摘要
目的探讨可溶性程序性死亡配体1(sPD—L1)对人T淋巴细胞增殖的影响及其机制。方法健康人外周血中分离得到T淋巴细胞,用植物血凝素(PHA)活化T淋巴细胞;实验分为5组:A:静止T淋巴细胞组、B:活化T淋巴细胞组、C:活化T淋巴细胞+PD-L1融合蛋白(sPD—L11g)组、D:活化T淋巴细胞+sPD-L1Ig+同型对照抗体(mIgG)组、E:活化T淋巴细胞+sPD—L1Ig+鼠抗人程序性死亡配体1(PD—L1)阻断型抗体(2H11)组。通过细胞计数试剂盒(CCK一8法)测定各组T淋巴细胞的吸光度(A)值,流式细胞术检测sPD—L1对T淋巴细胞凋亡和细胞周期的影响,Western印迹法分析程序性死亡受体1(PD-1)信号传导基序酪氨酸磷酸化程度,免疫沉淀技术进一步分析信号接头分子蛋白酪氨酸磷酸酶(SHP).1和SHP-2的募集量,探讨sPD—L1激发PD-1抑制信号的机制。结果CCK-8法体外实验结果显示:在sPD—L1Ig浓度为250ng/ml时,A~E组A值分别为0.42±0.03、1.20±0.06、0.87±0.05、0.78±0.05、1.11±0.09;C组显著低于B组(t=3.946,P=0.017),E组显著高于D组(t=3.139,P=0.035)。A~E组G,期细胞百分比分别为(94.49±0.50)%、(79.22±0.50)%、(89.62±0.33)%、(92.89±0.80)%、(87.94±0.87)%,C组显著高于B组(t=17.310,P〈0.001)。各组细胞凋亡率分别为(35.77±1.82)%、(35.20±2.70)%、(62.77±0.24)%、(64.47±0.44)%、(36.80±3.53)%;C组显著高于B组(t=10.160,P=0.001),E组显著低于D组(t=7.790,P=0.002)。SHP-1、SHP-2在各组细胞中的表达量差异均无统计学意义(均P〉0.05)。C组磷酸化SHP-1(p-SHP-1)、p-SHP-2表达量均显著高于B组(t=10.790,P〈0.001;t=13.051,P〈0.001);E组p-SHP.1显著低于D组(t=3.361,P=0.028)。结论sPD—L1可有效抑制T淋巴细胞的增殖,PD-1/sPD—L1的抑制信号可通过磷酸化SHP-1、SHP-2发挥作用,PD—L1阻断型抗体2H11可通过抑制19-SHP-1的表达部分恢复T淋巴细胞的增殖能力。
Objective To explore the effects of soluble programmed death ligand 1 (sPD-L1) on the proliferation of T lymphocytes and its mechanism. Methods T lymphocytes were isolated from healthy human peripheral blood and activated by phytohemagglutinin (PHA). The experiment had group A: resting T lymphocytes, group B : activated T lymphocytes, group C : activated T lymphocytes + sPD-L1Ig, group D : activated T lymphocytes + sPD-Lllg + membrance-bound immunoglobulin (mIgG) and group E: activated T lymphocytes + sPD-L1Ig + anti-PD-L1 antibody ( 2H11 ). The absorbance value (A) of T lymphocytes in each group was measured by cell counting kit ( CCK-8 ). The cell cycle and apoptosis of T lymphocytes induced by sPD-L1 were measured by flow cytometry. And the phosphorylation level of programmed death 1 ( PD-1 ) signaling motif tyrosine was measured by Western blot. Furthermore, the amounts of signal adaptor molecule Src homology 2 domain-containing tyrosine phosphatase (SHP)-I and SHP-2 were quantified by immunoprecipitation. And the exciting mechanism of sPD-L1 was explored for PD-1 inhibitory signals. Results CCK-8 study showed that A values in each group were 0. 42± 0. 03, 1.20± 0.06, 0. 87 ± 0. 05, 0. 78 ±0. 05 and 1.11 ± 0. 09 respectively when the concentration of sPD-L1Ig was 250 ng/ml. The proliferation of T lymphocytes in group C significantly decreased compared with group B ( t = 3. 946, P = 0.017) while group E significantly increased compared with group D (t = 3. 139, P =0.035). The percentage of cell number in G1 phase of the above-mentioned 5 groups were (94.49 +0. 50) %, (79. 22 + 0.50)%, (89.62 ±0.33)%, (92.89 ±0.80)% and (87.94 ±0.87)% respectively and group C significantly increased compared with group B ( t = 17. 310, P 〈 O. 001 ). The apoptotic rate of the abovementioned five groups were (35.77 ± 1.82)%, (35.20 ±2.70)%, (62.77 ±0.24)%, (64.47± 0.44)% and (36.80 ± 3.53)% respectively. And apoptotic rate in group C significantly increased compared with group B (t = 10. 160, P =0. 001 ) while group E significantly decreased compared with group D (t = 7. 790, P = 0. 002). The expressions of SHP-1 and SHP-2 showed no inter-group difference (all P 〉 O. 05). However, the expressions of p-SHP-1 and p-SHP-2 in group C was higher than those in group B (t = 10. 790, P 〈 0. 001 ; t = 13. 051, P 〈 0. 001 ) while the expression of p-SHP-1 decreased in group E compared with group D (t = 3. 361, P = O. 028). Conclusions Soluble PD-L1 can effectively inhibit the proliferation of T lymphocytes. The phosphorylation of SHP-1 and SHP-2 contributes to the inhibitory signaling of PD-1/sPD-L1 pathway. And anti-PD-L1 blocking antibody may partially restore the proliferation of T lymphocytes through a down-regulated expression of p-SHP-1.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2015年第6期449-452,共4页
National Medical Journal of China
基金
国家自然科学基金(81272610)
苏州市科技发展计划(SYS201467)