l of 1:50 dilution/106 cells) in FACS buffer, add to CD5+ lymphocytes and incubate for 30 min at 4 &#176;C in the dark.</li>
		<li>Wash the cells with 1 ml FACS buffer by centrifuging at 300 x g for 10 min at 4 &#176;C and resuspend in 4 ml FACS buffer at a final concentration of 107 cells/ml.</li>
		<li>Filter the cells through a 30 &#181;m cell strainer and place the cells on ice.</li>
		<li>Calibrate the flow cytometer for cell sorting according to the manufacturer&#8217;s recommendations 14,15. 
		Note: Calibration of a flow cytometer for cell sorting requires training and can be performed by an experienced operator, or a trained technician.</li>
		<li>Add 10 &#181;l of a 20 &#181;M working solution of DAPI per 106 cells and incubate at RT for 5 min. Acquire approximately 104 enriched CD5+ lymphocytes on the flow cytometer. Electronically gate on FSC-Wlo cells to exclude cell doublets and aggregates (Figure 2A). Then electronically gate out DAPI+ CD8+CD19+ cells (dump channel) within the FSC-Wlo population (Figure 2B), and use SSC-A and FCS-A to electronically select lymphocytes (Figure 2C) as described in 2.1.1.1.
		<ol>
			<li>Plot &#945;GalCer/CD1d tetramer by CD3&#949; and electronically gate &#945;GalCer/CD1d tetramer+CD3&#949;+ iNKT cells as shown in&#160; Figure 2D. Acquire no more than 2 x 105 CD5+ enriched lymphocytes to determine the percentage &#945;GalCer/CD1d tetramer+CD3&#949;+ iNKT cells present in the enriched cellular fraction.</li>
		</ol>
		</li>
		<li>Using the electronic gates created in 2.2.7., FACS sort &#945;GalCer/CD1d tetramer+CD3&#949;+ iNKT cells on the flow cytometer at 70 psi using a 70 &#181;m nozzle at a flow rate of &#39;1.5&#39;. Collect sorted iNKT cells in a FACS tube containing 1 ml 100% FCS. Subsequently rinse sorted iNKT cells from the side of the FACS tube using 10 ml RMPI 1640 medium and centrifuge the cells at 300 x g for 10 min at 4 &#176;C.</li>
		<li>Resuspend sorted iNKT cells in 1 ml RPMI 1640 medium and acquire 40 &#181;l thereof on a flow cytometer to assess the purity of sorted iNKT cells. Select FSC-Wlo DAPI- lymphocytes as described in 2.2.7. (Figure 2E-G) and electronically gate &#945;GalCer/CD1d tetramer+CD3&#949;+ iNKT cells as shown in Figure 2H.</li>
	</ol>
	</li>
</ol>
3. Culture and Expansion of Mouse iNKT Cells
Day 0
<ol>
	<li>Coat a flat-bottomed 96 well plate with purified anti-CD3&#949; (3 &#181;g/ml) per well for 1 hr at RT. Wash twice with 200 &#181;l 1x PBS and once with 200 &#181;l complete RPMI 1640 medium (10% vol/vol FCS, 100 U/ml penicillin&#8211;streptomycin, 5.5 &#181;M &#946;-mercaptoethanol).</li>
	<li>Count the number of viable sorted iNKT cells (step 2.2.8)&#160;using 0.4% a hemocytometer as in 1.11., centrifuge at 300 x g for 10 min at 4 &#176;C and resuspend at 5 x 105 cells/ml in complete RPMI 1640 medium containing recombinant murine IL-2 (10 ng/ml), recombinant murine IL-12 (1 ng/ml) and soluble anti-mouse CD28 (5 &#181;g/ml). Plate sorted iNKT cells at 105 cells/well in anti-CD3&#949; coated plates and incubate at 37 &#176;C in a CO2 incubator for 2 days.</li>
