Name: isolation and th17 differentiation of na&iuml;ve cd4 t lymphocytes
Uploaded: 2013-09-26T21:15:00
Description: Watch this Scientific Journal Video about Isolation and Th17 Differentiation of NaÃƒÂ¯ve CD4 T Lymphocytes at JoVE.com

这项工作是由美国国立卫生研究院/ NCATS临床与转化科学奖，以佛罗里达州的TL1 TR000066和UL1 TR000064，从父格兰特R01AI056152多样性的补充来自美国国立卫生研究院的BD Biosciences公司试剂授予大学和大学的支持部份佛罗里达州。

所有动物使用是根据批准的机构动物护理和使用委员会的协议进行的。 1。混音及媒体的制备 <ol><li> 无菌PBS pH值：7.3（1升） 0.23克磷酸二氢钠 1.15克的Na 2 HPO 4 9.0克氯化钠 占用体积去离子水。消毒用高压锅。 </li><li> 细胞培养基（100毫升） 89毫升RPMI 10毫升10％FBS的 1毫升抗生素抗...

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	<li>Chen, Z., Lin, 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=Foxp3+and+ROR&#947;T:+transcriptional+regulation+of+Treg+and+Th17.">Foxp3 and ROR&#947;T: transcriptional regulation of Treg and Th17.</a> International Immunopharmacology. 11, 536-542 (2011).</li><li>Cua, D. J., Sherlock, 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=Interleukin-23+rather+than+interleukin-12+is+the+critical+cytokine+for+autoimmune+inflammation+of+the+brain.">Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain.</a> Nature. 421, 742-748 (2003).</li><li>El-Behi, M., Rostami, 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=Current+views+on+the+roles+of+Th17+and+Th17+cells+in+experimental+autoimmune+encephalitis.">Current views on the roles of Th17 and Th17 cells in experimental autoimmune encephalitis.</a> J. 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D., Dabelic, R., 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+Kinase+Inhibitory+Region+of+SOCS-1+is+Sufficient+to+Inhibit+T+helper-17+Function+in+Experimental+Allergic+Encephalomyelitis.">The Kinase Inhibitory Region of SOCS-1 is Sufficient to Inhibit T helper-17 Function in Experimental Allergic Encephalomyelitis.</a> J. Neuroimmunol. 232, 08-18 (2011).</li><li>Kimura, A., Kishimoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-6:+regulator+of+Treg/Th17+balance.">IL-6: regulator of Treg/Th17 balance.</a> Eur. J. Immunol. 40, 1830-1835 (2010).</li><li>Korn, T., Bettelli, 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=IL-17+and+Th17+Cells.">IL-17 and Th17 Cells.</a> Annu Rev Immunol. 27, 485-517 (2009).</li><li>Kriegel, M. A., Seflk, 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=Naturally+transmitted+segmented+filamentous+bacteria+segregate+with+diabetes+protection+in+nonobese+diabetic+mice.">Naturally transmitted segmented filamentous bacteria segregate with diabetes protection in nonobese diabetic mice.</a> PNAS. 108, 11548-11553 (2011).</li><li>Langrish, C. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th17+and+regulatory+T+cells+in+mediating+and+restraining+inflammation.">Th17 and regulatory T cells in mediating and restraining inflammation.</a> Cell. 140, 845-858 (2010).</li><li>O'Connor, W., Kamanaka, 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=A+protective+function+for+interleukin+17A+in+T+cell-mediated+intestinal+inflammation.">A protective function for interleukin 17A in T cell-mediated intestinal inflammation.</a> Nat Immunol. 10, 603-609 (2009).</li><li>Picca, C. C., Larkin, 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=Role+of+TCR+specificity+in+CD4+CD25++regulatory+T+cell+selection.">Role of TCR specificity in CD4+CD25+ regulatory T cell selection.</a> Immunol Rev. 212, 74-85 (2006).</li><li>Shah, K., Lee, W. 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=Disregulated+balance+of+Th17+and+Th1+cells+in+systemic+lupus+erythematosus.">Disregulated balance of Th17 and Th1 cells in systemic lupus erythematosus.</a> Arthritis Research &amp; Therapy. 12, R53 (2010).</li><li>Sospedra, M., Martin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+of+Multiple+Sclerosis.">Immunology of Multiple Sclerosis.</a> Annu. Rev. 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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=TGF&#946;+in+the+context+of+an+inflammatory+cytokine+milieu+supports+de+novo+differentiation+of+IL-17-producing+T+cells.">TGF&#946; in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.</a> Immunity. 24, 179-189 (2006).</li><li>Zou, Y., Zhang, H., 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=Strain-dependent+production+of+interleukin-17/interferon-&#947;+and+matrix+remodeling-associated+genes+in+experimental+Candida+albicans+keratitis.">Strain-dependent production of interleukin-17/interferon-&#947; and matrix remodeling-associated genes in experimental Candida albicans keratitis.</a> Mol Vis. 18, 1215-1225 (2012).</li></ol>

