Name: in vitro differentiation of mouse granulocyte-macrophage-colony-stimulating factor (gm-csf)-producing t helper (thgm) cells
Uploaded: 2018-09-10T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells at JoVE.com

这项研究得到新加坡国立大学卫生系统 (T1-2014 Oct-12 和 T1-2015 Sep-10) 赠款的支持。

所有在本议定书中使用的老鼠都是在 C57BL/6 的遗传背景下, 并被安置在新加坡国立大学特定的无病原体条件下。所有实验都使用新加坡国立大学机构动物保育和使用委员会批准的协议进行。
1. 试剂和材料制备
<ol><li>制备500毫升磷酸盐缓冲盐水 (PBS), 含2% 胎牛血清 (血清) 和1毫米乙二胺四乙酸 (EDTA)。 注意: 在整个过程中保持缓冲区在冰的最佳效果。</li>
	<li>准备50毫升完整的 ...

<ol>
	<li>Zhu, J., Paul, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4+T+cells:+fates,+functions,+and+faults.">CD4 T cells: fates, functions, and faults.</a> Blood. 112, 1557-1569 (2008).</li><li>Kara, E. E., 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=Tailored+immune+responses:+novel+effector+helper+T+cell+subsets+in+protective+immunity.">Tailored immune responses: novel effector helper T cell subsets in protective immunity.</a> PLoS Pathogens. 10, e1003905 (2014).</li><li>Bou Nasser Eddine, F., Ramia, E., Tosi, G., Forlani, G., Accolla, 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=Tumor+Immunology+meets...Immunology:+Modified+cancer+cells+as+professional+APC+for+priming+naive+tumor-specific+CD4++T+cells.">Tumor Immunology meets...Immunology: Modified cancer cells as professional APC for priming naive tumor-specific CD4+ T cells.</a> Oncoimmunology. 6, e1356149 (2017).</li><li>Dong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TH17+cells+in+development:+an+updated+view+of+their+molecular+identity+and+genetic+programming.">TH17 cells in development: an updated view of their molecular identity and genetic programming.</a> Nature Reviews. Immunology. 8, 337-348 (2008).</li><li>Korn, T., Bettelli, E., Oukka, M., Kuchroo, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-17+and+Th17+Cells.">IL-17 and Th17 Cells.</a> Annual Review of Immunology. 27, 485-517 (2009).</li><li>Sheng, W., 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=STAT5+programs+a+distinct+subset+of+GM-CSF-producing+T+helper+cells+that+is+essential+for+autoimmune+neuroinflammation.">STAT5 programs a distinct subset of GM-CSF-producing T helper cells that is essential for autoimmune neuroinflammation.</a> Cell Research. 24, 1387-1402 (2014).</li><li>Codarri, L., 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=RORgammat+drives+production+of+the+cytokine+GM-CSF+in+helper+T+cells,+which+is+essential+for+the+effector+phase+of+autoimmune+neuroinflammation.">RORgammat drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation.</a> Nature Immunology. 12, 560-567 (2011).</li><li>El-Behi, 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=The+encephalitogenicity+of+T(H)17+cells+is+dependent+on+IL-1-+and+IL-23-induced+production+of+the+cytokine+GM-CSF.">The encephalitogenicity of T(H)17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF.</a> Nature Immunology. 12, 568-575 (2011).</li><li>Ivanov, I. I., 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=The+orphan+nuclear+receptor+RORgammat+directs+the+differentiation+program+of+proinflammatory+IL-17++T+helper+cells.">The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells.</a> Cell. 126, 1121-1133 (2006).</li><li>Zhang, J., 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+novel+subset+of+helper+T+cells+promotes+immune+responses+by+secreting+GM-CSF.">A novel subset of helper T cells promotes immune responses by secreting GM-CSF.</a> Cell Death and Differentiation. 20, 1731-1741 (2013).</li><li>Croxford, A. L., Spath, S., Becher, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM-CSF+in+Neuroinflammation:+Licensing+Myeloid+Cells+for+Tissue+Damage.">GM-CSF in Neuroinflammation: Licensing Myeloid Cells for Tissue Damage.</a> Trends in Immunology. 36, 651-662 (2015).</li></ol>

在这里我们描述了一个从小鼠天真的 CD4+ 细胞的体外TH基因分化的协议, 其次是对分化细胞的分析, 以验证该方法。注意, 脾脏和淋巴结均可用于幼稚的 CD4+ t 细胞纯化和 tH基因的分化。细胞内细胞因子染色与流式细胞术相结合的细胞因子表达表明, 在 gm (图 1A) 下, 约有55% 的神经细胞被诱导成为 gm-脑脊液表达细胞。与 th17 分化条件下的细...

