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The preliminary research of immune function monitoring before and after allogeneic hematopoietic stem cell transplantation in children with aplastic anemia TONG Chun1 , 2 ,GUO Zhi2,LOU Jing-Xing2,LIU Xiao-Dong2,YANG Kai2,HE Xue-Peng2, ZHANG Yuan2,CHEN Peng2,CHEN Hui-Rren2* 1 Clinical Medical College of Anhui Medical University,General Hospital of Beijing Military Command,Beijing 100700,China. 2Department of Hematology,General Hospital,Beijing Military Area,Beijing 100700,China *Corresponding author: CHEN Hui-Ren, Senior Physician, Professor, Tutor of Doctorial Postgraduated. Email: [email protected] Abstract Objective: To explore the clinical significance of the relationship between the immune function and the pathogenesis of aplastic anemia in children with aplastic anemia, along with the incidence of graft-versus-host disease (GVHD) by monitoring the changes of T lymphocyte subsets dynamicly cell transplantation. on +1, +3, +6, +12 month Methods: 12 cases after allogeneic hematopoietic stem received allogeneic hematopoietic stem cell transplantation in the department of Hematology of General Hospital of Beijing Military Region from January 2013 to January 2014, including 4 males and 8 females, average age of 7.92 (3-14 ) years old with 5 cases of HLA matched and 7 cases of HLA mismatched. Monitoring the level of T lymphocyte subsets including CD3+, CD4+, CD8+, CD4+/CD8+, CD56+, CD4+CD25high+FOXP3+ with flow cytometry (FCM) on +1, +3, +6, +12 month after and before transplantation dynamicly in the peripheral blood. While monitoring T lymphocyte subsets level in 12 cases healthy children at the same period as control. Results: Follow up to March 2015, 10 cases have abnormal cellular immunity (CD4+/CD8+ inversion) in the 12 cases. Compared with the control group, CD3+ slightly higher, CD4+ decreased, CD8+ increased, CD4+/CD8+ decreased and CD56+ decreased, CD4+CD25high+FOXP3+ decreased in AA patients before transplantation, with the difference were statistically significant (P< 0.05). The lever of CD3+, CD4+, CD8+, CD4+/CD8+, CD56+, CD4+CD25high+FOXP3+ had a different degree of recovery after transplantation for all cases and returned to normal on +12 month basically. The level of CD4+CD25high+FOXP3+ for patients with acute GVHD was lower than that of non acute GVHD cases. Conclusion: 1.Compared with healthy control group, abnormal cell immune function in some cases with AA; 2.Compared with pre-transplantation, the level of CD3+, CD4+, CD8+, CD4+/CD8+, CD56+ and CD4+CD25high+FOXP3+ reduced after allogeneic hematopoietic stem cell transplantation; CD8+ T cells recovered earlier than CD4+ T cells, and returned to normal on +12 month gradually, then achieved stable on +18 month finally; While the decrease of CD4+ T cells lasted more than 1 year; The proportion of CD4+/CD8+ inversion also lasted for more than 1 year; 3.After transplantation, the level of CD4+CD25high+FOXP3+ in GVHD positive group was significantly lower than the negative group, which can be used to predict the occurrence of GVHD. Key words: Aplastic anemia; Allogeneic hematopoietic stem cell transplantation; cellular immune function; flow cytometry; T lymphocyte subsets; graft versus host disease Aplastic anemia (AA) is a bone marrow failure disease, which mainly involving inner defects of hematopoietic stem cells, abnormal immune response in hematopoietic stem cells, hematopoietic microenvironment defect[1], especially for AA (severe aplastic anemia, SAA) with poor clinical prognosis. It should be performed blood component transfusion and special treatment immediately when diagnosed, otherwise most children died of infection or complications such as bleeding about one year [2]. At present, the immune