</ol>
Day 2&#160;
<ol start="3">
	<li>Transfer the cells to a fresh uncoated flat-bottomed 96 well plate and culture the cells under resting conditions in complete RPMI 1640 medium at 37 &#176;C in a CO2 incubator for 2 days.</li>
</ol>
Day 4&#160;
<ol start="4">
	<li>Dislodge the cells from the plate by gentle pipetting and transfer the cells to a fresh 15 ml tube. Centrifuge at 300 x g for 10 min at 4 &#176;C, resuspend at 5 x 105 cells/ml&#160;in complete RPMI 1640 medium containing recombinant murine IL-7 (5 ng/ml), and plate 105 cells/well in a flat-bottomed 96 well plate for 4 days at 37 &#176;C in a CO2 incubator.</li>
</ol>
Day 8
<ol start="5">
	<li>Collect the cells in a fresh tube, centrifuge at 300 x g for 10 min at 4 &#176;C and resuspend at 5 x 105 cells/ml in complete RPMI 1640 medium containing soluble anti-CD28 (5 &#181;g/ml).</li>
	<li>Coat a flat-bottomed 96 well plate with purified anti-CD3&#949; (3 &#181;g/ml) per well and wash as in 3.1. Plate 105 cells/well in anti-CD3&#949; coated plates and incubate at 37 &#176;C in a CO2 incubator until day 11.</li>
</ol>
Day 11 &#8211; 18
<ol start="7">
	<li>Repeat steps 3.4 to 3.6.</li>
</ol>
Day 19-20
<ol start="8">
	<li>Transfer the cells to a fresh 15 ml tube, centrifuge at 300 x g and resuspend at 5 x 105 cells/ml in complete RPMI 1640 medium.</li>
	<li>Plate cells at 105 cells/well in a flat-bottomed 96 well plate and allow the cells to rest for 2 days at 37 &#176;C in a CO2 incubator prior to use in an experiment.</li>
</ol>
4. Cytokine Production by Mouse iNKT Cells Using a CD1d Plate-bound Assay
<ol>
	<li>Coat a flat-bottomed 96 well plate with 100 &#181;l of recombinant murine CD1d (10 &#181;g/ml in 1x PBS) per well and incubate for 1 hr at 37 &#176;C.</li>
	<li>Wash four times with 200 &#181;l 1x PBS, add 200 &#181;l of 2% FCS in 1x PBS (blocking solution) per well and incubate for 2 hr at 37 &#176;C.</li>
	<li>Remove the blocking solution, add 200 &#181;l of &#945;GalCer (5 ng/&#181;l) per well, and incubate the plate at 37 &#176;C for 16 hr. [For preparation of the &#945;GalCer solution, add 1,085 &#181; 1x PBS to 55 &#181;g of &#945;GalCer dissolved in vehicle containing 96 mg/ml&#160;sucrose, 10 mg/ml sodium deoxycholate and 5 mg/ml tween 20].</li>
	<li>After incubation, discard the &#945;GalCer solution and wash the well twice with 200 &#181;l 1x PBS followed by 200 &#181;l complete RPMI 1640 medium.</li>
	<li>Collect the in vitro expanded iNKT cells at day 21, centrifuge the cells at 300 x g for 10 min at 4 &#176;C and resuspend in complete RPMI 1640 medium at a concentration of 0.5 x 106 cells/ml.</li>
	<li>Add 200 &#181;l of the iNKT cell resuspension per well and incubate for 72 hr at 37 &#176;C in a CO2 incubator.</li>
	<li>Collect the supernatant and measure cytokines of interest by enzyme-linked immunosorbant assay (ELISA) according to manufacturer&#8217;s protocol.</li>
</ol>

