The Effects And Mechanisms Of Regulatory γδ T Cells In Acute Graft-versus-host Disease

Background and ObjectivesAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is the primary means for treatment of hematologic and non-hematologic malignancies and other diseases. Graft-versus-host disease (GVHD) was the systemic autoimmune disease, damaged multisystem (including skin, esophagus, stomach, liver, etc.) after allo-HSCT, affected the prognosis quality of life, and one of the most important factors that constrain the efficacy of allo-HSCT. According to the disease progression and clinical manifestations, it was divided into acute GVHD (aGVHD) and chronic GVHD (cGVHD). Some of related literature reported, in the human leukocyte antigen (HLA) consistent compatriots after allo-HSCT, the incidence of GVHD was 30%-50%; in HLA not consistent compatriots and unrelated donor transplantation, the incidence of aGVHD can be as high as 60% to 80%. The pathogenesis of GVHD is complex and influenced in part by the major or minor histocompatibility antigenic (MHC) disparities between donor and recipient and the presence of host-derived antigen-presenting cells. At present, the first-line treatment drugs of aGVHD is mainly glucocorticoids. related literature reports that the remission rate was only 50%. Due to glucocorticoid resistance aGVHD, second-line treatment drugs are mainly tacrolimus (FK506), CD25 monoclonal antibody, mycophenolate mofetil (MMF), antilymphocyte globulin (ATG), cyclophosphamide (CTX), etc, but some literature reported that the efficient of these drugs was only about 30%. And the mechanism of action of these immunosuppressive drugs to prevention and therapy of aGVHD mainly by inhibiting the body immune response related cells (T cells, B cells, macrophages, etc) of the proliferation and differentiation, effectively reduce the body’s immune response, and thus effectively reduce tissue and organ damage, but these strategies are hampered by the high rates of opportunistic infections、 increased risk of tumor relapse、 and the second tumor. So, we need to seek a new kind of immune regulation mechanism to control the occurrence and development of GVHD, while maintaining the body’s anti-tumor and anti-infection activity.In 1995, Sakaguchi et al found a group phenotype and function specific T cell in the peripheral blood, thymus, spleen of normal people and mice at the first time, this is regulatory T cells (Treg), and was named CD4+CD25+Treg. As there are many research on Treg, found that there were a lot of molecular markers in Treg surface, including GITR、CD62L、CTLA-4(CD152) and so on, but these molecules are not specific markers of Treg.Then, Fontenot et al found that such as fork sample transcription factor (the forkhead/winged helix transcription, Foxp3) is a special marker of Treg cells. So, Foxp3 is recognized as the most specific cd4+cd25+treg markers. Now, many research have proved that such cells by inhibiting the proliferation of T cells and production of anti-inflammatory cytokines to suppress immune reaction. Treg are pivotal for the maintenance of self tolerance, regulate the homeostasis of the peripheral T cell pool, contribute to tolerance induction after solid organ transplantation and protect from GVHD lethality in allo-HSCT. However, as for further study of Treg, there have been increasing reports that challenge this view by demonstrating that Treg had plasticity (transfer to Thl,Th2,Thl7) and might lead to autoimmune diseases. Treg inducing autoimmune diseases might be associated with the plasticity of Treg, and the plasticity was related to the heterogeneity of Treg population. At present, some study found that the function of immune adjustment Treg are not confined to CD4+ T cells、 CD8+T cells、 CD4-CD8-T, yδ Treg cell also has immune regulating function of groups. Therefore, a further subdivision of Treg population is quite necessary.Lately, investigators found a more homogeneous cell population in nomal mice and lung cancer、gastric cancer and carcinoma of kidney patients, this is regulatory yδ T cells (yδ Treg), which belonged to yδ T cells, expressed Foxp3 and CD25, and can suppressive effect on the proliferation of autologous naive CD4+T cells. Compared to common Treg (αβ T cells), both of expressed Foxp3 and CD25, and had suppressive effect on the proliferation of autologous naive CD4+T cells. γδ Tregs belonged to γδ+ T cells,recognize specific antigen without need antigen