从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生
Summary
The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.
Abstract
体外过继转移扩大了自体肿瘤浸润淋巴细胞（肿瘤浸润淋巴细胞）可以调节的转移性黑色素瘤患者显著的子集耐用和完整的答复。这种方法的主要障碍是转移的T细胞，引起端粒缩短的可行性减少的，并且从患者获得的肿瘤浸润淋巴细胞的数量有限。分化少T细胞端粒长就过继性T细胞治疗的理想T细胞亚群;然而，产生大量的这些分化较少的T细胞是有问题的。过继性T细胞疗法的这种限制可以通过使用诱导多能干细胞（iPS细胞）在理论上克服自我更新，维持多能性，具有细长的端粒，并提供自体T细胞的免疫治疗的一个无限源。在这里，我们提出了一个协议，以产生使用仙台病毒载体的重编程因子转导入肿瘤浸润淋巴细胞iPS细胞。该协议产生S完全重新编程，免费矢量克隆。这些TIL衍生的iPSC可能能够产生用于过继性T细胞疗法分化少患者 – 和肿瘤特异性T细胞。
Introduction
细胞重新编程的技术，通过一组已定义的转录因子的过表达允许产生诱导性多能干细胞（iPS细胞）的保持在基于细胞的疗法1,2-领域大有希望。这些iPS细胞表现出转录和后生特征和具有自我更新和多能性的能力，类似于胚胎干细胞（ESC）3-5。在重编程技术在过去十年取得了显着的进步，使我们能够产生甚至从终末分化细胞，如T细胞6-8人的iPS细胞。 T细胞来源的iPS细胞（TiPSCs）保留的T细胞受体（TCR）链基因作为原始T细胞相同的重排结构，它允许从TiPSCs 9-11抗原特异性T细胞的再生。
近80％的黑色素瘤浸润淋巴细胞（的TIL）中获得来自病人的肿瘤特异性识别肿瘤相关抗原的ND保持对原有的肿瘤细胞杀伤12。值得注意的是，在的TIL细胞程序性死亡蛋白-1（PD-1）的表达被发现以识别自体肿瘤反应性的剧目，包括突变的特定新抗原-CD8 +淋巴细胞13。在组合离体扩增的自体肿瘤浸润淋巴细胞的过继转移用制备lymphodepleting方案和白细胞介素-2（IL-2）的全身给药会引起患者14亚转移性黑素瘤的实质性退化。尽管取得了令人鼓舞的临床前模型和患者中的结果，注入的T细胞的存活差和免疫抑制途径的存在似乎妥协过继性T细胞疗法的潜能。目前临床协议需要自体T细胞的广泛离体操作，以便获得大的数字。这导致具有不良存活终末分化的T细胞，降低海峡的产生iferative容量，以及高含量的PD-1 15。
继T细胞疗法的这种限制可以通过使用iPS细胞，可以提供自体T细胞免疫治疗无限源理论上克服。我们最近已报道黑素瘤的TIL的表达由仙台病毒（SeV载体）的四个转录因子介导的转导，OCT3 / 4，SOX2，KLF4和c-Myc的16个高层次的PD-1的重编程。虽然逆转录病毒载体需要整合到宿主染色体以表达重编程基因，SeV载体是非整合并最终被从细胞质淘汰。重新编程的效率是与SeV的系统高得多慢病毒或逆转录病毒载体6-8相比。此外，SeV载体能特异性重新编程在外周血单核细胞（PBMC）的T细胞，而通过慢病毒或逆转录病毒载体产生的一些的iPSC克隆可从非淋巴谱系6-8。在这里，我们详细介绍实施了人体黑素瘤的TIL的分离和活化和用于使用的SeV重新编程系统的TIL衍生的iPSC的生成的程序。
Protocol
Representative Results
Discussion
在这里，我们证明了通过四个转录的SeV介导转导重新编程黑素瘤的TIL到的iPSC一个协议因素OCT3 / 4，SOX2，KLF4和c-MYC。这种方法，采用了SeV载体系统重新编程的T细胞，提供了一个非整合方法7的优点。
先前的研究表明，重编程的SeV体系是高效可靠的改编不仅成纤维细胞，而且外周血T细胞7,17。此外，我们最近表明，黑色素瘤的TIL是更加分化和表达更高水平的抑制?…
Declarações
The authors have nothing to disclose.
Acknowledgements
We thank Ms. Deborah Postiff and Ms. Jackline Barikdar in the Tissue Procurement Core and Dr. Cindy DeLong in the Pluripotent Stem Cell Core Laboratory at the University of Michigan for her technical assistance. This study was supported by University of Michigan startup funding and grants from the Central Surgical Association, American College of Surgeons, Melanoma Research Alliance, and NIH/NCI (1K08CA197966-01) to F. Ito.
Materials
| gentle MACS C Tubes | Miltenyi Biotec | 130-093-237 | |
| gentle MACS Dissociator | Miltenyi Biotec | 130-093-235 | |
| Tumor Dissociation Kit, human | Miltenyi Biotec | 130-095-929 | |
| RPMI 1640 | Life technologies | 11875-093 | |
| Falcon 70 um Cell Strainer | BD | 352350 | |
| BD Falcon 50ml Conical Cntrifuge tubes | BD | 352070 | |
| IMDM | Life technologies | 12440053 | |
| human AB serum | Life technologies | 34005100 | |
| L-glutamine (200mM) | Life technologies | 25030-081 | |
| 2-mercaptoethanol (1000x, 55mM) | Life technologies | 21985-023 | |
| Penicillin-Streptomycin | Life technologies | 15140-122 | |
| gentamicin | Life technologies | 15750-060 | |
| Ficoll-Paque PLUS | GE | 17-1440-02 | |
| D-PBS (-) | Life technologies | 14040-133 | |
| recombinant human (rh) IL-2 | Aldesleukin, Prometheus Laboratories Inc. | ||
| Purified NA/LE Mouse Anti-Human CD3 | BD | 555329 | |
| Purified NA/LE Mouse Anti-Human CD28 | BD | 555725 | |
| X-VIVO 15 | Lonza | 04-418Q | |
| FBS | Gibco | 26140-079 | |
| HEPES | Life technologies | 15630-080 | |
| N-Acetylcysteine | Cumberland Pharmaceuticals Inc. | NDC 66220-207-30 | |
| Falcon Tissue Culture Plates (6-well) | Corning | 353046 | |
| Falcon Tissue Culture Plates (24-well) | Corning | 353047 | |
| Sendai virus vector | DNAVEC | ||
| SNL feeder cells | Cell Biolabs, Inc | CBA-316 | |
| mitomycin C | SIGMA | M4287 | soluble in water (0.5 mg/ml) |
| gelatin | SIGMA | G1890 | |
| Primate ES Cell Medium | Reprocell | RCHEMD001 | warm in 37 ℃ water bath before use |
| basic fibroblast growth factor (bFGF) | Life technologies | PHG0264 | |
| ReproStem | Reprocell | RCHEMD005 | warm in 37 ℃ water bath before use |
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