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The authors have no competing interests to disclose.

Critical steps in the current protocol include the isolation and subsequent enrichment of CD5+ lymphocytes (Section 1 &#38; 2), FACS sorting (Section 3) and the initial plating of iNKT cells (Section 4). Of the steps performed in Section 1, remember to carefully layer the splenic cellular suspension over the density gradient medium such that a distinct cellular interphase is generated following centrifugation. The subsequent enrichment for CD5+ lymphocytes by magnetic cell separation (Section 2) minimizes the reagents and time required to stain for and FACS sort iNKT cells (Section 3), which helps increase iNKT cell yields post-sort. Finally, it is important to seed no more than 105 iNKT cells per well for the first 2 days of culture (Section 4) as seeding wells with higher numbers of iNKT cells can result in cellular apoptosis.
A number of considerations are important when modifying and/or troubleshooting this protocol: Firstly, the age of the animals used is important. Animals that are either too old, or have accessory infections, may hamper your ability to isolate, distinguish, and FACS sort sufficient cells from the spleen. Secondly, when staining with &#945;GalCer/CD1d tetramers, it is important to keep the volume of cells resuspended in PBS buffer low as too high a volume dilutes out the staining efficiency of the &#945;GalCer/CD1d tetramer, which in turn reduces the clustering of tetramer+ iNKT cells by FACS. Ideally, the presence of tightly clustered &#945;GalCer/CD1d tetramer+ iNKT cells during FACS acquisition allows for more accurate gating by FACS and yields higher percentage purity iNKTs during post-sort analysis. This is important as lower sorted iNKT cell yields will result in the outgrowth of other contaminating T cell populations during culture. Thirdly, continuously refresh medium that becomes yellow during the culture and expansion of iNKT cells. For example, replace 50% of yellowed medium with new medium containing the appropriate cytokines, particularly during days 8 and day 11 of culture. iNKT cell yields can be maximized in this way.
To our knowledge this is the first iNKT expansion protocol to include a density gradient and CD5+ lymphocyte enrichment step for the isolation and enrichment of iNKT cells from mouse spleen. A number of alternative approaches have been explored to enrich for splenic iNKT cells including &#945;GalCer/CD1d dimers 13, V&#945;14 TCR Tg mice 10 as well as depletion of non-T cells by magnetic cell separation 9. However, although iNKT cells can be successfully expanded in vitro using these approaches, such methods require either large volumes of &#945;GalCer/CD1d dimers to enrich for iNKTs 13, only generate transgenic iNKT cells 10, or simultaneously give rise to some non-iNKT cell lines 9. Furthermore, the use of plate-bound anti-CD3&#949; to maintain and expand sorted iNKT cells (Section 4) precludes a requirement for activated APCs during iNKT expansion in vitro 9. In this context, an APC-free iNKT expansion protocol has the advantage of not having to separate APCs from expanded iNKT cells prior to injection in vivo. We should however also add that the protocol described is limited by the fact that the investigator requires easy access to, as well as the relevant training, to calibrate and operate a FACS sorter. Unfortunately FACS sorters are expensive and they are not always available in every laboratory. Nevertheless, although the current protocol has been optimized to isolate and culture murine iNKT cells, the enrichment steps described herein can also be used to isolate and study iNKT recent thymic emigrants (RTEs) 16 and can be adapted to enrich for and characterize other rare T cell populations by FACS.
Collectively, we define an optimized approach that can be used to efficiently generate up to 108 iNKT cells from the spleens of three mice within 3 weeks. iNKT cells generated in this manner retain the ability to secrete cytokines upon stimulation, making them useful for adoptive transfer experiments and the study of iNKT cell biology in vivo.

Murine invariant natural killer T (iNKT) cells are a distinct population of innate T lymphocytes selected in the thymus by CD1d-expressing cortical thymocytes 1,2. iNKT cells express a T cell receptor (TCR) comprised of an invariant V&#945;14-J&#945;18 TCR chain paired with either V&#946;8, V&#946;7 or V&#946;2 TCRs 3, which is capable of recognizing endogenous as well as foreign lipid antigens in the context of CD1d. For example, murine iNKT cells recognize and are activated by an endogenous lipid antigen called isoglobotrihexosylceramide (iGb3) 4, as well as &#945;-galactosylceramide (&#945;GalCer) 5,6, a glycolipid isolated from marine sponges. TCR-dependent activation of iNKT cells promotes the priming of adaptive immune responses, and as a result, iNKT cells have been shown to be functionally involved in the amelioration or development of a range of pathologies including rheumatic disease 7 and cancer 8. Currently, synthetic iNKT cell ligands constitute promising new vaccine adjuvants that may be capable of regulating a number of immunopathological conditions.
It has previously been demonstrated that iNKT cells can be generated in vitro following isolation from mouse tissue however; many of these studies employ the use of primary antigen-presenting cells (APCs) and/or cell lines 9, V&#945;14 TCR transgenic (Tg) mice 10, or thymomas for the generation of iNKT cell-derived hybridomas 11,12. Furthermore, large numbers of mice, high volumes of reagents such as &#945;GalCer-loaded CD1d dimers, and lengthy culture times make some published protocols less ethically and economically appealing 9,13.
In this report we describe an adapted method for the isolation and in vitro expansion of iNKT cells from mouse spleen. More specifically, the protocol describes a method for enriching iNKT cells from mouse spleen which reduces the mice, reagents and time required for FACS cell sorting, and proposes an optimized approach for expanding sorted splenic iNKT cells in vitro.

