扩展人外周血γδT细胞使用Zoledronate
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，在生理条件下 ，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
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?…
Disclosures
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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