The study of the role of blockade of CD40 ligand-CD40 costimulatory pathway on T lymphocytes typing and cytokines

Objective:To investigate the role of blockade of CD40 ligand-CD40 costimulatory pathway by anti-CD40L mAb on T lymphocytes typing and cytokines, and provide the experimental clues of inducing donor-specific T cell anergy.Methods:To monitor primary MLR, C57BL/6 H-2b spleen T cells were isolated as responder cells, and BALB/C H-2d spleen cell as stimulator cells. Anti-CD40L mAb was added to primary MLR cultures(termed anti-CD40L mAb group), and in control group(no anti-CD40L mAb). Incubated for 7 days, the cells responsiveness rates were detected by 3 H-TdR methods at the indicated time points, and supernatants were assayed for IFN-γ,IL-2,IL-4,IL-10 cytokines levels by commercial ELISA kits. Day 5 MLR-cultured cells were assessed for the expressions of CD4, CD8, CD25, CD69, CD40L and CD45RA with flow cytometry(FCM).Cells proliferation for 5 days and cytokines in secondary MLR were assayed according above-mentioned method.Results:Blockade of the CD40-CD40L pathway by anti-CD40L mAb can induce the hyporesponsiveness to alloantigen in primary and secondary MLR culture. In primary MLR culture, the expressions of CD4 +T and CD8 +T cells and CD4 +CD25 +T, CD4 +CD69 +T, CD4 +CD40L +T, CD8 +CD25 +T and CD8 +CD69 +T cells in anti-CD40L mAb group were lower than that in control group ( P 0.05). The expression of CD8 +CD40L +T were similar in two groups( P 0.05).The expression of CD4 +CD45RA + T in anti-CD40L mAb-cultured group was higher than that in control group( P 0.05). The levels of IL-4 and IL-10 in supernatant of the primary MLR culture were undetectable in two groups. The levels of IFN-γ and IL-2 in supernatants of the anti-CD40L mAb-cultured primary MLR were significantly lower than that of the control group ( P 0.01). The levels of IFN-γ 、 IL-2 and IL-4、IL-10 in supernatants of the anti-CD40L mAb-cultured secondary MLR were lower than that of the control group ( P 0.05).Conclusion:In a MLR system,anti-CD40L mAb cocultured with donor spleen T cells could act on the CD4 + and CD8 +T cells, decrease the expressions of CD40L +, CD25 + and CD69 + and the proliferation of T cells, interfere the early activation and maturation of T cells, and mainly inhibit the Th_1 cytokines and the Th_2 cytokines production.These indicated donor T cell tolerance was induced in vitro.