<table><tbody><tr><td>Material</td><td></td><td></td><td></td></tr><tr><td>recombinant murine IL-2</td><td>eBiosciences</td><td>14-8021</td><td></td></tr><tr><td>recombinant murine IL-12</td><td>eBiosciences</td><td>39-8122-65</td><td></td></tr><tr><td>recombinant murine IL-7</td><td>eBiosciences</td><td>14-8071</td><td></td></tr><tr><td>purified anti-mouse CD3e (145-2C11)</td><td>eBiosciences</td><td>16-0032-86</td><td></td></tr><tr><td>purified anti-mouse CD28 (37.51)</td><td>eBiosciences</td><td>16-0281-85</td><td></td></tr><tr><td>e450-conjugated anti-mouse CD19 (eBio1D3)</td><td>eBiosciences</td><td>48-0193-82</td><td></td></tr><tr><td>e450-conjugated anti-mouse CD8&amp;alpha; (53-6.7)</td><td>eBiosciences</td><td>48-0081-82</td><td></td></tr><tr><td>FITC-conjugated anti-mouse CD5 (53-7.3)</td><td>eBiosciences</td><td>11-0051-81</td><td></td></tr><tr><td>V500-conjugated anti-mouse CD3&amp;epsilon; (500A2)</td><td>BD biosciences</td><td>560771</td><td></td></tr><tr><td>anti-mouse CD5 (Ly-1) microbeads</td><td>Macs Miltenyi Biotec</td><td>130-049-301</td><td></td></tr><tr><td>purified anti-mouse CD16/32 (2.4G2)</td><td>Macs Miltenyi Biotec</td><td>130-092-575</td><td></td></tr><tr><td>MACS MS Columns</td><td>Macs Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>Trypan Blue, 0.4% (wt/vol)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Gibco</td><td>12633-020</td><td></td></tr><tr><td>1x PBS</td><td>Gibco</td><td>10010-056</td><td>[Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt; - free]</td></tr><tr><td>Fetal calf serum</td><td>Gibco</td><td>10270</td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030-123</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Sigma-Aldrich</td><td>P4458-100ML</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906-100MG</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>EDS-1KG</td><td></td></tr><tr><td>Ficoll-Paque plus</td><td>GE Healthcare</td><td>71-7167-00 AF</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>96-well plate F-bottom</td><td>Greiner-bio one</td><td>657-160</td><td></td></tr><tr><td>24-well plate</td><td>Greiner-bio one</td><td>665-180</td><td></td></tr><tr><td>6-well plate</td><td>Greiner-bio one</td><td>655-180</td><td></td></tr><tr><td>15 ml falcon tube</td><td>Greiner-bio one</td><td>188-271</td><td></td></tr><tr><td>50 ml falcon tube</td><td>Greiner-bio one</td><td>227-261</td><td></td></tr><tr><td>70 &amp;micro;m filter</td><td>Greiner-bio one</td><td>542-070</td><td></td></tr><tr><td>30 &amp;micro;m filter</td><td>Millipore</td><td>SVGP01050</td><td></td></tr><tr><td>MiniMACS separator</td><td>Macs Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>MS Columns</td><td>Macs Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>Water-jacketed CO2 incubator</td><td>VWR</td><td></td><td></td></tr><tr><td>Hemocytometer</td><td>VWR</td><td></td><td></td></tr><tr><td>Dissection Kit</td><td>VWR</td><td></td><td></td></tr><tr><td>BD FACSAria III</td><td>BD Biosciences</td><td></td><td></td></tr></tbody></table>

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.

