Huang Shao-liang, Chen Chun, Duan Lian-ning, Li Hao-wei, Wen Guan-mei, Li Lin, Zhan Mei-yi, Wei Jing
Department of Pediatrics, the Second Affiliated Hospital, Sun Yat-sen University, Guangzhou 510120, China.
Zhonghua Xue Ye Xue Za Zhi. 2003 Jun;24(6):290-4.
To investigate the effect and its mechanism of reducing graft-versus-host disease (GVHD) by in vitro blockade of CD(40)-CD(40)L pathway in vitro, the donor T lymphocytes cultured in vitro with anti-CD(40)L mAb were transfused in bone marrow transplantation (BMT) GVHD mouse model.
C57BL/6(H-2b) spleen T cells were isolated as responder cells, and BALB/c(H-2d) spleen cells as stimulator cells. They were cocultured with or without Anti-CD(40)L mAb as anti-CD(40)L mAb group and control group, respectively. At day 5, the mixed lymphocyte response (MLR)-culture cells mixed with bone marrow cells and transfused respectively into the TBI conditioned recipient mice. The mice were divided into two groups: group A, bone marrow cells (2 x 10(6)) and spleen T lymphocytes (2 x 10(6)) from MLR control group; group B, bone marrow cells (2 x 10(6)) and spleen T lymphocytes (2 x 10(6)) from MLR anti-CD(40)L mAb group. The GVHD incidence and hematopoietic reconstitution were observed. Peripheral blood sera and spleen cells of the recipients mice were harvested at scheduled time points for the measurement of cytokines and T cell immunophenotyping with flow cytometry.
The incidence of GVHD in group A was 100% (10/10), and in group B was 20% (2/10). The percentage of H-2D(b) positive cells in group B (n = 8) was (93.54 +/- 2.32)% at day 40 after transplantation. The levels of cytokines in serum from group B were significantly lower than those from group A (P < 0.05). The expressions of CD(4)(+), CD(8)(+), CD(4)(+)CD(25)(+), CD(8)(+)CD(25)(+), CD(4)(+)CD(69)(+), CD(8)(+)CD(69)(+) and CD(4)(+)CD(40)L(+) were lower in group B than in group A (P < 0.05). The expressions of CD(8)(+)CD(40)L(+) and CD(4)(+)CD(45)RA(+) were similar in the two groups (P > 0.05).
Blockade of CD(40)-CD(40)L interaction in vitro could induce immune tolerance in vivo, reduce aGVHD in aGVHD mice model and form chimerism, which was mediated by inhibiting the Th1 and Th2 cytokines production, inducing tolerance of CD(4)(+) and CD(8)(+) cells to alloantigens. The obstruction of T cells activation after tolerance happened mainly at the early and mature phase of T cells activation. These provided the experimental basis for the use of anti-CD(40)L mAb in the clinical transplantation to prevent aGVHD.
通过体外阻断CD(40)-CD(40)L途径研究其减轻移植物抗宿主病（GVHD）的作用及机制，将体外培养的抗CD(40)L单克隆抗体处理的供体T淋巴细胞输入骨髓移植（BMT）GVHD小鼠模型。
分离C57BL/6(H-2b)脾T细胞作为反应细胞，BALB/c(H-2d)脾细胞作为刺激细胞。分别在有或无抗CD(40)L单克隆抗体的情况下共培养，分别作为抗CD(40)L单克隆抗体组和对照组。第5天，将混合淋巴细胞反应（MLR）培养细胞与骨髓细胞混合后分别输入经全身照射预处理的受体小鼠。小鼠分为两组：A组，来自MLR对照组的骨髓细胞（2×10(6)）和脾T淋巴细胞（2×10(6)）；B组，来自MLR抗CD(40)L单克隆抗体组的骨髓细胞（2×10(6)）和脾T淋巴细胞（2×10(6)）。观察GVHD发生率和造血重建情况。在预定时间点采集受体小鼠外周血血清和脾细胞，用流式细胞术检测细胞因子和T细胞免疫表型。
A组GVHD发生率为100%（10/10），B组为20%（2/10）。移植后第40天，B组（n = 8）中H-2D(b)阳性细胞百分比为(93.54±2.32)%。B组血清中细胞因子水平显著低于A组（P < 0.05）。B组中CD(4)(+)、CD(8)(+)、CD(4)(+)CD(25)(+)、CD(8)(+)CD(25)(+)、CD(4)(+)CD(69)(+)、CD(8)(+)CD(69)(+)和CD(4)(+)CD(40)L(+)的表达低于A组（P < 0.05）。两组中CD(8)(+)CD(40)L(+)和CD(4)(+)CD(45)RA(+)的表达相似（P > 0.05）。
体外阻断CD(40)-CD(40)L相互作用可在体内诱导免疫耐受，减轻急性移植物抗宿主病（aGVHD）小鼠模型中的aGVHD并形成嵌合体，其机制是通过抑制Th1和Th2细胞因子产生，诱导CD(4)(+)和CD(8)(+)细胞对同种异体抗原的耐受。耐受发生后T细胞活化的阻断主要发生在T细胞活化的早期和成熟阶段。这些为临床移植中使用抗CD(40)L单克隆抗体预防aGVHD提供了实验依据。
