Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells
Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells
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DOI：
10.3791/2540
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发表时间：
2011-02-01
影响因子：
1.2
通讯作者：
Lee, Dean A.
中科院分区：
文献类型：
--
作者：
Somanchi, Srinivas S.;Senyukov, Vladimir V.;Lee, Dean A.
自然杀伤（NK）细胞在针对多种感染性微生物和肿瘤的免疫监视中起重要作用。NK细胞的有限可用性和体外扩增能力限制了NK细胞免疫疗法的发展。在这里，我们描述了一种使用表达膜结合的IL 21的K562细胞作为人工抗原呈递细胞（aAPC）来有效地体外扩增大量功能性NK细胞的方法。NK细胞过继疗法迄今为止已经利用了通过供体的稳态白细胞去除术随后耗尽T细胞或NK细胞的阳性选择而获得的细胞产物。该产品通常在IL-2中活化过夜，然后在第二天给药（1）。由于NK细胞在外周血中的频率较低，因此在临床试验中递送的NK细胞数量相对较少，不能在体外增殖NK细胞一直是产生足够数量的细胞以获得最佳临床结果的限制因素。仅通过高剂量IL-2可实现NK细胞的一些扩增（1-2周内扩增5-10倍）（2）。自体T细胞的活化可以介导NK细胞扩增，推测也是通过释放局部细胞因子（3）。间充质基质或人工抗原呈递细胞（aAPC）的支持可以支持外周血和脐带血NK细胞的扩增（4）。通过抗体包被的珠粒的组合的NKp 46和CD 2活化目前已上市用于NK细胞扩增（Miltenyi Biotec，奥本CA），导致在21天内扩增约100倍。使用aAPC扩增或活化的NK细胞的临床试验正在进行中，其中一项使用白血病细胞系CTV-1来引发和活化NK细胞（5）而没有显著扩增。第二项试验利用EBV-LCL进行NK细胞扩增，在21天内平均扩增490倍（6）。第三种方法使用了基于K562的aAPC，其用4-1BBL（CD 137 L）和膜结合IL-15（mIL-15）转导（7），其在21天内实现了平均NK扩增277倍。尽管与未扩增的NK细胞相比，使用K562- 41 BBL-mIL 15 aAPC扩增的NK细胞在体外和体内具有高度细胞毒性，并且参与ADCC，但其增殖受到归因于端粒缩短的衰老的限制（8）。最近报道了使用表达云母、4-1BBL和IL 15的K562的NK细胞的350倍扩增（9）。本文所述的我们的NK细胞扩增方法产生NK细胞的快速增殖而不衰老，在21天内实现中值21，000倍扩增。
Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigenpresenting cell (aAPC).NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day (1). Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone (2). Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine (3). Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood (4). Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells(5) without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days(6). The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)(7), which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening(8). More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL15(9).Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.