Isolation of splenic mononuclear cells using a density gradient takes approximately 1 hr and eliminates the use of reagents required to lyse red blood cells (RBCs). A high yield of viable cells is obtained using this method and debris generated during straining of the organ is removed. Typically, the frequency of iNKT cells within the splenic lymphocyte pool ranges between 1 and 5% of total T lymphocytes however, this can vary depending on the age, sex and health status of the animals used. Approximately 106 iNKT cells can be acquired from the pooled spleens of 3 mice and the highest yield of iNKT cells are obtained from spleens of 6-8 week old mice.
The use of mouse anti-CD5 magnetic beads significantly enriches for iNKT cells within the splenic MNC fraction and, apart from a minor population of B lymphocytes that express the CD5 antigen, the majority of cells isolated using this procedure constitute CD5+ lymphocytes. For example, 5-8% of the MNCs obtained after CD5 enrichment are CD5-negative (Figure 1H). For iNKT cell FACS sorting, combining &#945;GalCer/CD1d tetramer and anti-mouse CD3&#949; yields iNKT cell purities above 98% however, FACS dump channels for splenic B and CD8 T cells should be used to eliminate &#8216;sticky&#8217; lymphocyte populations during the sort. In addition, gate out any doublets that may have formed during the CD5 enrichment step. Using this FACS setup, we obtained iNKT cell purities post-sort in the order of 99% (Figure 2H).
Stimulating 106 sorted iNKT cells with plate-bound anti-CD3&#949; in the presence of IL-2, IL-12 and soluble anti-CD28 resulted in a 3-fold expansion of iNKT numbers following 2 days of culture (Figure 3). Subsequent expansion iNKT cells in the presence of IL-7 results in a 3- to 4-fold increase in the number of iNKT cells present at day 4 in culture. Notably, repeating these culturing conditions can yield an average of 7 x 107 iNKT cells after two rounds of expansion without any visible loss of cells between changes in culture media on different days (Figure 3). Thus, murine splenic iNKT cells can be expanded at least 70-fold using these conditions in 18 days.
We also assessed the cytokine producing capacity of expanded iNKT cells by stimulation with plate-bound recombinant murine CD1d loaded with &#945;GalCer. At 48 hr post-stimulation, IL-2, IL-4, IFN-&#947;, TNF-&#945; and IL-6 could be readily detected in culture supernatants by ELISA (Figure 4). Expanded iNKT cells therefore retain the ability to produce Th1 and Th2 cytokines post-expansion.
<img alt="Figure 1" src="/files/ftp_upload/53256/53256fig1.jpg" /> 
Figure 1.&#160;Enrichment for CD5+ lymphocytes from mouse splenic MNCs.&#160;Gating strategy for analyzing the percentage of CD5+ lymphocytes present in pre-enriched (A-D) and magnetic cell separation-enriched (E-H) mouse splenic MNC suspensions. Cell doublets (A, E) and dead cells (B, F) have been excluded using FSC-H versus FCS-W and DAPI versus FSC-A plots, respectively. The percentage CD5+ cells present within the lymphocyte gate (C, G) both before and after enrichment are shown in D and H, respectively. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53256/53256fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53256/53256fig2.jpg" />

Figure 2.&#160;FACS sorting iNKT cells from CD5-enriched splenic MNCs.&#160;Gating strategy for analyzing the percentage &#945;GalCer/CD1d tetramer+CD3&#949;+ iNKT cells present before (A-D) and after (E-H) FACS sorting. The gating strategy eliminates cell doublets (A, E), dead cells and CD8+CD19+ lymphocytes (B, F) from the analysis by plotting FSC-H versus FCS-W and DAPI, CD8 and CD19 (dump channel) versus FSC-A, respectively. The percentage &#945;GalCer/CD1d tetramer+CD3&#949;+ iNKT cells present within the lymphocyte gate (C, G) before and after FACS sorting are shown in (D) and (H), respectively. CD1d-Tet., &#945;GalCer/CD1d Tetramer. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53256/53256fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>


<img alt="Figure 3" src="/files/ftp_upload/53256/53256fig3.jpg" /> 
Figure 3.&#160;Absolute number of iNKT cells generated in vitro. Fold increase in the number of iNKT cells at the indicated time points after 3 weeks of expansion in vitro. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53256/53256fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53256/53256fig4.jpg" /> 
Figure 4.&#160;Cytokine producing capacity of in vitro expanded iNKT cells. Day 20 expanded iNKT cells were stimulated in vitro with mouse &#945;GalCer-loaded CD1d. After 72 hr supernatants were collected and analyzed for the presence of IFN-&#947;, IL-4, TNF-&#945;, IL-2 and IL-6 by ELISA. Bars represent means SEM (n = 2). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53256/53256fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

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Immunology and Infection

11.9K Views.  Ghent University Hospital. 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. 