CD4+ (tH) 细胞是免疫系统的重要组成部分, 在宿主防御微生物病原体、癌症监测和自身免疫性1、2、3中具有重要作用。在 t 细胞受体 (TCR) 激活, 天真的 CD4+ t 细胞可以被区分为t h1,t h2, th 17, 或调节 t (Treg) 细胞在不同的细胞因子环境传道2,4,5. 最近, 发现了一种主要生产 gm-CSF 的 th细胞的新子集, 并命名为 gm6。IL-7 通过激活信号传感器和转录激活剂 5 (STAT5) 来驱动 THGM 细胞的分化。这些细胞表达了大量的 GM-脑脊液, 同时低表达其他 TH细胞特征因子, 如 IFNγ和 IL-176。在 CD4+ T 细胞介导的 neuroinflammation7、8的发展中, 基因型脑脊液被发现发挥了关键作用。与 IFNγ或 IL-17-expressing 自身反应性 t 细胞相比, 转基因脑脊液表达 t 细胞转移到野生型 (小鼠) 导致早期疾病发作和更高的疾病严重性。此外, Csf2细胞没有诱导实验性自身免疫脑脊髓炎 (EAE) 后被过继转输转移到脑细胞受体, 而缺乏 IFNγ或 IL-17A 的 T 淋巴细胞保留了调解 EAE7的能力。此外, 使用中和抗体对 GM-CSF 的封锁改善了 EAE 疾病严重程度8。此外, 小鼠 t 细胞中 STAT5 的缺失导致了转基因一代的减少, 因此, 在小鼠对 EAE 发展6的抵抗力。这些发现强调了 GM-脑脊液表达 TH细胞在自身免疫性 neuroinflammatory 疾病中的重要性。因此, 建立一种从天真的 CD4+ t 细胞中鉴别 GM-脑脊液表达 tH细胞的方法, 对于研究自身免疫 neuroinflammation 和 T 细胞介导的免疫应答的发病机制具有重要意义。然而, 从小鼠天真的 CD4+ 有效地产生 TH基因电池的协议尚未建立。
在这里, 我们提出了一种方法, 区分小鼠 tHGM 细胞从天真的 CD4+ t 细胞。该协议描述了整个过程, 包括从小鼠中提取脾脏、单细胞悬浮液的制备、CD4 阳性选择、荧光活化细胞分选和 TH细胞。分化和分析。用细胞内细胞因子染色结合流式细胞仪对分化的 T 辅助细胞进行分析, 以确定单细胞水平的细胞因子表达, 采用定量实时 PCR 方法测定 mRNA 水平的细胞因子表达, 并通过 ELISA 对蛋白质水平的细胞因子表达进行评估。该方法可应用于 EAE 等不同条件下的转基因细胞生物学研究中, 其在发病机制中起着重要作用。

<table>
	<tbody>
		<tr>
			<td>RPMI 1640</td>
			<td>Biowest</td>
			<td>L0500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Heat Inactivated</td>
			<td>Capricorn</td>
			<td>FBS-HI-12A</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>1st Base</td>
			<td>BUF-2040-10</td>
			<td>Diluted to 1x PBS in sterile water</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>1st Base</td>
			<td>BUF-1053-100ml-pH8.0</td>
			<td></td>
		</tr>
		<tr>
			<td>10X ACK buffer</td>
			<td>Biolegend</td>
			<td>420301</td>
			<td>diluted in sterile water</td>
		</tr>
		<tr>
			<td>cell strainer</td>
			<td>SPL Life Sciences</td>
			<td>93070</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4 microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-201</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>LS column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>magnetic stand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml syringe</td>
			<td>Terumo</td>
			<td>SS+01T</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Eppendorf 5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Sony SY3200 cell sorter</td>
			<td>Sony</td>
			<td>Sony SY3200 cell sorter</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml conical centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>210261</td>
			<td></td>
		</tr>
		<tr>
			<td>15m conical centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tube</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well cell culture plate</td>
			<td>Greiner Bio-One</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD4 PerCP-eFluor 710</td>
			<td>eBioscience</td>
			<td>46-0041-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PE conjugated anti-mouse CD25 (IL-2Ra, p55)</td>
			<td>eBioscience</td>
			<td>12-0251-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human/mouse CD44 APC</td>
			<td>eBioscience</td>
			<td>17-0441-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD62L FITC</td>
			<td>eBioscience</td>
			<td>11-0621-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer-improved counting chamber</td>
			<td>Marienfeld</td>
			<td>640010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone Trypan Blue Solution</td>
			<td>GE healthcare Life Sciences</td>
			<td>SV30084.01</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope</td>
			<td>Nikon</td>
			<td>Nikon Elipse TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified&#160;anti-mouse CD3e&#160;antibody</td>
			<td>Biolegend</td>
			<td>100314</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified hamster anti-mouse CD28</td>
			<td>BD Biosciences</td>
			<td>553295</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat&#160;anti-mouse IFN&#947; &#160;</td>
			<td>eBioscience</td>
			<td>16-7312-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified anti-mouse IL-4 Antibody</td>
			<td>Biolegend</td>
			<td>504102</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-7 Protein</td>
			<td>R&#38;D system</td>
			<td>407-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL-6</td>
			<td>R&#38;D system</td>
			<td>406&#8209;ML</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human TGF-beta</td>
			<td>R&#38;D system</td>
			<td>240-B-010</td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Merck Millipore</td>
			<td>19-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>GolgiPlug protein transport inhibitor</td>
			<td>BD Biosciences</td>
			<td>51-2301KZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracellular Fixation&#38;Permeabilization buffer set</td>
			<td>eBioscience</td>
			<td>88-8824-00</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse GM-CSF PE</td>
			<td>eBioscience</td>
			<td>12-7331-82</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse IL-17A</td>
			<td>Biolegend</td>
			<td>506908</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse IFN-gamma</td>
			<td>Biolegend</td>
			<td>505810</td>
			<td></td>
		</tr>
		<tr>
			<td>GO Taq qPCR master mix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse GM-CSF ELISA Ready-SET-Go!</td>
			<td>invitrogen</td>
			<td>88-7334-88</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-17A Uncouted ELISA</td>
			<td>invitrogen</td>
			<td>88-7371-22</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro differentiation of mouse granulocyte-macrophage-colony-stimulating factor (gm-csf)-producing t helper (thgm) cells

