| Allogeneic hematopoietic stem cell transplantation (HSCT) is an importanttreatment modality in the management of a number of malignant and nonmalignanthematopoietic disorders.Unfortunately, the utility of HSCT is limited bytransplant-related complications, including graft-versus-host disease(GVHD).Currently available post transplantation immunosuppressive therapies areinsufficient and GVHD remains a major cause of morbidity and mortality inallogeneic HSCT. The pathophysiology of aGVHD is believed to be a multistepprocess. The initial step involves the development of an inflammatory milieu resultingfrom damage to host tissues induced by preparative chemotherapy or radiotherapy.Damaged tissues secrete inflammatory cytokines, including tumor necrosis factor-a(TNF-a), interferon-γ(IFN-γ) and interleukin (IL)-1. In the second step,antigen-presenting cells from both the recipient and the donor, in concert withinflammatory cytokines, trigger the activation of donor-derived T cells, which expandand differentiate into effector T cells. In the third step, activated donor T cells mediatecytotoxicity targeted towards host tissues through Fas–Fas ligand interactions,perforin–granzyme B, and the production of cytokines, such as TNF-a. aGVHDpredominantly affects the skin, upper and lower gastrointestinal tracts, liver, andoccasionally the eyes and oral mucosa.Regulatory T (Treg) cells are T cell subsets different from Th1and Th2cells,which have immune regulation function. Broadly speaking, CD4+T cellspossessing the ability to suppress immune responses can be divided into two types:naturally occurring (nTreg) and inducible (iTreg) or adaptive regulatory cells.Naturally occurring thymus-derived CD4+CD25+Tregs are a subset of T cells whichhave immunosuppressive properties and are5%–10%of the total peripheral CD4+Tcells. Tregs express the a chain of the IL-2receptor (CD25) and a nuclear transcription factor termed forkhead box P3(FoxP3). Treg cell have Immune suppression andinvolved in the pathological process that occurs in a variety of autoimmunediseases,which recently become an important part of the field of immunology. Human Treg isolated from peripheral blood have been shown to suppressalloresponses in the MLR.Several previous reports have indicated a role for murineTreg in tolerance induction to alloantigens. It is therefore not surprising that the roleof Treg in protection against GVHD has been studied extensively. Taylor etal showedin an allogeneic BMT model that depletion of CD25+cells from the donor CD4+T-cell population completely abrogated ex vivo tolerization to host alloantigens andresulted in loss of protection against GVHD. The same model was used to investigatethe capacity of naturally occurring, unmanipulated, CD4+CD25+T cells to suppressor prevent GVHD after allogeneic transplantation.The C57BL/6'BALB/ctransplantation model demonstrated that un-manipulated donor-type Treg inducedsignificant protection from GVHD lethality when co-transferred with CD4+CD25+conventional effector T cells, which otherwise induce uniform lethal GVHD. Over90%of BALB/c recipients were rescued from lethal GVHD by injecting the animalswith a high number of freshly isolated CD4+CD25+T cells from the C57BL/6donors.Three clinical trials have been reported that are evaluating the safety and efficacyof Tregs in treating GVHD, all demonstrating potentially safe and reliable efficacyprofiles. The first-in-man trial involved two patients.The first patient had cGVHD twoyears after transplantation. After receiving0.1×106/kg fluorescence-activated cellsorting (FACS) purified ex vivo-expanded Tregs from the donor, the symptomssubsided and the patient was successfully withdrawn from immunosuppression. Thesecond patient had acute disease that progressed despite three infusions with anaccumulative dose of3×106/kg expanded donor Tregs. A larger scale phase1trialhas recently been concluded in which it was shown that ex vivo-expanded Treginfusions from a third party could be used as supplemental GVHD prophylaxis afterdouble umbilical cord blood transplantation.Twenty-three patients with advancedhaematological malignancies were enrolled and treated with two units of umbilicalcord blood as source of stem cells and Teffs. Tregs were isolated using anti-CD25immunomagnetic bead selection from third-party cord blood samples that had4–6matches of human leukocyte antigen (HLA) with the recipient. Tregs infused with thegraft were detectable in the peripheral blood up to day14after transplantation.Duringthe1-year period after Treg infusion, the investigators observed no dose-limitingtoxicities or increase in adverse events when compared with historical controls.Incidences of severe aGVHD were significantly reduced in patients who received Treg therapy. In the third trial, freshly isolated anti-CD25immunomagneticbead-enriched donor Tregs without ex vivo expansion were infused4days beforethe infusion of CD34+cells and conventional T cells from the same donors in28patients with high-risk haematological malignancies undergoing haploidenticaltransplantation.No adjunct immunosuppression was given after transplant. Patientsdemonstrated accelerated immune reconstitution, reduced CMV reactivation, and