Hoyle C, Bangs C D, Chang P, Kamel O, Mehta B, Negrin R S
Division of Bone Marrow Transplantation, Department of Medicine; the Department of Pathology, Stanford University Medical School, Stanford, CA, USA.
Blood. 1998 Nov 1;92(9):3318-27.
We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMNCs) by the timed addition of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and the monoclonal antibody (MoAb) OKT3. These cells, termed cytokine-induced killer (CIK) cells, are composed primarily of T cells, and the population of cells with the greatest cytotoxic activity is an otherwise rare population of CD3(+)CD56(+) cells that expand dramatically under these culture conditions. CIK cells were expanded from PBMNCs from 13 patients with chronic myeloid leukemia (CML). These cultures contained a variable number of T cells at the start of the culture (median 44%, range 1% to 64%), yet after 21 to 28 days of culture, virtually all of the cells were CD3(+) T cells (median 97%, range 90% to 99%). The CD3(+)CD56(+) subset of cells expanded significantly (median 25-fold, range 2.2- to 525-fold). CIK cells from all patients showed cytotoxicity against the tumor cell lines OCI-LY8 and K562. In four patients the expanded CIK cells suppressed colony growth of autologous CML blast cells and myeloid progenitor cells. Allogeneic CIK cells from normal donors also suppressed CML colony growth but did not inhibit growth of normal hematopoietic colonies. Twelve of the 13 cultures were exclusively composed of Philadelphia (Ph)-negative cells and one culture had 1 out of 20 Ph-positive metaphases after 4 weeks in culture. Intracellular cytokine production was assayed by fluorescence-activated cell sorter (FACS), and the expanded T-cell cultures produced IL-2, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha), but not IL-4. Both the CD4(+) and CD8(+) subsets secreted this cytokine profile. To test the in vivo activity of the expanded CIK cells, CML was engrafted into severe combined immunodeficiency disease (SCID) mice using matrigel. After 4 weeks, 4 x 10(7) autologous CIK cells were injected intravenously by tail vein injection into groups of mice, and the animals were sacrificed after a total of 18 weeks. Bcr-abl was detected in the bone marrow or spleen of 5 out of 6 control mice and only 2 out of 13 mice who received the autologous CIK cells (P = .02). In an additional series of animals, the mice did not engraft with CML but instead developed large human Epstein-Barr virus-associated lymphomas by 12 weeks. The mice who received autologous CIK cells at 4 weeks had either no tumor (5) or small tumors (5), whereas all 10 mice that received CIK cells at week 8 developed lymphomas; however, these were not as large as in the 10 control mice who did not receive CIK cells (P = . 03). This study shows that CIK cells, which are Ph chromosome-negative, can be expanded from patients with CML and have potent in vitro and in vivo efficacy against autologous tumor cells.
我们通过定时添加干扰素-γ（IFN-γ）、白细胞介素-2（IL-2）和单克隆抗体（MoAb）OKT3，开发了从外周血单个核细胞（PBMNCs）高效扩增细胞毒性效应细胞的培养条件。这些细胞被称为细胞因子诱导的杀伤（CIK）细胞，主要由T细胞组成，具有最大细胞毒性活性的细胞群体是在这些培养条件下能显著扩增的原本罕见的CD3(+)CD56(+)细胞群体。CIK细胞从13例慢性髓性白血病（CML）患者的PBMNCs中扩增得到。这些培养物在培养开始时含有数量不等的T细胞（中位数为44%，范围为1%至64%），但在培养21至28天后，几乎所有细胞都是CD3(+) T细胞（中位数为97%，范围为90%至99%）。CD3(+)CD56(+)细胞亚群显著扩增（中位数为25倍，范围为2.2至525倍）。所有患者的CIK细胞对肿瘤细胞系OCI-LY8和K562均表现出细胞毒性。在4例患者中，扩增的CIK细胞抑制了自体CML原始细胞和髓系祖细胞的集落生长。来自正常供体的同种异体CIK细胞也抑制了CML集落生长，但不抑制正常造血集落的生长。13个培养物中有12个完全由费城（Ph）阴性细胞组成，1个培养物在培养4周后20个中期相中出现1个Ph阳性中期相。通过荧光激活细胞分选仪（FACS）检测细胞内细胞因子的产生，扩增后的T细胞培养物产生IL-2、IFN-γ和肿瘤坏死因子-α（TNF-α），但不产生IL-4。CD4(+)和CD8(+)亚群均分泌这种细胞因子谱。为了测试扩增后的CIK细胞的体内活性，使用基质胶将CML移植到严重联合免疫缺陷病（SCID）小鼠体内。4周后，通过尾静脉注射将4×10(7)个自体CIK细胞静脉注射到小鼠组中，共18周后处死动物。在6只对照小鼠中的5只的骨髓或脾脏中检测到Bcr-abl，而在接受自体CIK细胞的13只小鼠中只有2只检测到（P = 0.02）。在另一系列动物中，小鼠未移植CML，而是在12周时发展为大型人类爱泼斯坦-巴尔病毒相关淋巴瘤。在4周时接受自体CIK细胞的小鼠要么没有肿瘤（5只），要么有小肿瘤（5只），而在第8周接受CIK细胞的所有10只小鼠都发展为淋巴瘤；然而，这些淋巴瘤没有未接受CIK细胞的10只对照小鼠中的淋巴瘤大（P = 0.03）。这项研究表明，Ph染色体阴性的CIK细胞可以从CML患者中扩增得到，并且对自体肿瘤细胞具有强大的体外和体内疗效。
