Exley Mark A, Wilson Brian, Balk Steven P
Cancer Biology Program, Hematology-Oncology Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
Curr Protoc Immunol. 2010 Aug;Chapter 14:Unit 14.11. doi: 10.1002/0471142735.im1411s90.
This unit details methods for the isolation, in vitro expansion, and functional characterization of human iNKT cells. The term iNKT derives from the fact that a large fraction of murine NKT cells recognize the MHC class I-like CD1d protein, are CD4+ or CD4-CD8- (double negative), and use an identical "invariant" TCRalpha chain, which is generated by precise Valpha14 and Jalpha281 (now renamed Jalpha18) rearrangements with either no N-region diversity or subsequent trimming to nearly identical amino-acid sequence (hence, 'iNKT'). Basic Protocol 1 and Alternate Protocol 1 use multi-color FACS analysis to identify and quantitate rare iNKT cells from human samples. Basic Protocol 2 describes iNKT cell purification. Alternate Protocol 2 describes a method for high-speed FACS sorting of iNKT cells. Alternate Protocol 3 employs a cell sorting approach to isolate iNKT cell clones. A Support Protocol for secondary stimulation and rapid expansion of iNKT cells is also included. Basic Protocol 3 explains functional analysis of iNKT.
本单元详细介绍了人类iNKT细胞的分离、体外扩增及功能特性鉴定方法。iNKT这一术语源于以下事实：大部分小鼠NKT细胞识别MHC I类样CD1d蛋白，为CD4+或CD4-CD8-（双阴性），并使用相同的“恒定”TCRα链，该链由精确的Vα14和Jα281（现重新命名为Jα18）重排产生，无N区多样性或随后修剪为几乎相同的氨基酸序列（因此称为“iNKT”）。基本方案1和替代方案1使用多色荧光激活细胞分选（FACS）分析从人类样本中鉴定和定量稀有iNKT细胞。基本方案2描述了iNKT细胞纯化方法。替代方案2描述了一种iNKT细胞的高速FACS分选方法。替代方案3采用细胞分选方法分离iNKT细胞克隆。还包括一个iNKT细胞二次刺激和快速扩增的支持方案。基本方案3解释了iNKT的功能分析。
