Aim:(5R)-5-hydroxytriptolide(LLDT-8)displayed anti-arthritis and anti-allogenictransplantation rejection activities in our previous studies.Here,we aim to furtherclarify the effect of LLDT-8 on the pro-inflammatory cytokine IFN-γ.Methods:Tcells were activated with anti-CD3 antibody or concanavalin A(ConA).The ex-pression of cell surface molecules was detected with flow cytometry.Cells werelabeled with carboxyfluorescein diacetate succinimidyl ester(CFSE)to test celldivision.IFN-γ production was determined by enzyme-linked immunosorbentassay.Cell proliferation was evaluated by[~3H]-thymidine uptake.Mice wereimmunized with ovalbumin to assess the in vivo immune response.RT-PCR andReal-time PCR were applied to determine the mRNA expression.The protein phos-phorylation levels were detected by Western immunoblot assay.Results:LLDT-8 at 100 nmol/L did not change the CD25,CD69,and CD154 expressions in anti-CD3-stimulated T cells.LLDT-8 markedly blocked the cell division of CD4 andCD8 T cells after ConA stimulation.LLDT-8 inhibited T cell-derived IFN-γproduction.Moreover,LLDT-8 suppressed the ovalbumin-specific T cell prolif-eration and IFN-γ generation.In anti-CD3-activated T cells,LLDT-8 abrogated themRNA expression of signal transducer and activator of transcriptionl(STAT1),T-box transcription factor,IL-12 receptor β2,STAT4,and interferon regulatory fac-tor 1 in the IFN-γ expression pathway.Western blot analysis showed that LLDT-8 blocked the phosphorylation levels of extracellular signal-regulated kinase,stress-activated protein kinase(SAPK)/c-Jun N-terminal kinase,and p38 mitogen-activated protein kinase in anti-CD3 plus anti-CD28-activated T cells.In addition,LLDT-8 reduced the transcripts of macrophage inflammatory protein(Mip)-1α,Mip-1β,regulated upon activation normally T-cell expressed and secreted,induc-ible protein-10,IFN-inducible T cell a chemoattractant,and monokine induced byIFN-7 in IFN-γ-stimulated murine macrophage cell line Raw 264.7 cells.Conclusion:LLDT-8 was a potential inhibitor for IFN-γ-associated signaling. Aim: (5R) -5-hydroxytriptolide (LLDT-8) displayed anti-arthritis and anti-allogenic transplantation rejection activities in our previous studies. Here, we aim to further Clarify the effect of LLDT-8 on the pro-inflammatory cytokine IFN-γ . Methods: Tcells were activated with anti-CD3 antibody or concanavalin A (ConA). The ex-pression of cell surface molecules was detected with flow cytometry. Cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) to test celldivision. was determined by enzyme-linked immunosorbent assay. Cell proliferation was evaluated as [~ 3H] -thymidine uptake. Mici were immunized with ovalbumin to assess the in vivo immune response. RT-PCR and Real-time PCR were applied to determine the mRNA expression. Phos-phorylation levels were detected by Western immunoblot assay. Results: LLDT-8 at 100 nmol / L did not change the CD25, CD69, and CD154 expressions in anti-CD3-stimulated T cells. CD4 andCD8 T cell s after ConA stimulation. LLDT-8 inhibited T cell-derived IFN-γ production. Moreover, LLDT-8 suppressed the ovalbumin-specific T cell prolif-eration and IFN- γ generation. abrogated themRNA expression of signal transducer and activator of transcription1 (STAT1), T-box transcription factor, IL-12 receptor β2, STAT4, and interferon regulatory fac- tor 1 in the IFN- γ expression pathway. Western blot analysis showed that LLDT- 8 blocked the phosphorylation levels of extracellular signal-regulated kinase, stress-activated protein kinase (SAPK) / c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase in anti-CD3 plus anti-CD28- activated T cells. addition, LLDT-8 reduced the transcripts of macrophage inflammatory protein (Mip) -1α, Mip-1β, regulated upon activation normally T-cell expressed and secreted, induc-ible protein-10, IFN-inducible T cell a chemoattractant, and monokine induced byIFN-7 in IFN-γ-stimulated murine macrophage cell line Raw 264.7 cells.Concl usion: LLDT-8 was a potential inhibitor for IFN-γ-associated signaling.