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

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&#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 Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

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Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

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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.

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 ...

Isolating Invariant Natural Killer T Cells from a Mouse Model

Source: Meng, M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/60048">Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice.</a>&#160;J. Vis. Exp.&#160;(2020)
This video illustrates a method for isolating invariant natural killer T cells, also known as iNKT cells. The procedure begins with injecting a synthetic glycolipid into a mouse to stimulate the activation and proliferation of iNKT cells in the spleen. Subsequently, single-cell suspensions are prepared from the spleen and incubated with iNKT-cell-recognizing complexes labeled with fluorophores, followed by magnetic bead-based separation, to isolate the iNKT cells.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Cellular Immunotherapy

T-Cell Enrichment: A Technique to Isolate T-Cells from Mixed Cell Population by Magnetic Separation

Source: Nguyen, H. D., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/t/60083">Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease.</a> J. Vis. Exp. (2020).
This video describes T-cells enrichment from the spleen of a mouse by negative selection using a magnetic separator. The technique involves targeting the other cells in the sample for depletion using antibodies or ligands directed against specific cell surface antigens.

Source: Nguyen, H. D., et al. Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease. J. Vis. Exp. ...

Cancer Research

Leukemia

Cell isolation & characterization

Induction of Graft Versus Host Disease in Mouse Models via Allogeneic Bone Marrow and Splenic Cell Transplantation

Source: Buchele, V., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58871">Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice.</a> J. Vis. Exp. (2019).
This video demonstrates the induction of intestinal graft-versus-host disease, GvHD, in a lethally-irradiated recipient mouse using allogeneic hematopoietic stem cells and GvHD-inducing splenic alloreactive T lymphocytes. This protocol helps establish a severe acute intestinal GvHD model to understand the mechanism of the disease and develop innovative therapeutic options.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. GvHD ...

Immunopathology

Host-Pathogen Interaction Models

Natural Killer Cell Isolation from Liver Tissue for Selective Expansion

Begin by identifying and sectioning the viable tissue areas using sterile surgical equipment to obtain lymphocytes. Then, place the sectioned tissues in 30 milliliters of HBSS without calcium or magnesium, and keep the tissue on ice until ready for isolation.
Working inside a biosafety cabinet, mince the tissue into less than 0.5-centimeter cubes with sterile razor blades and forceps. Place the minced tissue pieces &#8212; not more than 4 grams &#8212; in the tissue dissociator tubes, and add 10 milliliters of collagenase IV to the tissue pieces.
To mince the tissue thoroughly, treat the tissue dissociator tubes into a tissue dissociator at 37 degrees Celsius. After removing the tubes from the tissue dissociator, triturate the minced tissue through a 40-micron nylon cell strainer using the back end of a 5-milliliter syringe. Discard the large, undigested fragments.
Spin down the collected eluent at 400 g for 5 minutes at room temperature, and aspirate the supernatant before resuspending the cell pellets in 30% polyvinylpyrrolidone-coated silica to remove the fat cells. Spin down the cells as described, before resuspending the cell pellet in 9 milliliters of R-10 media.
To separate lymphocytes from red blood cells and polymorphonuclear cells, carefully layer the cell suspension over 4 milliliters of Ficoll or lymphocyte separation media. Separate the layers by centrifuging at 400 g for 23 minutes at room temperature with the acceleration and brakes off. Then, carefully decant the upper medium layer and harvest the interphase containing tissue-infiltrating lymphocytes.
Rinse the cells with 10 milliliters of media and proceed for analysis of primary Natural Killer, or NK, cells expansion protocol.

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

Summary

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Chapters in this video

Mouse Naïve CD4⁺ T Cell Isolation and In vitro Differentiation into T Cell Subsets

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

An In Vivo Mouse Model to Measure Naïve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

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

Isolating Invariant Natural Killer T Cells from a Mouse Model

T-Cell Enrichment: A Technique to Isolate T-Cells from Mixed Cell Population by Magnetic Separation

Induction of Graft Versus Host Disease in Mouse Models via Allogeneic Bone Marrow and Splenic Cell Transplantation

Natural Killer Cell Isolation from Liver Tissue for Selective Expansion