前肿瘤反应性T细胞的体外扩增Bryostatin 1/Ionomycin手段和共同的γ链细胞因子配方
Summary
为有效的协议<em>体外</em>描述从肿瘤引流淋巴结或其他淋巴组织的继发性肿瘤轴承主机的肿瘤反应性T细胞的扩张。该协议有选择地扩大肿瘤特异性T细胞过继免疫治疗乳腺癌。
Abstract
据报道，乳腺癌患者既存的免疫反应，对他们的肿瘤1,2。然而，这种免疫反应，未能提供完整的保护，对发展或复发的乳腺癌。为了克服这个问题，通过增加肿瘤反应性T细胞的频率，过继免疫治疗已聘用。扩大肿瘤特异性T细胞已被用于多种协议。然而，这些协议限制的肿瘤抗原体外抗原特异性T细胞的活化使用。最近，常见的伽玛链细胞因子如IL – 2，IL – 7，IL – 15和IL – 21已单独或联合使用的抗肿瘤免疫反应的3的增强。不过，目前尚不清楚制定工作，为扩大肿瘤反应性T细胞的最佳。在这里，我们提出了一个从FVBN202在收养的T细胞治疗乳腺癌HER-2/neu的阳性乳腺癌的转基因小鼠模型的肿瘤反应性T细胞的选择性激活和扩张的协议。该协议包括bryostatin-1/ionomycin（B / I）和IL – 2激活T细胞，在16个小时的肿瘤抗原的情况下。 /我的激活模仿细胞内的信号，导致T细胞的活化蛋白激酶C活性和细胞内钙离子增加， 分别为4。该协议具体激活肿瘤特异性T细胞，而造成不相关的T细胞。与IL – 7和IL – 15的B /我激活T细胞培养24小时，然后用IL – 2脉冲。 24小时后，T细胞被洗净，分割，并培养与IL – 7 + IL – 15增加4天。 前体外扩增的T细胞特异性肿瘤和抗肿瘤疗效是确定的。
Protocol
1。分离淋巴细胞5 分离肿瘤引流淋巴结或荷瘤FVBN202转基因小鼠的脾脏和单细胞悬液，准备在冰冷的RPMI1640含10％胎牛血清的补充。 /我在激活50毫升聚丙烯锥形管，结果在一个更大的T细胞产量相比，聚苯乙烯管。氯胺酮和甲苯噻嗪注射麻醉IP。颈椎脱位是用来作为一个“安乐死”的方法。 文化在完全培养基中的细胞（10 6细胞/ mL）bryostatin – 1（5纳米）和离子霉素（1μM…
Discussion
肿瘤反应性T细胞的选择性扩张效应的抗肿瘤作用，可建议使用B协议实现/我的激活和γ链细胞因子IL – 2，IL – 7和IL – 15的体外扩增。 IL – 2是T细胞的生长因子，可支持抗原特异性T细胞的分化和扩张，而IL – 7可以抑制T细胞的凋亡，并支持在扩张他们的生存能力。 IL – 15可以支持，收养T细胞治疗9-11后产生长期的抗肿瘤反应的重要的记忆性T细胞。改变这些细胞因子的顺序和组合，可能影?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院R01 CA104757格兰特（MH Manjili）的支持。我们非常感谢VCU梅西癌症中心的支持和英联邦基金会为癌症研究。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Bryostatin 1 | Sigma | B7431-10ug | ||
| Ionomycin | Calbiochem | 407950 | ||
| Mouse IL-7 | PeproTech | 217-17 | ||
| Mouse IL-15 | PeproTech | 210-15 | ||
| Human IL-2 | PeproTech | 200-02 | ||
| RPMI1640 | Invitrogen | 11875 | ||
| FBS | Gemini | 100-106 | ||
| Penicillin/Streptomycin | Cellgro | 30-002-CI | ||
| L- glutamine | Invitrogen | 25030081 | ||
| β- mercaptoethanol | Sigma | M7522 | ||
| anti-CD16/32 antibody | Biolegend | 101302 | ||
| Annexin V-FITC Apoptosis Detection Kit | BD Pharmingen | 556547 | ||
| FITC-CD4 | Biolegend | 100406 | ||
| PE-CD8 | Biolegend | 100708 | ||
| anti-c-Erb2/c–Neu | Calbiochem | OP16 | ||
| PE- anti mouse IgG | Biolegend | 405307 | ||
| formaldehyde | Polysciences | 04018 | ||
| Hemocytometer | Hycor | 87144 | ||
| Light microscope | VWR | V200073 | ||
| Mouse IFN-γ ELISA set | BD Pharmingen | 555138 | ||
| Cell culture flasks | Greiner | 658175 |
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Cite This Article
Kmieciak, M., Toor, A., Graham, L., Bear, H. D., Manjili, M. H. Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation. J. Vis. Exp. (47), e2381, doi:10.3791/2381 (2011).