阻断B7／CD28及CD40／CD154共刺激信号对致敏小鼠免疫功能的影响

本研究旨在通过阻断致敏小鼠的B7／CD28及CIM0／CD154共刺激信号途径，探讨其免疫功能的变化，为应用于异基因骨髓移植的免疫耐受提供实验依据。将致敏的BALB／c小鼠分成4组：①CTLA4Ig＋anti—CD154鼠源性同型对照抗体；②anti—CD154单克隆抗体＋CTLA4Ig鼠源性同型对照抗体；③CTLA4Ig＋anti—CD154单克隆抗体；④CTLA4Ig及anti—CD154单克隆抗体的鼠源性同型对照抗体。正常BALB／c小鼠应用CTLA4Ig及anti—CD154单克隆抗体的鼠源性同型对照抗体。给予每只小鼠CTLA4Ig和anti—CD154单克隆抗体或相对应的同型对照抗体各500μg，于移植前7天尾静脉输注，每组小鼠各5只。移植当天处死各组小鼠，应用流式细胞仪检测脾细胞中CD19＋CD69＋B细胞数量、CD44high／CD62Lhigh／及CD44high／CD62Llow-T细胞数量。用流式细胞仪或ELISA法检测血清中抗体及细胞因子的变化。结果表明：小鼠致敏后CD19＋CD69＋B细胞数量与正常小鼠相比明显升高，差异有统计学意义（P〈0．01），阻断B7／CD28或／和CIM0／CD154共刺激信号通路后可见抑制效应（P〈0．01），两者同时阻断具有协同作用（P〈0．01）。小鼠致敏后记忆性T细胞CD44high／CD62Lhigh及效应性T细胞CD44high／CD62Llow/-与正常相比亦明显增多（P〈0．01）。阻断共刺激信号通路后可使其明显抑制，CTLA4Ig和anti-CD154单克隆抗体联合应用具有协同效应（P〈0．01）。各组小鼠细胞因子及IgG、IgM抗体均无明显差异（P〉0．05），但血清中致敏抗体检测显示致敏后特异性抗体升高，阻断共刺激信号通路后其明显被抑制（P〈0．01）。结论：阻断B7／CD28或CD40／CD154共刺激信号途径均能抑制致敏小鼠的细胞免疫功能及体液免疫功能；同时阻断有协同作用。

Influence of Blocking B7/CD28 and CD40/CD154 Co-stimulatory Signals on Immune Function of Sensitized Mice

This study was aimed to explore the effects of blocking B7/CD28 and CD40/CD154 co-stimulatory signals on immune function of sensitized mice＇, and provide the evidences of acquired immune tolerance for ailogeneic bone marrow transplantation. The mice sensitized on 7 day before transplant were divided into 4 groups ： （ 1 ） CTLA4Ig ＋ anti-CD154 isotype control IgG; （ 2 ） anti-CD154 ＋ CTLA4Ig isotype control IgG; （ 3 ） CTLA4Ig and anti-CD154 ; （ 4 ） isotype control IgG of CTLA4Ig and anti-CD154. CTLA4Ig and anti-CD154 used in normal BALB/c mice as isotype control lgG. Each mouse in all groups received CTLA4Ig and anti-CD154 （or corresponding isotype control IgG） 500 μg respectively, and was injected via tail vein on 7 day before transplant. There were 5 mice in each group. The mice were sacrificed on day 0, then the number of CD19 ＋ CD69 ＋ B cells, CD44high/CD62Lhigh and CD44high/CD62LloW/- T cells were measured by flow cytometry. Changes of cytokines and sensitized antibody were tested by ELISA or flow cytometry. The results showed that the numbers of CD19 ＋ CD69 ＋ B cells were significantly increased in comparison with the normal group （ P 〈 0.01 ）, whereas the numbers of cells were significantly decreased when blocking B7/CD28 or / and CD40/CD154 co-stimulatory signals（ P 〈0.01 ）. Blocking these 2 signals together displayed a synergistic effect（ P 〈 0. 01 ）. The central memory and effector T cells were defined as CD44high/CD62Lhigh and CD441aigh/CD62Llow/- respectively, those increased significantly after sensitized in comparison with those in normal group, whereas their numbers decreased when blocking B7/CD28 or/and CD40/CD154 co-stimulatory signals. Blocking these two signalstogether, displayed a synergistic effect （P 〈 0. 01 ）. Cytokines, IgG and IgM in all groups were not significantly different. Sensitizing antibody test showed that the fluores-cence intensity of sensitized group significantly increased as compared with normal group, whereas fluorescence indesity of CTLA4Ig or/and anti-CD154 treated groups significantly decreased as compared with sensitized group （ P 〈 0. 01 ）. It is concluded that blocking the B7/CD28 or/and CD40/ CD154 co-stimulatory signal can inhibit the cellulor and humoral immune function, whereas blocking these two signals together displays a synergistic effect.