在这里，我们所描述的协议，实现在体外 Th17细胞分化。 Th17细胞分化的研究是重要的，因为的T淋巴细胞分化成Th17细胞子集，是有效消除人类病原体13关键。相反，IL17生产已与自身免疫性疾病的进展13有关。因为它可以适用于免疫调节和自身免疫的许多不同的鼠模型中的Th17分化的方法适用于许多研究设置。此方法有助于回答有关的T淋巴细胞的能力，分化成Th17细胞的细胞，?...

CD4 + T淋巴细胞（T细胞）在与感染性微生物的免疫系统介导的防御中发挥至关重要的作用。相反，T细胞还密切与自身免疫性疾病如1型糖尿病，系统性红斑狼疮，类风湿关节炎的发病和进展相关。 CD4 + T淋巴细胞成为通过T细胞受体（TCR）与同源抗原/主要组织相容性复合体II（MHCII）的分子相互作用的结合活性，并与B7.1/B7.2的CD28受体相互作用的配体15。除了提供TCR刺激和CD28共刺激的，抗原呈递细胞还提供一种细胞因子环境，这就决定了T淋巴细胞的分化状态，从而指示该T淋巴细胞的反应，给定抗原。不同的病原体/抗原呈递细胞相互作用创造独特的细胞因子环境，这歪斜T淋巴细胞向下聚焦于ELI不同的途径税款发起病原体。不幸的是，T淋巴细胞效应的途径，原本旨在消除入侵的病原体，可错误地针对自身组织15。因此，更好地了解每个不同的CD4 + T细胞亚群的分化状态的是我们如何来调节消除病原体和宽容自我之间的平衡的理解是至关重要的。 除了TH1，TH2，以及诱导型调节性T细胞的分化途径，幼稚T淋巴细胞还可以通过细​​胞因子向下的Th17通路驱动。而Th1细胞战斗细胞内的病原体，Th2细胞消除细胞外的病原体，调节性T细胞（Treg细胞）减少炎症反应的1，16; Th17细胞在消除胞外细菌和真菌中发挥重要作用。 Th17细胞是通常用的谱系特异性转录因子RORγT和产生IL的表达-17A，促进巨噬细胞和嗜中性粒1,7的激活。 Th17细胞有牵连的一些自身免疫性疾病，以及它们相关的啮齿类动物模型。例如，已经证明了IL-23（它是必需的，以维持Th17细胞表型），而不是IL-12，是在中央的罪魁祸首在实验性自身免疫性脑炎（EAE），用于MS的啮齿动物的疾病模型。它随后被显示，减少的IL-17的产生是相关的，以预防EAE 2，6，17。此外，Th17细胞已与其他自身免疫性疾病，包括关节炎和系统性红斑狼疮（SLE）的10，16相关联。 IL-23缺陷的p19 - / -小鼠显示出具有非常低的数字Th17细胞，并具有耐显影不仅EAE，而且胶原诱导的关节炎，型号为风湿性关节炎10，18。此外，小鼠中和IL-17A抗体治疗船尾呃胶原诱导的关节炎的发病中也发现有关节损伤18号决议。应当指出的是Th17细胞在自身免疫性疾病的进展中的作用仍有待表征为最近的研究还表明Th17细胞在1型糖尿病9,11和肠道炎症14的保护作用。这些研究证实Th17细胞分化的自体免疫的重要性。 体外 Th17细胞分化为T细胞研究的一个必要方法，因为有至少两个需要进一步调查令人困惑的问题：1）究竟如何IL-6的调节Treg细胞和Th17细胞分化之间的平衡，以及2）什么是确切机制IL-17诱导的炎性疾病背后？我们的方法使用的CD4 + CD25-T细胞从C57BL / 6小鼠的脾和淋巴结。要注意的是很重要的，虽然有可能使用不纯的诱导Th17细胞分化人群中，获得至少80％纯的CD4 + CD25-T细胞群否定任何污染的忧虑，并确保更成功的Th17细胞分化的结果。为了实现正确的Th17分化，CD4 + CD25-T细胞在抗-CD3和抗-CD28，提供活化信号，1和2分别存在下，与IL-6和TGF-β。虽然已经报道了IL-23单独使用，可用于实现Th17细胞分化，但后来被证明IL-23是必要的Th17细胞的细胞群的稳定性，但IL-6和TGF-β对Th17细胞分化所必需3，18，19。鼠的研究表明，该IL-23受体表达于CD4 + T细胞，他们已经被刺激的IL-6和TGF-β13，18之后。此外，Th17细胞将成功地在IL-23阻断性抗体的存在下培养，只要IL-6和TGF-β的存在18,19。因此，本Th17的分化方案省IDES在适当的条件下，成功地诱导Th17细胞分化。开发更好的了解相关Th17细胞分化和IL-17的产生提供了机会为更好的治疗目的是自身免疫性疾病13发展的机制。