present、without MHC-restriction and more uniformity. Apparently, yδTreg and common CD4+CD25 Foxp3+Treg are two completely different immune regulating cell, we were known little about this kind of new biological characteristics of yδTreg subsets so we need further study. There are some studies reported that yδ Treg plays an important role in the occurrence and development of GVHD, but it was still unclear the mechanism of γδTreg supress GVHD, Hu et al suggested that it maybe related to the yδTreg secretion of IL-10. Our previous study found that the expression levels of γδTreg after G-CSF mobilization were significantly increased than that before mobilization, the incidence of GVHD in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) was negatively correlated with the number of yδ Treg subsets in transplant donors, suggesting that yδ Treg might have inhibitory effect to GVHD. Therefore, the purpose of this study is to indentify the effects and mechanisms of yδ Treg on aGVHD. This study compared the no aGVHD group and aGVHD group and different disease conditions of aGVHD in peripheral blood, the percentages of functional subsets of y8 Treg cells and the related expression of cytokines.Methods1. Using flow cytometry compared 24 patients had aGVHD incidence with 24 patients had not aGVHD incidence after allo-HSCT, these patients had the similar basic characteristic. To detect the peripheral blood specimens the percentages of functional subsets of y8 Treg cells (included total yδ T cell、V81 T、Vδ2 T、Foxp3+y8 T、Foxp3+Vδ1T、Foxp3+Vδ2T、CD25+γδ8T、CD25+Vγδ1T、CD25+Vδ2、 CD27+γδT、CD27+ Vδ1T、CD27+ Vδ2 T)and CD4+CD25+Foxp3+(Treg) Combined with their clinical data, the correlation between the percentages of functional subsets of y8 Treg cells and GVHD, relapse as well as survival in recipients was analyzed.2. Using flow cytometry to patients had different disease conditions (aGVHD incidence, improve and progressed),the function of y8 Treg cells subsets (included totalγδT cell、Vδ1T、Vδ2T、Foxp3+γδT、Foxp3+Vδ1T、Foxp3+Vδ2T、CD25+γδ T、CD25+Vδ1T、CD25+Vδ2T、CD27+γδT、CD27+γδT、CD27+Vδ2T) and CD4+CD25+Foxp3+(Treg) in peripheral blood, and combined with their clinical data, the correlation between the percentages of functional subsets of y8 Treg cells and GVHD, relapse as well as survival in recipients was analyzed.3. Using real-time polymerase chain reaction (RT-PCR) compared 24 patients had aGVHD incidence with 24 patients had not aGVHD incidence after allo-HSCT, these patients had the similar basic characteristic. To detect the expression levels of Foxp3、CD25、RORc、CTLA4、GATA3、GITR, IL-17A、IL-23R、STAT1、STAT3、 T-bet、TLR8 genes.4. Using RT-PCR to detect patients had different disease conditions (aGVHD incidence, improve and progressed) the expression levels of Foxp3、CD25、RORc、 CTLA4、GATA3、GITR、IL-17A、IL-23R、STAT1、STA3、T-bet、TLR8 genes.5. Used Liquichip technology to analysis patients had different disease conditions (aGVHD incidence, improve and progressed) after allo-HSCT, the concentration of γδ Treg cells telated cytokines (interferon-y, interleukin-10, interleukin-17, interleukin-4, IL-4,TNF-α) in cultural supernatant.6. Used Liquichip technology to analysis 24 patients had aGVHD incidence with 24 patients had not aGVHD incidence after allo-HSCT, the concentration of γδ Treg cells telated cytokines (interferon-γ, interleukin-10, interleukin-17, interleukin-4, IL-4,TNF-α) in cultural supernatant.7. Statistical analysis:all data was analyzed using SPSS 19.0. Paired-samples t test was used to compare the 24 patients had aGVHD incidence with 24 patients had not aGVHD incidence after allo-HSCT, these patients had the similar basic characteristic, the expression levels of Foxp3、CD25、RORc、CTLA4、GATA3、 GITR、IL-17A、IL-23R、STAT1、STAT3、T-bet、TLR8、IFN-γ、IL-10、IL-4、 IL-17A、TNF-α. Nonparametric tests was used to compare patients had different disease conditions (aGVHD incidence, improve and progressed) after allo-HSCT, the expression levels of Foxp3、CD25、RORc、CTLA4、GATA3、GITR、IL-17A、 IL-23R、STAT1、STAT3、T-bet、TLR8、IFN-γ、IL-10、IL-4、IL-17A、TNF-α. Spearman’s correlation analysis was used to estimate the correlation between the percentages of yδ Treg cells and aGVHD.P<0.05 was considered as statistically significant.Results1. The flow cytometry results of 24 paired matched patients. Compared with that developed aGVHD patients, the expression proportions of total yδ T cell, V82 proportion was significantly increased in not developed aGVHD