粒-巨噬细胞集落刺激因子 (gm-csf) 生产的 t 帮助器 (tHGM) 细胞是一个新发现的 t 辅助细胞子集, 主要分泌的 GM-脑脊液不产生干扰素 (干扰素) γ或白细胞介素 (IL)-17, 并发现在自身免疫性 neuroinflammation 中起重要作用。从单纯的 CD4+ t 细胞中分离出单纯的脾和 tH基因 CD4+ t 细胞, 是研究 t 细胞介导免疫和自身免疫性疾病的有效方法。这里我们描述了一种方法, 将小鼠天真 CD4+ t 细胞与 IL-7 促进的 tH基因细胞区分开来。用不同的方法分析细胞因子的表达, 包括细胞内细胞因子染色结合流式细胞术、定量实时聚合酶链反应 (PCR), 对分化结果进行评估, 并酶联免疫吸附测定法 (ELISA)。根据这里所描述的 THgm 分化协议, 大约55% 的细胞表达了 IFNα或 IL-17 的最小表达。通过对基因型脑脊液、IFNα和 IL-17在 mRNA 和蛋白质水平上表达的分析, 进一步证实了转基因脑脊液的主要表达。因此, 该方法可用于体外诱导单纯 CD4+ t 细胞与 th基因细胞的分离, 对转基因细胞生物学研究有一定的参考价值。

从两个8周大的雄性 C57BL/6 小鼠中分离出的朴素 CD4+ T 细胞分为三部分。在描述的协议之后, 细胞的一部分被区分成 THGM 细胞。另一部分是在 thgm 条件下培养的 anti-IL-4 抗体 (10 µg/毫升), 以测试 IL-4 封锁的影响, 在分化的 thgm。最后一部分是培养在 TH17 分化条件 (3 µg/毫升 anti-CD3e, 1 µg/毫升 anti-CD28, 10 ng/毫升 TGFβ, 30 ng/毫升 IL-6, 10 µg/毫升反 IFNγ...

Watch this Scientific Journal Video about In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells at JoVE.com

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

体外小鼠粒细胞-巨噬细胞集落刺激因子 (gm-CSF) 的分化--生成 t 辅助器 (tHgm) 细胞

在这里, 我们提出了一种区分小鼠粒细胞-巨噬细胞集落刺激因子产生的 t 型CD4+ t 细胞, 包括天真的 CD4+ t 细胞的分离, th基因的分化和差异性 TH基因细胞的分析。该方法可用于转基因细胞的调节和功能研究.

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Research

JoVE Journal

Immunology and Infection

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

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

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

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 ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of NaÃƒÆ’Ã‚Â¯ve CD4 T Lymphocytes

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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 ...

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

Mouse NaÃƒÆ’Ã‚Â¯ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

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

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells iTregs from NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve CD4+ T Cells Using a TGF-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-containing Protocol

在从幼稚的CD4 + T细胞的人CD4 + FOXP3 +诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...