<table border="1">
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 <td>Reagent/Material</td>
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 <td>Sterile Polyestrene Petri Dish</td>
 <td>Fisher Scientific</td>
 <td>0875713A</td>
 <td>60 mm x 15 mm</td>
 </tr>
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 <td>autoMACS Running Buffer</td>
 <td>Miltenyi Biotec</td>
 <td>130-091-221</td>
 <td></td>
 </tr>
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 <td>Premium Microscope Slides, Frosted</td>
 <td>Fisher Scientific</td>
 <td>12-544-3</td>
 <td>3" x 1"1 mm</td>
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 <td>5 ml 21G1 Latex Free Syringe and Needle</td>
 <td>BD Biosciences</td>
 <td>309632</td>
 <td></td>
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 <td>Corning 15 ml Centrifuge Tubes</td>
 <td>Sigma-Aldrich</td>
 <td>CLS430791</td>
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 <td>Nylon 40 microns</td>
 <td>Miami Aquaculture</td>
 <td>Nylon 40/26</td>
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 <td>Microtest tissue culture plate 96 well U bottom </td>
 <td>BD Biosciences</td>
 <td>35-3077</td>
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 <td>Corning Costar 24 well cell culture plates</td>
 <td>Sigma-Aldrich</td>
 <td>CL3524</td>
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 <td>Eppendorf Tubes 1.5 ml</td>
 <td>Fisher Scientific</td>
 <td>05-408-129</td>
 <td></td>
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 <td>Purified NA/LE Hamster Anti-Mouse CD3e</td>
 <td>BD Biosciences</td>
 <td>553057</td>
 <td></td>
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 <td>Purified NA/LE Hamster Anti-Mouse CD28</td>
 <td>BD Biosciences</td>
 <td>553294</td>
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 <td>Mouse IL-6 Recombinant Protein</td>
 <td>eBioscience</td>
 <td>14-8061-62</td>
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 <td>TGFbeta</td>
 <td>R&amp;D Systems</td>
 <td>240-B-002</td>
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 <td>Trypan blue solution 0.4 %</td>
 <td>Sigma-Aldrich</td>
 <td>66H2364</td>
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 <td>Pac Blue Rat Anti-Mouse CD4</td>
 <td>BD Biosciences</td>
 <td>558107</td>
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 <td>APC Rat Anti-Mouse CD8a</td>
 <td>BD Biosciences</td>
 <td>553035</td>
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 <td>PE Conjugated Anti-mouse CD25</td>
 <td>eBioscience</td>
 <td>E01155-516</td>
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 <td>Alexa Fluor 700 Rat Anti-mouse IL-17</td>
 <td>BD Biosciences</td>
 <td>560820</td>
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 <td>Intracellular Cytokine Staining Starter Kit-Mouse</td>
 <td>BD Biosciences</td>
 <td>51-2041AK (559311)</td>
 <td></td>
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 <td>MACS CD4+CD25+ regulatory T cell isolation kit, mouse</td>
 <td>Miltenyi Biotec</td>
 <td>130-091-221</td>
 <td></td>
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 <td>ABAM </td>
 <td>Cellgro</td>
 <td>30-004-CI</td>
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 <td>RPMI</td>
 <td>Corning, Cellgro</td>
 <td>10-040-CM</td>
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 <td>B 2-MercaptoEthanol</td>
 <td>MP Biomedical</td>
 <td>194834</td>
 <td>Hazardous</td>
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 <td>Phorbol 12-Myristate 13-Acetate (PMA)</td>
 <td></td>
 <td></td>
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 <td>Ionomycin Calcium Salt</td>
 <td>Sigma-Aldrich</td>
 <td>13909-1ML</td>
 <td>Hazardous</td>
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 <td>Brefeldin A (BFA)</td>
 <td>MP Biomedicals</td>
 <td>159027</td>
 <td></td>
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 <td>ELISA IL-17A Capture mAb </td>
 <td>BD Biosciences</td>
 <td>555068</td>
 <td></td>
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 <td>ELISA IL-17A Detection mAb </td>
 <td>BD Biosciences</td>
 <td>555067</td>
 <td></td>
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 <td>ELISA IL-17A Standard</td>
 <td>eBioscience</td>
 <td>14-8171-80</td>
 <td></td>
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 <td>IL-2 ELISA Kit</td>
 <td>BD Biosciences</td>
 <td>555148</td>
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 <td>TMB Substrate Reagent Set </td>
 <td>BD Biosciences</td>
 <td>555214</td>
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 <td>Equipment </td>
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 <td>autoMACS Pro Cell Separator</td>
 <td>Miltenyi Biotec</td>
 <td>130-092-545</td>
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 <td>Sorvall Legend RT+ Centrifuge</td>
 <td>ThermoScientific</td>
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 <td>Napco series 8000 WJ CO2 Incubator</td>
 <td>ThermoScientific</td>
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 <td>PTC-200 Peltier Thermal Cycler</td>
 <td>Biorad</td>
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</table>

