Study Of BET Inhibitor I-BET151in Prophylaxis Of Acute Graft-versus-host Disease

Part: Impact of I-BET151on Bone Marrow Dendritic Cells(BMDCs) and T cellsObjective: To study the impact of I-BET151on BMDCs and T cells in vitro.Methods:1). Bone marrow dendritic cells (BMDCs) were generated fromC57BL/6(B6, H-2b) mice. Bone marrow cells were isolated from mouse femurs and tibiasand cultured in RPMI supplemented with10%FCS,4mM L-glutamine,10U/ml penicillin,100μg/ml streptomycin,0.5mM2-ME and20ng/mL recombinant murine GM-CSF for7days. CD11c+cells were isolated with autoMACS cell separation system using CD11cmicrobeads. Cells purity was determined by FACS analysis.2). Impact of I-BET151onBMDCs viability. BMDCs were incubated with DMSO or I-BET151at250nM,500nM,1μM and5μM for4h,8h or24h. Apoptosis in BMDCs was detected using the7-aminoactinomycin D and Annexin V kit.3). Effect of I-BET151on the expression ofsurface costimulatory molecules and MHC II on BMDCs. BMDCs from B6wereincubated with DMSO or I-BET151for6hours with or without LPS and then wereexamined cell surface expression of CD40, CD80, CD86, PDL1and MHC II through flowcytometry.4). Effect of I-BET151on the cytokine production profile of BMDCs that aretriggered by LPS. After concomitant treatment of BMDCs with DMSO/I-BET151and250ng/ml LPS, culture supernatants were collected and stored at-20°C until analysis.Concentrations of cytokines, like IL-1β, IL-6, IL-10, IL-12and TNF-α in samples weremeasured by Enzyme-linked Immunosorbent Assay. Or, after3h stimulation with LPS,total RNA was extracted using RNeasy Mini Kit and first-strand cDNA was synthesizedusing the High Capacity cDNA Reverse Transcription Kits. Real-time PCR was performedwith a SYBR Green PCR mix on a Mastercycler realplex to determine the relative mRNAexpression levels of corresponding genes.5). Effect of I-BET151on BMDCs asstimulators of naive T cells. Mixed leukocyte reaction (MLR): splenic T cells (1×105/well) were magnetically purified from BALB/c (H-2d) mice by autoMACS using CD90.2microbeads and subsequently co-cultured with irradiated (25Gy) B6BMDCs for96hours.BMDCs were first preincubated with I-BET151or DMSO for6hours, thoroughly washedand then used as stimulators in the assay. Cells were pulsed with3H-thymidine for the last16-hour and proliferation was determined on a TopCount NTX. Or using CFSE labeled Tcells in the MLR system, proliferation was determined by FACS analysis. Apoptosis of Tcells was detected using Annexin V.6). Effect of I-BET151on T cell proliferation andcytokine productions. CFSE-labeled BALB/c T cells were treated with CD3/CD28antibodies and250nM I-BET151/DMSO for2days and analyzed by CFSE dye dilution ordetermined on TopCount NTX after pulsed with3H-thymidine. Concentration of cytokines(IFN-γ, IL-2, IL-4, IL-10and IL-17) in supernatant were measured by ELISA.Results:1). After7days of proliferation in vitro and isolated with CD11c microbeads,probably25×106cells could be harvested from each mouse and purity could reach96%.2). I-BET151influence the viability of BMDCs in a dose and time dependent manner.Treatment of BMDCs with I-BET151up to1μM for8hours did not induce greatapoptosis. However, when the drug is used at higher concentrations for24hours, asignificant amount of cell death could be observed,40%vs15%for1μM I-BET151andDMSO, respectively.3). Treatment with I-BET151markedly reduced the surfaceexpression of CD40, CD80, CD86, PDL1and MHC-II in a dose dependent mannerwhether the BMDCs are in resting status or activated by LPS, and CD86down regulationis more significant compared with others.4). I-BET151significantly inhibited theproinflammatory cytokines production of BMDCs triggered by LPS and the effects ofI-BET151were somewhat selective. IL-12concentrations were potently reduced while theinhibition effect on TNF-α production was relatively moderate.5). After pretreatment withI-BET151, the ability of BMDCs to induce proliferation of allogeneic T cells wassignificantly reduced. Compared to control group, no significant elevation in apoptosis wasobserved.6). I-BET151significantly