alower incidence of tumour relapse and GVHD when compared with historical controls.Only2(7%) patients developed aGVHD. These encouraging early experiencessupport further investigation of the efficacy of Treg therapy in controlling GVHD andapplying it in other disease settings.However, the paucity of Tregs in the peripheralblood is one of the major obstacles for the application of these models and eventualclinical translation. The number of nTregs that can be isolated from the periphery istoo small to be clinically effective. Clinical trials with ex vivo-expanded humanFoxP3+Tregs isolated from the peripheral blood are currently under way mainlybecause purified CD25+T cells may contain contaminant non-Tregs.Recent studiesshow that anti-CD4monoclonal antibody (GK1.5) could significantly enhance thepercentage of CD4+CD25+Foxp3+Treg cells in the periphery while keeping these cellsfunctional.Therefore, this study was designed to validate the anti-CD4monoclonal antibodypreconditioning whether can inhibiting GVHD occurred via modulating host Tregcells. First, we establish a stable, mature GVHD model, and then recipients(BALB/c mice) received an intraperitoneal injection of0.2mg GK1.5orphosphate-buffered saline (PBS) as a control on day0. Recipients were givensublethal TBI on day10. Then the recipients received transplanted donor bonemarrow cells (5×106) and whole spleen cells (5×106). the body weight andsurvival time of mice were observed in post operation and further verified via therecipients tissue pathology. Finally, to explore the molecular mechanisms by RT-PCR,flow cytometry and other detection methods.Part I A murine model of graft-versus-host disease induced byallogeneic bone marrow transplantationObjective: To establish a stable and reliable allogeneic hematopoietic stem celltransplantation mouse model of GVHD. Methods: C57BL/6mice as donor mice,bone marrow cells isolated from mouse femur and tibia, broken red, washing andcounting spare. Mouse spleen aseptically removed, placed in a200mesh sieve filtration grinding spleen cells were collected, broken red, washed count spare.Receptor BALB/c mice receive donor two kinds of cells mixed in differentproportions after irradiated.Then the recipients were monitored for fatigue, anorexia,weight loss, arched, hair loss, diarrhea and other clinical symptoms of GVHD andcompare the median survival time and histopathological changes in each group.Results: Injecting5×106bone marrow cells can guarantee the success of bonemarrow transplantation, while the input at least5×106spleen cells can induceoccurrence of GVHD, but different survival time. Conclusion: When the input bonemarrow cells5×106and5×106spleen cells can be the establishment of a stableand reliable mouse model of GVHD, the median survival time of mice was24d, thatsuitable for the study of this experiment.Part II Anti-CD4monoclonal antibodies preconditioningprevents GVHD and Preliminary its molecular mechanism.Objective: anti-CD4monoclonal antibody preconditioning whether can preventthe occurrence of GVHD in mice and explore its molecular mechanisms. Methods:24weight similar eight weeks BALB/c mice were randomly divided in two groups,namely, control group (A) and experimental group (B).Each group has12mice.Group B received intravenous injection of anti-CD4monoclonal antibodies, groups Awere given intravenous injection of isotype control Ig, All mice receive8.0.Gyirradiated after10d, Group A.B receive bone marrow cells（5×106）and spleen cell（5×106）. Compare of the severity of GVHD clinical symptoms, median survivaltime, histopathological changes and the percentage and yield of T cells in GVHDtarget tissues in each group. In4hours and7day after transplantation,we detectedthe percentages of Treg in CD4T cells in splenocytes and peripheral bloodmononuclear cells (PBMCs) in mice treated with or with-out anti-cd4(0.2mg/mouse)by Flow cytometric. Immune response of treg cells were detected by mixedlymphocyte reaction assay and the cell proliferation to Mitogen Concanavalin A.Thenin5day and10day after transplantation,we measured INF-γ, TGF-β and IL-10expression in CD25cell in splenocytes in mice treated with or with-out anti-cd4byRT-PCR. Results: compared with control group, anti-CD4monoclonal antibodypreconditioning mice can significantly improve in the clinical scores of GVHD、prolong the median survival time and histopathology confirmed T cell infiltrationand tissue damage is reduced in the liver, skin, gastrointestinal. After anti-CD4monoclonal antibody preconditioning, the outer peripheral CD4+cells in the recipient mice was decreased, but CD4+CD25+FoxP3in the CD4cells significantincrease. Furthermore, enriched CD4+CD25+Treg cells in GK1.5-treated mice showimmunosuppressive ability on the immune response of T effector cells to alloantigensor mitogen as efficiently as those from the control mice in vitro. while RT-PCRdetection TGF-β and IL-10expression increased in CD25+cells. Conclusion:Anti-CD4monoclonal antibodies preconditioning can inhibit T cell inflammatoryinfiltration and migration to prevent the occurrence of GVHD via increasing host Tregcells and its secretion TGF-β and IL-10. |
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