mediated hematopoietic function inhibition plays an important role in the pathogenesis of AA, and T lymphocytes are the major effector cells in the immune response[3]. The first-line treatment of SAA includes immunosuppressive therapy (IST) and hematopoietic stem cell transplantation (HSCT). IST could make blood completely back to normal in about 20%~40% patients, but which effects slowly, and still need blood transfusion, prevention and treatment of infection and so on, with greater risk. HSCT has rapid hematopoietic recovery, complete remission generally which needs no maintenance treatment, can reduce the mortality rate of graft-versus-host disease (GVHD) with survival rate can be improved distinctly[4]. In 2000, Horowitz MM[5] reported that allogeneic stem cell transplantation could make the 5 years survival rate of AA to 77%, and 90% for children. It is important to monitor the reconstruction of immune function in SAA children dynamically after transplantation. It was confirmed[6]that the T lymphocyte subsets are the main regulatory cells in the reconstruction of immune function. This study by monitoring T lymphocyte subsets level of patients with SAA pre-transplantation and on + 1, + 3, + 6 and 12 months post-transplantation with flow cytometry and explore the change rule. By comparing T lymphocyte subsets of AA children and healthy control group to investigate immune factors in the pathogenesis of AA, also compared between GVHD positive and negative to explore the relationship between GVHD and T lymphocyte subsets. 1 materials and methods 1.1 Basic data 12 cases with SAA underwent allogeneic hematopoietic stem cell transplantation in the department of hematology of General Hospital of Beijing Military Region from January 2013 to January 2014, including 4 males and 8 females, average age of 7.92 (3-14)years old, randomly selected 12 cases healthy children in our hospital physical examination center as control over the same period, 6 males and 6 females, with the median age of 6.83 (2-12) years old. SAA diagnostic criteria is in accordance with the third edition "blood disease diagnosis and efficacy standards" which edited by Zhang Zhinan[7]. The transplantation methods included 5 cases of HLA matched, other 7 cases of HLA mismatched. According to the graft-versus-host disease (GVHD) occurrence after transplantation, cases were divided into GVHD positive group and negative group, and the gender, age, diagnosis, HLA matching, pre-treatment regimen and GVHD prophylaxis had comparability in the two groups. All cases were examed the functions of important organs comprehensively before transplantation and were not associated with Basic disease or important organ dysfunction. Children's families were fully informed and signed informed consent, and the transplantation program was approved by the hospital ethics committee. The basic information of the children is in table 1. Table 1 Group Gender Age (years) The basic information of cases HLA matching Infusion of Follow-up CD34+ cells time (×106/kg) (month) 3.25 6 Complication outcome Pneumonia、 Dead Infusion of nuclear cells(×108/kg) 1 M 3 5/10 8.47 Intestinal GVHD 2 F 2 6/10 5.38 2.52 12 3 F 12 10/10 10.84 2.97 12 Pneumonia Alive Pneumonia、 Dead Intestinal GVHD 4 M 8 10/10 5.56 6.81 14 Pneumonia Alive 5 M 1 5/10 7.99 3.37 15 Hemorrhagic Alive cystitis、 Septicemia 6 F 8 5/10 15.54 14.73 16 Septicemia、Skin Alive GVHD 7 M 9 10/10 14.22 10.16 17 Hemorrhagic Alive cystitis、Skin GVHD 8 F 11 10/10 16.39 9.09 18 cytomegalovirus Alive viremia、 Intestinal GVHD 9 F 14 6/10 8.96 4.12 22 Hepatic GVHD Alive 10 F 6 10/10 11.16 2.90 24 Pneumonia、Oral Alive infection 11 F 10 5/10 12.45 8.63 25 Skin GVHD Alive 12 F 11 7/10 11.17 9.37 26 Pneumonia、 Alive Intestinal GVHD 1.2 Preprocessing