Lind D S, Tuttle T M, Bethke K P, Frank J L, McCrady C W, Bear H D
Division of Surgical Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.
Surg Oncol. 1993 Oct;2(5):273-82. doi: 10.1016/s0960-7404(06)80002-2.
Current adoptive immunotherapy strategies in cancer patients require large numbers of activated T-cells and are limited by the availability of autologous tumour. We describe a novel method of T-cell activation that produced relatively rapid, high-fold expansion of stored, frozen lymphocytes obtained from the lymph nodes of 20 breast cancer patients during axillary dissection but does not require autologous tumour. In vitro exposure of thawed cells to bryostatin-1 (B), a non-tumour promoting protein kinase C activator and ionomycin (I), a calcium ionophore, at day 0 followed by culture in low dose interleukin-2 (IL-2 20 units ml-1) and restimulation again on day 10 results in 269-28,206 fold (geometric mean = 2254) expansion in cell numbers counted 17 days after initial stimulation. Analysis of cell surface markers revealed that B/I expanded human cells were predominantly T-cells (83-97%) and consisted of a mixture of CD8+ (46-74%) and CD4+ (4-30%) cells. B/I expanded cells did not lyse autologous tumour cells when tested in a 4-h 51Cr release assay, but murine studies reported previously have demonstrated specific and curative in vivo efficacy in MCA-105 tumour-bearing mice despite an inability to lyse autologous tumour in vitro. B/I expanded T-cells from five of six patients secreted the cytokines tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in response to co-culture with autologous tumour cells but not with irrelevant tumour. These results are analogous to findings in a murine model, in which non-cytolytic B/I expanded T-cells mediated specific, curative anti-tumour effects in vivo, and lay the groundwork for a clinical trial of this novel strategy for the adoptive immunotherapy of breast cancer patients.
目前癌症患者的过继性免疫治疗策略需要大量活化的T细胞，并且受到自体肿瘤可用性的限制。我们描述了一种新型的T细胞活化方法，该方法能使从20例乳腺癌患者腋窝清扫术中获取的储存、冷冻淋巴细胞实现相对快速、高倍数的扩增，但不需要自体肿瘤。在第0天将解冻的细胞体外暴露于苔藓抑素-1（B）（一种非促肿瘤的蛋白激酶C激活剂）和离子霉素（I）（一种钙离子载体），随后在低剂量白细胞介素-2（IL-2，20单位/毫升）中培养，并在第10天再次进行刺激，在初始刺激后17天计数细胞数量扩增了269 - 28206倍（几何平均值 = 2254）。细胞表面标志物分析显示，B/I扩增的人细胞主要是T细胞（83 - 97%），由CD8 +（46 - 74%）和CD4 +（4 - 30%）细胞混合组成。在4小时的51Cr释放试验中测试时，B/I扩增的细胞不裂解自体肿瘤细胞，但先前报道的小鼠研究表明，尽管在体外无法裂解自体肿瘤，但在携带MCA - 105肿瘤的小鼠中具有特异性和治愈性的体内疗效。来自六名患者中的五名患者的B/I扩增T细胞在与自体肿瘤细胞共培养而非与无关肿瘤细胞共培养时，分泌细胞因子肿瘤坏死因子-α（TNF-α）和干扰素-γ（IFN-γ）。这些结果类似于在小鼠模型中的发现，即非细胞溶解性的B/I扩增T细胞在体内介导特异性、治愈性的抗肿瘤作用，为这种用于乳腺癌患者过继性免疫治疗的新策略的临床试验奠定了基础。