patients (t=-2.244, t=-5.640; P=0.044,.P=0.000), whereas the expression proportions of Vδ1 subsets was similar between the two groups (t=-2.066; P=0.061). In addition, there was a significant decrease in the expression proportions of CD27+γδ T、 CD27+Vδ1 (t=-3.116,t=-2.778;P=0.009、0.018)in aGVHD patients and had no significant different in the proportions of CD27+Vδ2 (t=-1.613; P=0.120). The expression proportions of Foxp3+γδT、Foxp3+Vδ1、Foxp3+Vδ2 and Treg were increased in paired matched patients (t=-3.453,t=-2.862,t=-2.548,t=-5.088; P=0.005、P=0.014、 P=0.026, P=0.000). The expression proportions of CD25+y8 T、CD25+ Vδ1 and CD25+ Vδ2 subsets were also no significant difference. There was a negative correlation between the incidence of aGVHD and the percentages of Foxp3+y8 T、 Foxp3+Vδ1、Foxp3+Vδ2 subsets (P=0.014, r,=-0.477; P=0.001,rs-0.610; P=0.040, rs=-0.405).2. The RT-PCR results of 24 paired matched patients. Compared with that developed aGVHD patients, The mRNA expression level of FOXP3、T-bet、TLR8 were significantly increased in not developed aGVHD patients (t=2.169,t=2.551,t= 3.583; P=0.042, P=0.019,P=0.002); The Mrna expression level of CD25,RORc、 CTLA4、GATA3、GITR、IL-17A、IL-23R、STAT1、STAT3 was similar between the two groups (t=1.244,t=1.340,t=-2.026, t=-1.406, t=-0.650,t=1.877, r=-0.443, t-1.528,t=-0.290; P=0.234, P=0.194, P=0.056,P=0.174, P=0.524, P=0.073, P=0.663, P=0.142,P=0.775).3. We used Liquichip technology detection the five kinds of related cytokines, include IFN-γ、IL-10、IL-17A、IL-4、TNF-α. we found that the concentration of IL-10 and TNF-α was significantly increased in aGVHD patients (t=-2.512, t=-2.229; P=0.002,P=0.036); The concentration of IL-4. IL-17A and IFN-y was similar between the two groups (t=0.898,t=-0.833,t=-1.417; P=0.379,P=0.414,P=0.170).4. In 15 with aGVHD patients, among 12 patients with methylprednisolone and CSA treatment aGVHD was controlled (included complete remission in 8 patients, partial remission in 4 patients), but the left 3 patients condition not get fully controlled, the aGVHD was progressed. Compared with aGVHD improve patients, the expression proportions of Foxp3+yδ T、CD25+γδ T、 CD25+V81、Foxp3+V81 and Foxp3+Vδ2 were significant decrease(P=0.023, P=0.008, P=0.028,P=0.008, P=0.028); whereas the expression proportions of γδT 、Vδ1、Vδ2、CD27+γδT、 CD27+Vδ1、CD27+Vδ2、CD25+Vδ2 and Treg were similar between the two groups (P=0.308,.P=0.117,P=0.099, P=0.182,P=0.084,P=0.530,P=0.209,P=0.066).At the same time,we observed 3 patients with aGVHD who were treatment failure lead to progressed, we found that the expression proportions of Foxp3+yδ T、CD27+yδ T and CD25+Vδ1 had a downward tendency; but the expression proportions of γδT Vδ1、Vδ2、CD25+γδ1、CD27+Vδ1、CD27+Vδ2、CD25+ Vδ2 and Treg had no significant different.5. Compared with aGVHD improved patients, the mRNA expression level of Foxp3、T-bet、RORc、TLR8 were significant decrease(P=0.060,P=0.042,P=0.001, P=0.041);whereas the mRNA expression level of CD25、CTLA4、GATA3、GITR、 IL-17A、IL-23R、STAT1、STAT3 were similar between the two groups (P=0.95, P=421,P=0.051,P=0.605,P=0.911,P-0.547,P=0.186,P=0.809).In the aGVHD progressed, the mRNA expression level of Foxp3 was significant decrease tendency. But the mRNA expression level of CD25、RORc、CTLA4、GATA3、GITR、IL-17A、 IL-23R、STAT1、STAT3、T-bet and TLR8 had no significant different.6. Compared with aGVHD improve patients, found that the concentration of IL-10 was increased (t=3.161, P=0.005). The concentration of IFN-γ、IL-4、IL-17A and TNF-a was similar between the two groups (t=-0.445, t=1.033,t=1.494, t=1.168; P=0.662, P=0.314, P=0.152, P=0.254). In the aGVHD progressed, the concentration of IL-10 had a rise tendency; but the concentration of IFN-y、IL-4、IL-17A and TNF-α had no significant different.Conclusions1. Compared with the matched group patients without aGVHD at the same period, the expression proportions of Foxp3+γδ T、Foxp3+V81、Foxp3+Vδ2、 CD27+V81 and CD27+Foxp3+V81 subsets were significantly decreased in peripheral blood at aGVHD group patients. There was a negative correlation between the incidence of aGVHD and the percentages of Foxp3+γδ T、Foxp3+Vδ1、Foxp3+Vδ2 subsets.2. Compared with aGVHD improved patients, the expression proportions of Foxp3+y8 T、Foxp3+Vδ1 and Foxp3+Vδ2 will be decreased in aGVHD patients. If the aGVHD treatment in efficient, the expression proportions of Foxp3+γδ T will on the decline. It shows that γδ Treg participate in the occurrence and development of aGVHD.