可溶性CD160分子对荷瘤小鼠CD4＋T细胞免疫活性的调节作用

Regulatory effect of soluble CD160 on the immunoactivity of CD4＋T cells from the tumor - bearing mice

目的观察CDl60胞外段（即可溶性CD160分子）对荷瘤小鼠CD4＋T淋巴细胞的活悱和饱疫功能的影响。方法构建小鼠CD160分子胞外段（sCD160）的重组表达载体psCDl60，转让『j1Ij4仓鼠卵巢（CHO）细胞，Westernblot检测培养上清中的sCDl60水平。建立TC-1宫颈癌细胞简蜊小鼠模型，流式细胞术（FCM）检测CDl60存脾CD4＋T细胞上的表达；免疫磁珠分选荷瘤3周小鼠脾CD4＋T细胞，与负载肿瘤细胞抗原肽复合物的树突状细胞（DC）共培养6d，FCM检测CD4＋T细胞tCDl60的表达。负载抗原肽复合物的DC分别在转染pcDNA3．1或psCDl60的CHO培养卜清中孵育18h后，与分选的CD4＋T细胞共培养6d，羧基荧光素二醋酸盐琥珀酰亚胺脂（CFSE）标i0法枪测CD4＋T细胞增殖，酶联免疫吸附实验（ELISA）检测培养上清中白细胞介素-2（IL-2）和γ-干扰素（IFN-1）的浓度。结果转染psCDl60的CHO细胞表达并分泌可溶性CDl60分子。荷瘤3删小鼠CD4＋T细胞上CDl60分子的阳性率与对照组比较差异无统计学意义[（5．288±2．723）％比（3．213±1．461）％，P〉0．05]，在体外被负载特异性抗原肽复合物的DC再活化后，其CD160阳性著增高达（17．590±7．287）％（P〈0．05）。与未经sCD160作用的DC组相比，经sCD160封闭的负载特异性抗原肽复合物的DC再活化的CD4＋T细胞中增殖细胞百分数[（17．980±5．337）％比（12．610±4．431）％，P〈0．05]、上清巾的IL-2和IFN-γ浓度[（383．9±83．67）ng／L比（237．5±54．64）ng／L，（730．8±135．8）ng／L比（451．9±149．5）ng／L，P〈0．05]均显著升高。结论肿瘤特异性CD4＋T细胞在体外冉活化后CD160的表达升高，可溶性CD160阻断其作用可有效增强CD4＋T细胞增殖和细胞因子分泌的活性。

Objective To investigate the effects of CD160 extracellular domain （namely soluble CD160） on the immunoaetivity of CD4 ＋ T cells from the tumor - bearing mice. Methods Recombinant ex- pression vector psCDl60, expressing CD160 extracellular domain （sCD160）, was constructed and trans- leered into CHO cells, and the sCDI60 expression of CHO cells transfeeted with psCD160 was verified by Vcestern blotting. The expression of CD160 on splenetic CD4＋T ceils was detected by flow cytometry （ FCM ） after C57BIV6 mice were inoculated with TC - 1 cervical cancer cells. CD4 ＋T cells were separated from splenocytes with immunomagnetic beads 3 weeks after the inoculation of TC - 1 cells and were co- cullured with dendritic cells （DCs） for 6 days, which were loaded with tumor cells peptide complex, then FCM was performed to detect CD160 on CD4 ＋T ceils. The sorted CD4 ＋T cells were co -cultured with DCs from four different treatment groups for 6 days, which were loaded separately with different tumor cells peptide complex in the presence of culture supernatants of CHO cells transfected with pcDNA3. 1 or psCD160. The proliferation of CD4 ＋T cells was assayed by carboxyfluorescein sueeinimidyl amino ester （CFSE） labelling in combination with flow cytometry, and the concentrations of interleukin - 2 （ IL - 2 ） and γ-interferon （IFN- γ） were measured by enzyme linked immunosorbent assay （ELISA）. Results Soluble CD160 was expressed and secreted into culture supernatants of CHO cells transfected with psCl）160. On the week 3 after the inoculation with TC - 1 cells, the positive percentage of CD160 on CD4＋ T cells of tumor - bearing mice had no statistically significant difl＇erence as compared with that of control mice [ （5. 288 ± 2. 723 ） % vs. （ 3. 213 ± 1. 461 ） %, P 〉 0.05 ]. After reactivation by DCs loaded with specific antigen complex, the positive percentage＇of CD160 on CD4＋T cells was significantly rose to （ 17. 590 ± 7. 287） % （P 〈 0. 05 ）. Compared with those of CD4 ＋ T cells activated by DCs in the absence of sCD160, the proliferation percentage [ （ 17. 980 ± 5. 337 ） % vs. （ 12. 610 ±4. 431 ） %, P 〈 0. 05 ] and the concentrations of IL - 2 and IFN - γ [ （383.9 ± 83.67） ng/L vs. （237.5 ±54.64） ng/L, （730.8 ± 135.8） ng/L vs. （451.9±149.5 ） ng/L, P 〈 0.05 ] in supernatants of CD4＋T cells activated by DCs loaded with specific antigen complex in the presence of sCD160 significantly increased. Conclusion After reactivation in vitro, tumor specific CD4＋T cells up -regulate the expression of CD160. Soluble CD160, by blocking the pathway of CD160, enhances the activities on proliferation and secretion of CD4＋T cells.