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

我们感谢Paolo Dellabona和Giulia Casorati对手稿的科学支持和批判性阅读。我们还感谢NIH Tetramer Core Facility为小鼠CD1d四聚体。该研究由Fondazione Cariplo Grant 2018-0366（至M.F.）和意大利癌症研究协会（AIRC）奖学金2019-22604（至G.D.）资助。

这里描述的程序由圣拉斐尔科学研究所的机构动物护理和使用委员会（IACUC）（第1048号）审查和批准。
注意：所有程序必须在无菌条件下进行。所有使用的试剂都列在 材料表。
1. 脾脏加工
<ol><li>根据机构政策，通过吸入CO2 对iVα14-Jα18小鼠实施安乐死。 注意：iVα14-Jα18小鼠必须8周大或以上。为了避免从细胞的体内转移中排斥细?...

<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>

在这里，我们展示了一种可重复且可行的方案，以获得数百万个即用型iNKT细胞。由于这些细胞在体内的缺乏，因此非常需要一种扩增它们的方法。我们提出的协议既不需要特定的仪器，也不需要大量的小鼠。我们故意利用iVα14-Jα18转基因小鼠来减少手术所需的小鼠数量。
来自iVα14-Jα18转基因小鼠的iNKT细胞扩增的另一种成功方案在文献10中可用。该协议涉及?...

不变的自然杀伤性T细胞（iNKT细胞）是先天性T淋巴细胞，表达半不变的αβ T细胞受体（TCR），由不变的Vα14-Jα18链与一组有限的不同Vβ链1配对，其特异性地存在于小鼠中，该链特异性于MHC I类相关分子CD1d2呈递的脂质抗原。iNKT细胞经历激动剂选择程序，导致获得已经在胸腺中的活化/先天效应表型，这发生在几个成熟阶段3，4，产生CD4+和CD4-亚群。通过该程序，iNKT细胞获得不同的T辅助（TH）效应子表型，即TH1（iNKT1），TH2（iNKT2）和TH17（iNKT17），可通过转录因子T-bet，GATA3，PLZF和RORγt的表达来识别，分别为5。iNKT细胞识别一系列微生物脂质，但对内源性脂质也具有自我反应性，内源性脂质在细胞应激和组织损伤的病理情况下上调，例如癌症和自身免疫2。激活后，iNKT细胞通过直接接触和细胞因子产生调节其他先天性和适应性免疫效应细胞的功能2。
小鼠模型（包括CD1d缺陷小鼠或Jα18缺陷小鼠）以及抗原负载CD1d四聚体的产生以及人为半不变TCR特异性单克隆抗体（mAbs）的产生促进了iNKT细胞的研究。然而，原代小鼠iNKT细胞系的产生已被证明是困难的。为了更好地表征iNKT细胞的抗肿瘤功能并将其用于过继细胞治疗，我们建立了一个方案来纯化和扩增iVα14-Jα18转基因小鼠（iVα14Tg）6的脾iNKT细胞，其中iNKT细胞的频率是野生型小鼠的30倍。
扩增的iNKT细胞可用于体外测定，并在体内转移回小鼠体内。例如，在这种情况下，我们已经展示了它们有效的抗肿瘤作用7。此外，体外扩增的iNKT细胞在体内注射8之前可以通过基因转移或编辑进行功能修饰，从而可以对分子途径进行有见地的功能分析，并为先进的细胞疗法铺平道路。

