Relationship Between CD4~+CD25~+ Regulatory T Cells And Graft-Versus-Host Disease After Allogeneic Haematogenesis Stem Cell Transplantation

Human T regulatory cells include many subsets, such as CD4~+CD25~+ regulatory cells (CD4~+CD25~+Treg), Tr1 and Th3. CD4~+CD25~+ regulatory cells have been the object of intense study because their function appears critical in maintaining self tolerance. These cells still express CTLA-4(CD152),GITR,CD62L,CD45RB and so on. But none of them is unique for CD4~+CD25~+ regulatory cells. Recent experimental studies indicate that Foxp3, which encodes a forkhead-winged helix transcription factor,is a key regulatory gene for the development and suppressive activity of regulatory T cells, and represents a more specific marker for Treg. CD127, the chain of the interleukin-7 receptor, has recently been identified as a specific marker for CD4~+CD25~+ regulatory cells. Human CD4~+CD25~+ regulatory cells express lower level of CD127 than the other CD4+T cells. Expression of Foxp3 protein and the CD127low phenotype were highly correlated within the CD4~+CD25~+ population. These findings will help us to research the relation between CD4~+CD25~+ regulatory cells and human immune function. CD4~+CD25~+ regulatory cells fail to proliferate or secrete cytokines (IL-2 or IFN-γ) in response to polyclonal or antigen-sepcific stimulation via their T cell receptor, but their anergic state can partially be reversed by high doses of interleukin (IL)-2. Upon stimulation with anti-CD3 plus anti-CD28mAb, the CD4~+CD25~+ regulatory cells in coculture experiments suppress the activation, proliferation and cytokine production of CD4+CD25-responder T (T-resp) cells and CD8+ T cells. Studies also show that CD4~+CD25~+ regulatory cells can suppress anti-tumor immunity and tumor immunosurveillance which is mediated by tumor-specific CD8+ cytotoxic T lymphocytes (CTLs), T-resp and NK cells,with no MHC limited. Graft-Versus-Host Disease is the lifethreatening and frequent complication of allogeneic haematogenesis stem cell transplantation (Allo-HSCT), Previous studies showed that GVHD was possibly correlated to HLA compatibility, coenogenesis, sex, age, physiclal status in donor and recipient. Until now, people had not discovered a independent indicator which can predict generation and development of early GVHD in clinical practice. Many animal studies recently showed that the CD4~+CD25~+ Regs could also control alloreactive responses. Therefore, CD4~+CD25~+ regulatory cells play important roles during immune reponses, as in graft versus host disease.Objectives (1) To identify the relationship between CD4~+CD25~+ regulatory T cells (CD4~+CD25~+Tregs) and acute/chronic graft-versus-host disease(a/cGVHD) after allogeneic haematogenesis stem cell transplantation (Allo-HSCT), provided new laboratory indicator to predict effectively generation and prognosis of GVHD in clinical practice. (2) To investigate the relationship between cytokine tumor necrosis factor-α(TNF-α),transforming growth factor-β(TGF-β) and acute/chronic graft-versus-host disease(a/cGVHD) after Allo-HSCT. (3) To approach initially the relationship between immunodepressive and CD4~+CD25~+Tregs.Methods (1) three color monoclonal antibodies directly labeled with immunofluorescence were used to analyze the surface antigens (such as CD4, CD25, CD127 et al.) by multiparameter flow cytometry. (2) cytokine tumor necrosis factor-α(TNF-α) and transforming growth factor-β(TGF-β) were detected by enzyme linked immunosorbent assay. (3) four color monoclonal antibodies directly labeled with immunofluorescence were used to analyze the surface and cytoplasma antigens (such as CD4, CD25, CD127, FoxP3 et al.) by multiparameter flow cytometry.Results (1) All the patients achieved engraftment. (2) Compared with healthy adults, the levels of the other immunocytes including CD3+CD4+ T cells, NK cells and CD4 to CD8 ratio were reduced significantly in no-GVHD, aGVHD, cGVHD groups(all P <0.05) , but there was no difference in the level of CD3+ T cells(P>0.05). Further comparison discovered that the levels of the CD3+, CD3+CD4+, CD3+CD8+ T cells, NK cells , and CD4 to CD8 ratio were not significantly different in three groups of patients post-aHSCT(P>0.05). (3) The percentage of CD4+CD25high and CD4~+CD25~+CD127low T cells in aGVHD patients decreaseed significantly as compared with health adults, (P <0.05). However, the percentage of Treg cells was no significant difference between health adults and patients with cGVHD(P >0.05). Further comparison revealed that the levels of CD4~+CD25~+ Treg cells in patients without GVHD were higher than healthy adults, but there was no significance (P >0.05) .Compared with non-GVHD patients group, the percentage of Tregs for patients in aGVHD and cGVHD decreased significantly (all P <0.01). Decrease in CD4~+CD25~+ Treg was strongly correlated to severity degree of aGVHD. Accompanied with aggravation of the grade, expression of CD4~+CD25~+ Treg in aGVHD patients decreased obviously. Further comparison shown that there were significantly difference for percentages of CD4~+CD25~+ Treg cells in three groups. the percentage of CD4~+CD25~+Tregs in gradeⅠaGVHD group were highest, then was gradeⅡaGVHD group, and the lowest was gradeⅢandⅣaGVHD group. Compared with health adults, there were no obvious difference in patients with gradeⅠaGVHD (P >0.05), but there were significant difference in patients with gradeⅡ,Ⅲ～ⅣaGVHD( P <0.05, P <0.01 respectively).In comparison to healthy adults, expression of CD4~+CD25~+ T regulatory cells in patients with extensive cGVHD decreased markedly ( P <0.05), but there were no obvious difference in patients with limited cGVHD (P >0.05). (4) In contrast to levels of TGF–βin patients with GVHD dropping strikingly, levels of TNF-αin those group were elevated markedly. Levels of TGF–βin aGVHD and cGVHD group were significantly lower than that in health adults and non-GVHD group (all P < 0.01), on the contrary, levels of TNF-αin aGVHD group and cGVHD group were significantly higher than that in health adults and non-GVHD group(all P < 0.01). Levels of TGF–βin non-GVHD group declined slightly, simultaneously, levels of TNF-αin that group rose a little. Nevertheless, difference of the 2 cytokines between health adults and non-GVHD group was not significant(P>0.05). (5) In comparison to healthy adults ,The percentage of CD4~+CD25~+CD127low and CD4~+CD25~+foxp3+ T cells in no-GVHD which cpalended large dose immunodepressive(ciclosporin≥100mg/d ;≥half a year) elevated markedly(P < 0.01).Conclusions To summmarize, the levels of CD4~+CD25~+ T regulatory cells and cytokines in patients after Allo-HSCT were closely correlated to occurrence and serious degree of GVHD, especially aGVHD. Monitoring the levels of CD4~+CD25~+ T regulatory cells should be valuable for diagnosing early GVHD, predicting the onset, severity, and prognosis of GVHD, and guiding the application of immunosuppressant. In addition, ex vivo expansion and recurrent infusion of autoallergic Tregs may be a new therapeutic strategies to preclude and treat GVHD.