小鼠不变自然杀伤性T细胞的纯化和扩增用于体外和体内研究
Summary
我们描述了一种快速而强大的方案，以从小鼠脾脏中富集不变的自然杀伤T（iNKT）细胞，并在体外将其扩增至适合体外和体内研究的数量。
Abstract
不变的自然杀伤性T（iNKT）细胞是先天性样T淋巴细胞，表达保守的半不变T细胞受体（TCR），该受体对非多态性MHC I类相关分子CD1d呈递的自身或微生物脂质抗原具有特异性。iNKT细胞在整个物种中都非常保守，小鼠模型（包括CD1d缺陷或iNKT缺陷小鼠）促进了它们的研究，并且有可能在小鼠和男性中明确检测到它们，CD1d四聚体或mAbs特异性为半不变TCR。然而，iNKT细胞是罕见的，它们需要扩增以达到任何研究的可管理数量。由于原代小鼠iNKT细胞系的体外生成已被证明是困难的，因此我们已经建立了一个强大的方案来纯化和扩增来自iVα14-Jα18转基因小鼠（iVα14Tg）的脾iNKT细胞，其中iNKT细胞的频率是其30倍。我们在这里表明，原代脾iVα14Tg iNKT细胞可以通过免疫磁性分离过程富集，产生约95-98%纯iNKT细胞。纯化的iNKT细胞受到抗CD3 / CD28微球加IL-2和IL-7的刺激，导致培养物的天+14的30倍扩增，纯度为85-99%。扩增的iNKT细胞可以很容易地进行遗传操纵，为在体外解剖激活和功能机制提供了宝贵的工具，更重要的是，在体内过继转移时也是如此。
Introduction
不变的自然杀伤性T细胞（iNKT细胞）是先天性T淋巴细胞，表达半不变的αβ T细胞受体（TCR），由不变的Vα14-Jα18链与一组有限的不同Vβ链1配对，其特异性地存在于小鼠中，该链特异性于MHC I类相关分子CD1d2呈递的脂质抗原。iNKT细胞经历激动剂选择程序，导致获得已经在胸腺中的活化/先天效应表型，这发生在几个成熟阶段3，4，产生CD4+和CD4–亚群。通过该程序，iNKT细胞获得不同的T辅助（TH）效应子表型，即TH1（iNKT1），TH2（iNKT2）和TH17（iNKT17），可通过转录因子T-bet，GATA3，PLZF和RORγt的表达来识别，分别为5。iNKT细胞识别一系列微生物脂质，但对内源性脂质也具有自我反应性，内源性脂质在细胞应激和组织损伤的病理情况下上调，例如癌症和自身免疫2。激活后，iNKT细胞通过直接接触和细胞因子产生调节其他先天性和适应性免疫效应细胞的功能2。
小鼠模型（包括CD1d缺陷小鼠或Jα18缺陷小鼠）以及抗原负载CD1d四聚体的产生以及人为半不变TCR特异性单克隆抗体（mAbs）的产生促进了iNKT细胞的研究。然而，原代小鼠iNKT细胞系的产生已被证明是困难的。为了更好地表征iNKT细胞的抗肿瘤功能并将其用于过继细胞治疗，我们建立了一个方案来纯化和扩增iVα14-Jα18转基因小鼠（iVα14Tg）6的脾iNKT细胞，其中iNKT细胞的频率是野生型小鼠的30倍。
扩增的iNKT细胞可用于体外测定，并在体内转移回小鼠体内。例如，在这种情况下，我们已经展示了它们有效的抗肿瘤作用7。此外，体外扩增的iNKT细胞在体内注射8之前可以通过基因转移或编辑进行功能修饰，从而可以对分子途径进行有见地的功能分析，并为先进的细胞疗法铺平道路。
Protocol
Representative Results
Discussion
在这里，我们展示了一种可重复且可行的方案，以获得数百万个即用型iNKT细胞。由于这些细胞在体内的缺乏，因此非常需要一种扩增它们的方法。我们提出的协议既不需要特定的仪器，也不需要大量的小鼠。我们故意利用iVα14-Jα18转基因小鼠来减少手术所需的小鼠数量。
来自iVα14-Jα18转基因小鼠的iNKT细胞扩增的另一种成功方案在文献10中可用。该协议涉及?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Paolo Dellabona和Giulia Casorati对手稿的科学支持和批判性阅读。我们还感谢NIH Tetramer Core Facility为小鼠CD1d四聚体。该研究由Fondazione Cariplo Grant 2018-0366（至M.F.）和意大利癌症研究协会（AIRC）奖学金2019-22604（至G.D.）资助。
Materials
| Ammonium-Chloride-Potassium (ACK) solution | in house | 0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA, pH 7.2-7.4 | |
| anti-FITC Microbeads | Miltenyi Biotec | 130-048-701 | |
| anti-PE Microbeads | Miltenyi Biotec | 130-048-801 | |
| Brefeldin A | Sigma | B6542 | |
| CD19 -FITC | Biolegend | 115506 | clone 6D5 |
| CD1d-tetramer -PE | NIH tetramer core facility | mouse PBS57-Cd1d-tetramers | |
| CD4 -PeCy7 | Biolegend | 100528 | clone RM4-5 |
| Fc blocker | BD Bioscience | 553142 | |
| Fetal Bovine Serum (FBS) | Euroclone | ECS0186L | heat-inactivated and filtered .22 before use |
| FOXP3 Transcription factor staining buffer | eBioscience | 00-5523-00 | |
| H2 (IAb) -FITC | Biolegend | 114406 | clone AF6-120.1 |
| hrIL-2 | Chiron Corp | ||
| Ionomycin | Sigma | I0634 | |
| LD Columns | Miltenyi Biotec | 130-042-901 | |
| LS Columns | Miltenyi Biotec | 130-042-401 | |
| MACS buffer (MB) | in house | 0.5% Bovine Serum Albumin (BSA; Sigma-Aldrich) and 2Mm EDTA | |
| MS Columns | Miltenyi Biotec | 130-042-201 | |
| Non-essential amino acids | Gibco | 11140-035 | |
| Penicillin and streptomycin (Pen-Strep) | Lonza | 15140-122 | |
| PermWash | BD Bioscience | 51-2091KZ | |
| PFA | Sigma | P6148 | |
| Phosphate buffered saline (PBS) | EuroClone | ECB4004L | |
| PMA | Sigma | P1585 | |
| Pre-Separation Filters (30 µm) | Miltenyi Biotec | 130-041-407 | |
| Recombinat Mouse IL-7 | R&D System | 407-ML-025 | |
| RPMI 1640 with glutamax | Gibco | 61870-010 | |
| sodium pyruvate | Gibco | 11360-039 | |
| TCRβ -APC | Biolegend | 109212 | clone H57-597 |
| αCD3CD28 mouse T activator Dynabeads | Gibco | 11452D | |
| β-mercaptoethanol | Gibco | 31350010 |
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