<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

不变的自然杀伤性T（iNKT）细胞是先天性样T淋巴细胞，表达保守的半不变T细胞受体（TCR），该受体对非多态性MHC I类相关分子CD1d呈递的自身或微生物脂质抗原具有特异性。iNKT细胞在整个物种中都非常保守，小鼠模型（包括CD1d缺陷或iNKT缺陷小鼠）促进了它们的研究，并且有可能在小鼠和男性中明确检测到它们，CD1d四聚体或mAbs特异性为半不变TCR。然而，iNKT细胞是罕见的，它们需要扩增以达到任何研究的可管理数量。由于原代小鼠iNKT细胞系的体外生成已被证明是困难的，因此我们已经建立了一个强大的方案来纯化和扩增来自iVα14-Jα18转基因小鼠（iVα14Tg）的脾iNKT细胞，其中iNKT细胞的频率是其30倍。我们在这里表明，原代脾iVα14Tg iNKT细胞可以通过免疫磁性分离过程富集，产生约95-98%纯iNKT细胞。纯化的iNKT细胞受到抗CD3 / CD28微球加IL-2和IL-7的刺激，导致培养物的天+14的30倍扩增，纯度为85-99%。扩增的iNKT细胞可以很容易地进行遗传操纵，为在体外解剖激活和功能机制提供了宝贵的工具，更重要的是，在体内过继转移时也是如此。

本手稿中描述的方案能够通过图1A中总结的免疫磁性分离过程从iVa14-Ja18转基因小鼠的脾脏中富集iNKT细胞。首先通过消耗B细胞和单核细胞来对总脾T细胞进行阴性选择，然后用PBS-57脂质抗原负载的CD1d四聚体进行iNKT细胞阳性免疫磁性分选，从而能够特异性地仅染色iNKT细胞。该方案从单个iVa14-Ja18 Tg小鼠的脾脏中产生约2 x 106个95-98%纯iNKT细胞。在阴性部分中没有或实际上?...

