Effect of Interfering Tumor Microenvironment on Homing and Killing Activity of Cytotoxic T Lymphocytes
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摘要: 目的 探讨应用低剂量环磷酰胺（cyclophosphamide, CYC）干扰荷瘤小鼠体内微环境对改善过继回输的细胞毒性T淋巴细胞（cytotoxic T lymphocyte, CTL）瘤体内归巢和杀伤活性的作用。方法 建立C57BL/6荷瘤小鼠模型并分为对照组、CYC注入（CYCI）组、0.9%NaCl溶液注入（NSI）+CTL组和CYCI+CTL组，每组40只，分别给CYCI组、CYCI+CTL组每只小鼠腹腔注入CYC （20 mg/kg），NSI+CTL组小鼠注入等量0.9%NaCl溶液。检测注射前后不同时间小鼠瘤体内调节性T细胞（Treg）、白细胞介素-10（IL-10）和转化生长因子-β（TGF-β）水平变化。于注射后第4天回输CFSE-CTL，每只0.2 ml，含5×l06个细胞。流式检测对比NSI+CTL组和CYCI+CTL组瘤体内CFSE-CTL 比例。结果 NSI+CTL组小鼠Treg、IL-10、TGF-β水平随着瘤体增长而逐渐增高；CYCI+CTL组比NSI+CTL组回输的CFSE-CTL在肿瘤内归巢明显增加和持久（P<0.05）。各组肿瘤体积除对照组和CYCI组外，其余各组间差异均有统计学意义（P<0.05）。结论 提前应用低剂量CYC干预荷瘤小鼠体内微环境，可降低瘤体内Treg、IL-10和TGF-β水平，使过继回输的CTL在肿瘤中的归巢聚集明显增强，杀伤肿瘤效率提高。Abstract: Objective To explore the effect of interfering tumor microenvironment with low-dose cyclophosphamide (CYC) administration on homing and killing activity of adoptive transfused cytotoxic T lymphocytes in adoptive immunotherapy. Methods Melanoma-bearing mice models were established and randomly divided into four groups: control group, CYC injection group (CYCI), the solution of 0.9% w/v of NaCl injection plus CTL infusion group (NSI+CTL) and CYC injection plus CTL infusion group (CYCI+CTL), 40 cases in each group. CYC (20 mg/kg) were intraperitoneally injected in mice of CYCI group and CYCI+CTL group. The same amount of the solution of 0.9% w/v of NaCl was injected in mice of NSI+CTL group. The levels of Treg, TGF-β and IL-10 were detected at the different time points before and after CYC or the solution of 0.9% w/v of NaCl injection. The cultured CFSE-CTLs were transfused at the 4th day after injection, 5×l06 cells per mouse. Then, CFSE-CTLs ratio was analyzed by FCM. Results In NSI+CTL group, the levels of Treg, TGF-β and IL-10 were all increased continuously with tumor growth. CFSE-CTLs ratio in CYCI+CTL group was significantly higher and lasted longer than that in NSI+CTL group (P<0.05). There was significant difference among every two group, except control group and CYCI group(P<0.05). Conclusion Tumor microenvironment in tumor-bearing mice could be effectively intervened by low-dose CYC administration in advance, with decreased Tregs, IL-10, and TGF-β levels. As a result, more transferred CTLs homing leads to the strenthened killing efficacy.
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