Effects And Mode Of Action Of BM935, A Benzimidazole Derivative, On Inbition Of T Cell Proliferation

Objective: The purpose of the study was to explore the effects and mode of action ofBM935on inhibition of T cell proliferation.Methods:1. CD3⁺T cells were isolated by MACS system and the purity of T cells was detectedby using flow cytometry combined with fluorescently labeled anti-CD3.2. Human and mouse T cells were stimulated by mitogen or anti-CD3/CD28, and the Tcell proliferation was detected using CFSE labeling and flow cytometry.3. The absolute number of viable cells was counted by flow cytometry with PI staining.4. Cell cycle distribution and CD25expression in T cells were measured by flowcytometry.5. IL-2, IL-4, IL-6, IL-10, IL-17A and IFN-γ levels in anti-CD3/CD28-stimulatedhuman T cell culture supernatant were measured by ELISA.6. PBMC was cultured with IL-2to obtain high purity T cells. The effect of BM935onIL-2-stimulated T cell proliferation was measured by flow cytometry.7. Double staining method with Annexin and PI was used to detect the apoptotic anddead T cell.Results:1. The purity of human or mouse T cell isolated by MACS was more than95%.2. BM935significantly inhibited human and mouse T cell proliferation, stimulatedeither by mitogen or anti-CD3/CD28. The half inhibition concentration of BM935was between1and2micromolar.3. BM935did not affect the resting human or mouse T cell survival at the concentrationof inhibiting T cell proliferation.4. BM935inhibited cell proliferation by inducing cell cycle arrest at the G₀/G₁phase inactivated T cells. BM935did not affect T cell activtion and CD25expression. 5. BM935did not inhibit T cells to secrete IL-2, IL-4, IL-10, but inhibited T cells tosecrete IFN-γ, IL-6, IL-17A.6. A purity of more than95%of T cells can be obtained by culturing PBMC with IL-2for a long time. BM935can effectively inhibit IL-2-stimulated T cell proliferation.7. High concentration of BM935did not promote T cell apoptosis while inhibiting theIL-2-stimulated T cell proliferation. The proportions of apoptotic and dead cells inthe presence of BM935have no significant difference with positive control group.Conclusion:BM935selectively inhibits T cell proliferation without obvious cytotoxicity onresting T cell. BM935did not inhibit proliferation by inducing T cell apoptosis orinhibiting T cell activation. BM935can inhibit T cell proliferation by affectingdownstream signaling pathway of IL-2receptor and arresting cell cycle in activated Tcells. The effect of BM935on T cell cytokine profile was different with otherimmunosuppressants, suggesting BM935may have effect on a new target. Thus, BM935has the potential to be used as a lead compound for design and development of newimmunosuppressive agents.