Summary

扩展人外周血γδT细胞使用Zoledronate

Published: September 09, 2011
doi:

Summary

扩大γδT细胞从外周血单个核细胞(PBMC)的一个方法来描述。外周血单核细胞衍生的γδT细胞受到刺激和扩大使用zoledronate和白细胞介素2(IL – 2)。 γδT细胞的大规模扩张,可应用于自体肿瘤细胞免疫疗法。

Abstract

人类γδT细胞能够识别和响应到多种应激性抗原,从而开发先天广泛的抗肿瘤和抗感染活性。1外周血中大多数γδT细胞的Vγ9Vδ2T细胞受体。这些细胞识别抗原在主要组织相容性复合体独立的方式,并发展强大的杀伤和Th1样的效应功能。因此,γδT细胞是肿瘤免疫治疗的有吸引力的候选人的效应细胞。 Vγ9Vδ2T细胞响应,如phosphoantigens(E)4 -羟基- 3 -甲基- 2 -己烯焦磷酸(HMBPP),这是通过类异戊二烯生物合成的细菌合成;和异戊烯基焦磷酸(IPP),这是生产在真核细胞通过甲羟戊酸途径,在生理条件 ,3中,IPP的nontransformed细胞的生成是没有足够的γδ T细胞的活化。甲羟戊酸途径在肿瘤细胞失调, 导致3,因为IPP的积累和γδT细胞的活化。aminobisphosphonates(如帕米膦酸或zoledronate)抑制法尼基焦磷酸合成酶(制剂),酶的作用IPP的甲羟戊酸途径下游,细胞内水平IPP和sensitibityγδT细胞识别,可以治疗由aminobisphosphonates增加。 IPP的积累是低效率的nontransfomred细胞比肿瘤细胞,使我们对癌症与aminobisphosphonatesγδT细胞通过激活免疫药理相关浓度的aminobisphosphonates,4有趣的是,IPP的积累在单核细胞的PBMC与aminobisphosphonates治疗,因为高效,这些细胞的药物摄取。积累的IPP成为抗原提呈细胞和Vγ9Vδ2T细胞刺激外周血单核细胞。基于这些机制,我们制定了一个大规模扩张γδT细胞zoledronate文化和白细胞介素的技术-2(IL – 2)7其他方法扩大γδT细胞利用合成phosphoantigens bromohydrin焦(BrHPP)8或2 -甲基-3 -丁烯- 1 -焦磷酸(2M3B1PP)9,所有这些方法允许前体外扩增,γδT细胞过继免疫治疗中使用的大量产生。然而,只有zoledronate是一个FDA批准的市售试剂。 Zoledronate扩大γδT细胞显示CD27 CD45RA 效应记忆体的表型和它们的功能可以通过干扰素γ的生产检测7评估。

Protocol

1。隔离的PBMC 绘制成一个BD真空采血管彩管的细胞与肝素钠的制备管血(7.5-8.0毫升)。管含有钠抗凝和聚蔗糖Hypaque密度流体,加上聚酯凝胶屏障,这两种液体分开。在一个水平旋翼为20分钟1800 × 克 (摇摆头),在室温(18℃至25℃)离心管/血液样本。切换离心机刹车关闭。 离心后,层的顺序发生如下(从上到下看到):一)等离子体 – B)外周血单个核细胞(PBMC)和血小板…

Discussion

这里介绍的方法,使γδT细胞从外周血单个核细胞的有效扩张。 γδT细胞激活,并通过zoledronate扩大和IL – 2制定完整的效应功能,细胞因子的产生和细胞毒性反映。据报道,合成phosphoantigens bromohydrin焦(BrHPP)和2 – 甲基3 – 丁烯-1 – 焦磷酸(2M3B1PP)也扩大γδT细胞,但是,他们不市售。相比之下,zoledronate已经授权作为择泰的临床应用。因此,一个可靠的试剂是容易获得。

培养?…

Declarações

The authors have nothing to disclose.

Materials

Reagent name Company Catalogue number Comments (optional)
ZOMETA Novartis Pharma K. K   zoledronate
PROLEUKIN Novartis Pharmaceuticals   human recombinant IL-2
BD Vacutainer CPT Cell Preparation Tube with Sodium Heparin BD 362753  
RPMI1640 Invitrogen 21870-076  
ALyS203- medium Cell Science & Technology Institute 0301-7  
OpTmizer Invitrogen 0080022SA  
brefeldin A Sigma B5936-200UL  
phorbol 12-myristate 13-acetate (PMA) Sigma P1585-1MG  
ionomycin Sigma 13909-1ML  
IntraPrep BECKMAN COULTER A07803  
anti-human CD3-FITC or PE/Cy5 BECKMAN COULTER A07746 FITC
A07749 PE/Cy5
 
anti-human CD4-ECD BECKMAN COULTER 6604727  
anti-human CD8-PE/Cy5 BECKMAN COULTER 6607011  
anti-human CD14-PE/Cy5 BECKMAN COULTER A07765  
anti-human CD19-PE BECKMAN COULTER A07769  
anti-human CD45-ECD BECKMAN COULTER A07784  
anti-human CD56-PE/Cy5 BECKMAN COULTER A07789  
anti-human TCRαβ-PE BECKMAN COULTER A39499  
anti-human TCR Vγ9-FITC BECKMAN COULTER IM1463  
anti-human CD27-PE/Cy5 BECKMAN COULTER 6607107  
anti-human CD45RA-ECD BECKMAN COULTER IM2711  
anti-human CD69-PE BD 555531  
anti-human NKG2D-PE BECKMAN COULTER A08934  
Anti-humal IFNγ-PE BECKMAN COULTER IM2717U  
Mouse IgG1 isotype control-PE BECKMAN COULTER A07796  
Mouse IgG1 isotype control-ECD or PE/Cy5 BECKMAN COULTER A07797 A07798  

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Kondo, M., Izumi, T., Fujieda, N., Kondo, A., Morishita, T., Matsushita, H., Kakimi, K. Expansion of Human Peripheral Blood γδ T Cells using Zoledronate. J. Vis. Exp. (55), e3182, doi:10.3791/3182 (2011).

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