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

我们描述了一种快速而强大的方案，以从小鼠脾脏中富集不变的自然杀伤T（iNKT）细胞，并在体外将其扩增至适合体外和体内研究的数量。

小鼠不变自然杀伤性T细胞的纯化和扩增用于体外和体内研究

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

该协议能够纯化INK T细胞，这是非常罕见的，并允许其数量增加30倍。纯化可以在一天内进行，并在两周内产生大量即用型INK T细胞，用于体内和体外研究。演示该程序的将是实验室的博士后Gloria Delfanti。解剖小鼠脾脏后，通过70纳米细胞过滤器将其粉碎，以获得10毫升PBS和2%FBS的单细胞悬浮液。以300倍G离心五分钟。除去上清液并用一毫升无菌氯化铵钾赖氨酸缓冲液重悬细胞沉淀。在室温下孵育细胞三分钟，然后用5毫升PBS和2%FBS阻断。以300倍G离心五分钟。除去上清液后，将细胞沉淀重悬于3毫升PBS中，含2%FBS，并通过移液除去脂肪残留物。对于富集步骤，保持细胞冷，并使用预冷却在四摄氏度的溶液，然后保持在冰上。以300倍G离心五分钟。用2%FPS和Fc阻滞剂将所有细胞重悬于适量的PBS中，并在室温下孵育15分钟。每1000万个细胞用一到两毫升MACS分离缓冲液洗涤，并以300倍G离心10分钟。除去上清液后，用CD19-FITC和H2-IAb-FITC染色细胞，并在黑暗中以4至8摄氏度孵育15分钟。通过每1000万个细胞加入一到两毫升MACS缓冲液并以300倍G离心10分钟来洗涤细胞。除去上清液并将细胞沉淀重悬在每1000万个细胞的90微升MACS缓冲液中。每1000万个细胞加入10微升抗FITC微珠。充分混合，在4至8摄氏度的黑暗中孵育15分钟。通过每1000万个细胞加入一到两毫升MACS缓冲液来洗涤细胞，并以300倍G离心10分钟。除去上清液，将多达1.25亿个细胞重悬于500微升MACS缓冲液中。将LD柱放置在MACS分离器的磁场中。为避免堵塞，请在LD柱上应用预分离过滤器，并用两毫升MACS缓冲液冲洗。将细胞悬浮液施加到过滤器上，并收集通过色谱柱的未标记细胞。用一毫升MACS缓冲液洗涤空柱储液器三次。收集富含T细胞的流出物并计数细胞，保留50微升用于FACS分析。以300倍G离心5分钟后，除去上清液并用CD1d四聚体-PE染色细胞。搅拌均匀，在黑暗中在冰上孵育30分钟。通过每1000万个细胞添加一到两毫升MACS缓冲液来洗涤细胞。在以300倍G离心10分钟后，除去上清液并将细胞沉淀重悬在每1000万个细胞的80微升MACS缓冲液中。每1000万个细胞添加20微升抗PE微珠。充分混合，在4至8摄氏度的黑暗中孵育15分钟。通过每1000万个细胞加入一到两毫升MACS缓冲液并以300倍G离心10分钟来洗涤细胞。除去上清液，将多达1亿个细胞重悬于500微升MACS缓冲液中。根据细胞计数，将LS或MS色谱柱置于MACS分离器的磁场中，并用MACS缓冲液冲洗色谱柱。将细胞悬浮液施加到色谱柱上，收集通过的未标记细胞，并如前所述洗涤色谱柱三次。这是负数。将色谱柱从磁场中取出，并将其放在新的收集管上。将MACS缓冲液移液到色谱柱上，并将提供的柱塞推入色谱柱中，以冲洗掉富含INK T细胞的阳性组分。为了进一步提高INK T细胞的回收率，将阴性部分以300倍G离心10分钟，然后用新的LS或MS色谱柱重复先前的步骤。拉动阳性组分并确定细胞计数。保留50微升的正馏分和负组分，用于FACS分析，以检查纯度。要以一比一的比例激活INK T细胞，将适当体积的抗CD3和CD28磁珠转移到管中，并以等体积的PBS，然后涡旋五秒钟。将试管放在磁铁上一分钟，然后丢弃上清液。从磁体上取下磁管，将洗涤后的磁珠以适当体积的 RPMI 重悬。将纯化的INK T细胞以300倍G离心5分钟，并与小鼠T激活剂抗CD3或CD28磁珠混合。将一毫升细胞悬浮液，抗CD23和CD28磁珠放在48孔板中，每毫升IL-2有20个单位，然后在37摄氏度下孵育。五天后，每毫升IL-7添加10纳克。当细胞达到80%至90%汇合时，将细胞分成两半，每毫升IL-2始终添加20个单位，每毫升IL-7添加10纳克。在这些条件下，INK T细胞可以扩增长达15天。使用该协议，INK T细胞通过免疫磁分离过程从转基因小鼠的脾脏中富集。富集后，可以用抗CD3和CD28微球扩增细胞，从而在培养物的第14天平均扩增30倍。抗CD3和抗CD微球的强激活诱导了细胞表面IMK T细胞TCR表达的下调，并出现了双阴性群体。大多数扩增的INK T细胞是CD4阴性的。谱系特异性转录因子PLZF和ROR γ T的表征使得在第0天和第14天鉴定富集INK T细胞上的NKT1，NKT2和NKT17表型成为可能。富集的INK T细胞显示出T80样效应子表型，其在扩增14天后是保守的，这可以通过PMA和离子霉素刺激后干扰素γ和IL-4的分泌得到证实。在纯化步骤中，请记住使用预冷却溶液快速工作，并清除移液时可能看到的任何脂肪残留物。扩增的INK T细胞可用于体外测定和体内转移到小鼠中，即使在通过基因转移或编辑进行功能修饰之后也是如此。INK T细胞的体外扩增克服了匮乏的局限性，使它们可以在肿瘤免疫监测，传染病和自身免疫领域的临床前研究中加以利用。

Research

JoVE Journal

Immunology and Infection

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

T细胞受体在小鼠T细胞的逆转录病毒转导

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 ...

科研

免疫与感染

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

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 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

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac转座子初级人类T细胞系统改造

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

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

人工抗原提呈细胞（AAPC）的自然杀伤T细胞介导的​​激活和扩展

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

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

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

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

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

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

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

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

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

人全血中NK细胞杀伤活性的流式细胞仪分析

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

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

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

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

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...