可溶性程序性死亡配体1对T淋巴细胞增殖的影响及机制

Effects of soluble programmed death ligand 1 on regulating the proliferation of T lymphocytes and its mechanism

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.