reduced T cell proliferation when activated byCD3/CD28antibodies. It also suppressed the production of IL-17and IFN-γ, but increasedIL-4and IL-2expression. Importantly, I-BET151at250nM in these experiments did notincrease T cells apoptosis.Conclusion: I-BET151can induce apoptosis of BMDCs in a dose and time dependentmanner. However, pretreatment with1μM I-BET151for8hours did not induce great apoptosis. I-BET151can profoundly impair BMDCs immunostimulatory capacity:decrease the expression of major histocompatibility complex and a panel of costimulatorymolecules, inhibit cytokines secretion and reduced the ability of BMDCs to induceproliferation of allogeneic T cells. I-BET151can inhibit T cell expansion directly and alterT cells cytokine expression.Part П: Effect of I-BET151on acute graft-versus-host diseaseObjective: To examine whether short-term administration of I-BET151at the time oftransplantation could affect the severity of aGVHD and subsequent mortality.Methods:1). Using two mice myeloablative hematopoietic stem cells transplantation(HSCT) models, BALB/c (H-2d)→B6(H-2b) and B6(H-2b)→BALB/c (H-2d), to verifywhether administration of I-BET151alleviates the severity of GVHD.2). In B6(H-2b)→BALB/c (H-2d) model, assess the levels of proinflammatory cytokines, donor CD4+, CD8+as well as Treg cell proportion in the spleens, and aGVHD histopathologic severity of skin,bowel and liver of recipients after HSCT at day7and14.Results:1). I-BET151administration (20mg/kg) from day-1to day5was safe in thesetting of ablative conditioning and syngeneic SCT, demonstrating no impairment ofhematopoietic engraftment and other significant toxicities. I-BET151treated mice showedsignificantly alleviated aGVHD and improved survival rate compared to recipients thatreceived only vehicle.2). At day7, I-BET151treatment significantly decreased IL-6andTNF-α level in serum and donor-derived T cell numbers in spleen compared with controlgroup, however, donor chimerism, T cell activation and Treg cell proportion were similarbetween2groups. Meanwhile, I-BET151treated allogeneic animals showed significantlyreduced histopathological damage in the small and large bowel, as well as liver.Conclusion: Short-term administration of I-BET151at the time of transplantationreduced serum levels of the proinflammatory cytokines, decreased allo T cells proliferation,as well as clinical severity and mortality from GVHD in murine HSCT models comparedwith vehicle-treated mice. I-BET151had no impairment of hematopoietic engraftment. Part ПI: Effect of I-BET151on NF-κB pathway in BMDCs and TcellsObjective: To explore the potential common molecular pathways for the regulatoryeffects of I-BET151in BMDCs and T cells.Methods:1). Whole cell lysates were prepared from purified BMDCs treated with orwithout LPS (250ng/ml) and I-BET151(500nm). Signalling downstream of Toll-likereceptor4was evaluated by analysis of the kinetics and amplitude of LPS-induced IκBαdegradation and ERK phosphorylation.2). After stimulation with LPS for6hours,expression of BRD4, RelA and Acetyl-310RelA in DMSO or I-BET151treated BMDCswere detected by Western Blot.3). Disruption of interaction between acety-310RelA andBRD4with I-BET151in BMDCs was evaluated by immunoprecipitation andimmunoblotting analysis.4). Simlar experiments were repeated in T cells after stimulationwith CD3/CD28antibodies.Results:1). I-BET151didn’t alter the pattern of LPS-induced IκBα degradation andERK phosphorylation in BMDCs.2). Expression of acetylated RelA was highly induced ineither activated BMDCs or T cells, and I-BET151did not affect this acetylation process.3).Acetyl-310RelA was associated with BRD4in both activated BMDCs and T cells, whiletreatment with I-BET151disrupted this association.Conclusion: I-BET151had no effect on LPS induced activation of MAPK/ERKpathway and initiation of NF-κB activation. However, it could disrupt the binding of BRD4to acetylated RelA, thus removing the “read” signal of the acetylation dependent histonecode, which may be responsible for alterations in BMDCs and T-cell functions.