The Role And Mechanisms Of CD4～+CD25～+ Regulatory T Cells In The Suppression Of Allogeneic Immune Responses

Allogeneic hematopoietic stem cells transplantation (HSCT) is the treatmentof choice for many hematological malignancies and hematological disorders. Thisapproach relies on the elimination of the hematopoietic compartment, includingmalignant cells, by high-dose chemotherapy and irradiation and the reconstitutionof a new hematopoietic system provided by the donor graft containinghematopoietic stem cells. The transplant also contains mature T cells capable ofrecognizing allogeneic host antigens. The graft-versus-host reaction induced bydonor T cells is responsible for the main complication encountered afterallogeneic HSCT: graft-versus-host disease (GVHD). To date, an immuno-suppressive regimen based on cyclosporin A assocated with methotrexate and /orcorticosteroids administered with in the months following HSCT is the mostefficient preventive treatment but remains only partially effective. Procedures inwhich T cells are depleted from the transplant can prevent GVHD but have alsorevealed the important role that donor T cells play in the prevention of graftrejection, in lowering risks of infection, and in the graft-versus-host leukemia(GVL) and or the graft-versus-tumor (GVT) effects. Thus, administering donorallogeneic T cells without causing GVHD remains the key challenge of allogeneicHSCT.In the past few years, the development of non-myeloablative conditioningregimens has been spurred by two concepts: The first is that the curative potentialof hematopoietic-cell transplantation is due mainly to the killing of the recipientsmalignant cells by T lymphocytes in the graft (graft-versus-leukemia effect),rather than by the dose-intensity of the regimen. The second one is that highlyimmunosuppressive, nonmyeloablative conditioning-regimens can allow theengraftment of allogeneic cells and lessen epithelial-cell injury, thereby reducingthe risk of GVHD. Nonmyeloablative conditioning regimens are being usedincreasingly;today, they are used in 40 percent of all hematopoieticstem-celltransplantation procedures.Although nonablative conditioning reduces the risk of GVHD, thiscomplication of hematopoietic-stem-cell transplantation remains a clinicallysignificant problem. A new approach to its prevention takes advantage of theimmune system's regulatory T cells. In mice, natural killer T cells,CD3+CD4-CD8-ab-TCR+ (DN T) and CD4+CD25+ T cells not only have a vitalrole in the maintenance of tolerance to self-antigens but also, when present in thestem-cell graft, prevent GVHD without loss the graft-versus-leukemia effect. Itwas shown that if depletion of this Treg cells in several animal models, there willbe multi-organ autoimmune diseases and also it was demonstrated thatCD4+CD25+T cells can suppress CD4+ and CD8+ T cell proliferations. So it isreasonable to hypothesis that it will prevent or control GVHD after BMT. It couldbe a potential therapeutic applications of the cells themselves-an area ofpersonalized medicine. It has been proposed that, in many systems, regulation is aby-product of competition for the lymphoid niche. The use of allogeneic bonemarrow transplantation is a clinical setting in which there is lymphopaenia. So itis reasonable to consider that this clinical setting may be the first for Treg to goclinical trials. But there are several limitations to use them: one is the rarepopulations. And also we still do not sure what it will do on GVL or graftment.Our projects are focusing on 1. In vitro isolation, purification and expansionof CD4+CD25+T cells and in vitro function assays, related mechanism studies;2.The prevention of GVHD by CD4+CD25+T cells in semi-allogeneic murine BMTmodel;3. CD4+CD25+T cell effects on GVL and engraftment1. CD4+CD25+T cell isolation, purification and expansion in vitroWe use the micro-magnetic beads to purify the CD4+CD25+T cells. After thecolumns, the purity of the cells are usually about 85%-94%, and survival % ofcells after the purification is 98%. The percentage of the cells in na?ve mice isabout 5.7%.Next, we tested the suppression function in vitro by setting up with MLRculture systems. After 3 days culture, stimulated with allo-APC, CD4+CD25+Tcells are anergic to the stimulators but when put into the MLR ( allo-CD4+T cells,stimulated with all-APC), they suppress the proliferation of CD4+ T cells and isdose-dependent.We use the transwell system to test if the suppression is dependent oncell-cell contact or cytokine dependent. The data showed that CD4+CD25+T cellsneed cell-cell contact to function, and also they can suppress CD4+T cellsecreation of IL-2, which also depends on cell-cell contact when we analyzed thedata of IL-2 level in culture supernatant after 3days culture with or without CD25+T cells.In order to explore if we can activate and expand them in vitro to overcomethe low number issue for future, we adopted 2 protocols, one is stimulated purifiedCD4+CD25+T cells with plate-bound CD3mAb, another one is stimulated withallo-APC (irradiated splenocytes). Both the protocol IL-2 included in 100u/mlconcentration. The data showed that it can be expanded by both of protocols but,in terms of yield, plate-bound CD3mAb is better than another one. Expansion getsits peak on day 7, then goes down.2. CD4+CD25+T cells Prevent and control of GVHD mediated by CD4+TcellsOur data showed that in (C3H to C3HXB6) F1 BMT model when co-injectedCD25+ with CD4+ T cells, injected CD25+ alone to lethally irradiated recipientmice which is (C3HXB6) F1, no clinical GVHD were found, which is contrastwith CD4+T cells only in which all mice died about 7-14days. So it is obviouslyshown that CD4+CD25+T cells can prevent GVHD in this model.Further more, we tried to figure out if CD4+CD25+ T cells also can suppressCD4+ T cells in vivo. So, we labeled CD4+T cells with CFSE before injections.Then, on day 2 after BMT, we tested if CD4+CD25+T cells suppress CD4+T cellcytokine production by gated on CFSE after taken the recipient mouse spleenstained with IFN-gamma-PE antibody. The data showed that CD4+CD25+T cellsinhibit CD4+T cell IFN-gamma production in vivo, on day 4 after BMT, we tookrecipient spleen out, staining with CD4-PE, then gated on CFSE to look at theCFSE+ cell proliferation. The data showed that with CD25+ T cells, CD4+T cellproliferation decreased, compared with without CD25+ mice. It was first reportthat CD4+CD25+T cell inhibit CD4+T cell proliferation in vivo.Later, we moved on to delayed injection of CD4+CD25+T cells 2days afterBMT to see if they still can suppress GVHD. We used both freshly isolatedCD4+CD25+T cells and plate-bound CD3mAb expanded CD4+CD25+T cells. Thedata showed that expanded one has much stronger suppression function when welooked at the mouse survival data.3. Delayed transfusion of CD4+CD25+T cells can prevent GVHD whilemaintaining GVL and improving the engraftment in miHA mismatched modelwhich GVHD mediated by donor CD8+T cells.We used B10BR to CBA mouse combination, did the CD8+T cell titrationfirst. Then we tested if CD4+CD25+T cell injection on day 10 after BMT canprevent GVHD. The data showed that they really can control on-going GVHD.Further more, we IP injection MMCBA tumor cell line to CBA recipient one daybefore BMT. What we found is with CD25+ injection on day 10, the mice survivalwithout clinical GVHD signs, and no progressive tumor. Compared with othergroups, either the mice died of tumor or GVHD. We also analyzed theengraftments between ATBM and CD25+ mice. We found CD4+CD25+T cellimprove donor T cells, B cell and also NK cell engraftment. It is promising totranslate the mouse data into clinical setting later once the research works havebeen done more and found more about CD4+CD25+T cells.