Wallace P K, Palmer L D, Perry-Lalley D, Bolton E S, Alexander R B, Horan P K, Yang J C, Muirhead K A
Zynaxis Cell Science, Inc., Malvern, Pennsylvania 19355.
Cancer Res. 1993 May 15;53(10 Suppl):2358-67.
Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and lymphokine-activated killer cells has been demonstrated to mediate regression of tumors in murine models and in selected patients with advanced cancer. Improved methods for monitoring immune cell traffic, particularly to sites of tumor, are needed to elucidate mechanisms of antitumor activity and optimize treatment protocols. Traditional cell tracking methods such as fluorescent protein labeling and radiolabeling using 111In, 125I, or 51Cr are limited by isotope half-life, leakage or transfer of label from immune cells, and toxicity or altered cell function caused by the labeling process. Labeling with genetic markers allows long-term cell tracking but is laborious to perform and difficult to quantitate. We have used two recently described lipophilic cell tracking compounds (PKH26 and 125I-PKH95) which stably partition into lipid regions of the cell membrane to track immune cells in vivo. Concentrations of each tracking compound which had no adverse effects were determined for a variety of murine TIL and lymphokine-activated killer cell functions. Viability was unimpaired at labeling concentrations of up to 5 microM for PKH95 and 20 microM for PKH26. TIL proliferation was unaltered by labeling with up to 5 microM PKH95, 20 microM PKH26, or a combination of 15 microM PKH26 and 5 microM PKH95. In vivo cytotoxic effector function and in vivo therapeutic efficacy of lymphokine-activated killer cells and TIL were also unimpaired by labeling with 20 microM PKH26 or 1 microM 125I-PKH95. Subsequent studies in an adoptive transfer immunotherapy model used 125I-PKH95 to track the biodistribution of TIL in tumor and in non-tumor-bearing animals and PKH26 fluorescence to monitor microdistribution within tissues and distinguish TIL from host T-cells. The results suggest that differential accumulation, selective retention, or proliferation at the tumor site cannot account for the observed pattern of therapeutic efficacy. We hypothesize that a minimum number of TIL must reach the tumor site in order to achieve a demonstrable therapeutic effect.
采用肿瘤浸润淋巴细胞（TIL）和淋巴因子激活的杀伤细胞进行过继性免疫治疗已被证明在小鼠模型和部分晚期癌症患者中可介导肿瘤消退。需要改进监测免疫细胞转运的方法，尤其是肿瘤部位的转运，以阐明抗肿瘤活性机制并优化治疗方案。传统的细胞追踪方法，如荧光蛋白标记和使用铟 - 111、碘 - 125或铬 - 51进行放射性标记，受到同位素半衰期、标记从免疫细胞的泄漏或转移以及标记过程导致的毒性或细胞功能改变的限制。用遗传标记进行标记可实现长期细胞追踪，但操作繁琐且难以定量。我们使用了两种最近描述的亲脂性细胞追踪化合物（PKH26和125I - PKH95），它们稳定地分配到细胞膜的脂质区域以在体内追踪免疫细胞。针对多种小鼠TIL和淋巴因子激活的杀伤细胞功能，确定了每种追踪化合物无不良影响的浓度。对于PKH95，标记浓度高达5微摩尔/升以及对于PKH26高达20微摩尔/升时，细胞活力未受损害。用高达5微摩尔/升的PKH95、20微摩尔/升的PKH26或15微摩尔/升的PKH26与5微摩尔/升的PKH95组合进行标记，TIL增殖未改变。用20微摩尔/升的PKH26或1微摩尔/升的125I - PKH95进行标记，淋巴因子激活的杀伤细胞和TIL的体内细胞毒性效应功能及体内治疗效果也未受损害。随后在过继性转移免疫治疗模型中的研究使用125I - PKH95追踪TIL在肿瘤和无肿瘤动物中的生物分布，并使用PKH26荧光监测组织内的微观分布并区分TIL与宿主T细胞。结果表明，肿瘤部位的差异积累、选择性滞留或增殖不能解释观察到的治疗效果模式。我们假设必须有最少数量的TIL到达肿瘤部位才能实现可证明的治疗效果。