isolation and th17 differentiation of na&iuml;ve cd4 t lymphocytes

Th17细胞是T细胞已被发现产生白细胞介素17（IL-17）的一个独特的子集，并且不同的功能从其他T细胞亚群，包括Th1细胞，Th2细胞和调节性T细胞。 Th17细胞已与许多自身免疫性疾病相关的炎症过激的免疫反应成为中央罪魁祸首。在这个方法中，我们从净化C57BL / 6小鼠的脾脏和淋巴结T淋巴细胞，并刺激纯化的CD4 + T细胞在控制之下和Th17细胞诱导的环境。在Th17细胞诱导的环境包括在抗CD3和抗CD28抗体，IL-6和TGF-β的存在下刺激。培养至少72小时后长达五天在37℃，细胞随后通过流产生IL-17流式细胞仪，定量PCR和酶联免疫吸附能力进行分析。 Th17细胞分化的CD4 + CD25-T细胞可以被用来进一步阐明角色Th17细胞在自身免疫和宿主德的发生和发展过程中发挥FENSE。此外，CD4 + CD25-细胞从不同的小鼠基因敲除/疾病模型的Th17分化，可以促进我们的细胞命运可塑性的理解。

这Th17细胞分化的协议始于切除脾脏及腋窝，上臂，肠系膜，颈和腹股沟淋巴结肿大。每个位置的表示可以在图2和图3中找到。图1和5都提供在本协议中所述的方法的可视化表示。 此协议的Th17着重从CD4 + CD25-T淋巴细胞种群分化。要注意的是如何有效的autoMACS临细胞分离富集从总汇集淋巴结和脾（ 图4）我们所期望的...

Watch this Scientific Journal Video about Isolation and Th17 Differentiation of NaÃƒÂ¯ve CD4 T Lymphocytes at JoVE.com

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

与效应器功能从其它的T细胞亚群的不同的，Th17细胞已被集中牵连的炎性自身免疫性疾病。这种体外 Th17细胞分化协议提供了一种方法来确定初始CD4 + T淋巴细胞是否能够分化成Th17细胞，并进一步研究其在自身免疫和宿主反应的作用。

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 ...

Research

JoVE Journal

Immunology and Infection

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

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

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  ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

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 ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

静电粘附实验对整合素激活的T细胞研究

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

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 ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Isolation and Ex Vivo Culture of VÃƒÅ½Ã‚Â´1+CD4+ÃƒÅ½Ã‚Â³ÃƒÅ½Ã‚Â´ T Cells, an Extrathymic ÃƒÅ½Ã‚Â±ÃƒÅ½Ã‚Â²T-cell Progenitor

分离和<em&gt;离体</emVδ1的&gt;企业文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT细胞，胸腺外的αβT细胞祖

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...