scheme All patients accepted the reduced intensity conditioning regimen, which using fludarabine (Flu), cyclophosphamide (CTX) and anti lymphocyte globulin (ATG) for 5 cases had HLA haploidentical donor, specifically: Flu 30 mg/(m2·d), -5d, -4d, -3d, -2d; CTX 40 mg/(m2·d), -4d, -3d; ATG 5 mg/(m2·d), -4d, -2d, -3d, -1d; Bai Shufei (Bu), CTX, ATG and Flu was used in 7 cases with HLA mismatched donors, specifically: Bu 3.2mg/(kg·d), -6 day; Flu 30 mg/(m2·d), - 5d, - 4d, - 3d, - 2d; CTX 40 mg/(kg·d), - 5d, - 4 d; ATG 5 mg/(kg·d), - 4d, - 3d, - 2d, - 1 d. 1.3 Stem cell collection and GVHD prevention Transplantation method was bone marrow combined with peripheral blood stem cell transplantation. Donors received mobilization by granulocyte colony stimulating factor, 5-10 μ g/(kg·d), for 5-6 days and collected bone marrow suspension on the 5 day, 8-12 ml/kg, then separated peripheral blood stem cells by blood cell separator on the 6 day. Using flow cytometry counted CD34 + cells to ensure the mononuclear cell number ≥5×108/kg, and the median CD34 + cells ≥2.0×106 / kg. Cyclosporin A (CSA), methotrexate (MTX), mycophenolate mofetil (MMF) and tacrolimus (FK506) for GVHD prophylaxis. If there was any GVHD, according to the severity, added ATG, sugar cortical hormone, and CD25 monoclonal antibody. If with no GVHD the immunosuppressant reduced gradually in the half year after transplantation. 1.4 The method of lymphocyte subsets detection T lymphocyte subsets were detected by 3-4ml venous blood from children.The main reagents and instruments were monoclonal antibodies, red cell lysis and FACSCantoII type flow cytometry. Take two FACS tubes with anticoagulant blood 100μl, joined CD3-APC, CD8-APC, CD4-FITC, CD45-VioGREENA, CD56-PE monoclonal antibody each 10μl into one tube, mixed and incubated protect form light at room temperature for 20 min, then add the FACS Lysing solution lysis to be measured. CD4-FITC, CD45-VioGREENA, CD25-APC, 10μl respectively, were added into the other tube. All of the above antibodies were cell membrane antibodies. 1.5 Intracellular markers FOXP3 Using intracellular staining method for the detection of Foxp3 antibody, specific steps as follow, in accordance with the above methods with membrane labeled antibody, incubation, hemolysis, centrifugal, discard supernatant, then add 2ml PBS to mix, 1050rpm centrifugation for 5 minutes, add 750 mu IF/P/D and 250 mu IF/P to mix after discard supernatant, 4℃ for 30 minutes, and add 2 ml buffer to mix, then centrifuge 5 minutes to discard the supernatant, add 3ul cytoplasmic antibody FOXP3-PE, in dark at room temperature light incubation then add 2 ml buffer to mix after incubation for 30 minutes at room temperature. Finally, add 0.5ml PBS to mix then detected on the machine. 1.6 Follow up and statistics Follow up to March 2015, the average follow-up time of 17.3 months (6-26 months), the follow-up period of survival children were more than 1 year, and the longest follow-up period was up 26 months. Analyse implantation, transplantation related complications and disease-free survival situation of all children, then use flow cytometry (FCM) dynamic to monitor the lever of CD3+, CD4+, CD8+, CD4+/CD8+ and CD56+, CD4+CD25high+FOXP3+ in aplastic anemia patients before transplantation and on + 1, + 3, + 6 and + 12 month after transplantation, and statisticsthe overall survival rate of the whole group. Monitor T lymphocyte subsets in healthy children as control group at the corresponding period. Using SPSS13.0 statistical software for statistical processing. The measurement data were expressed by mean± standard deviation( x ±SD), P<0.05 was statistically significant, and survival analysis was used Kaplan-Meier method. 2 Result 2.1 The effect of transplantation Follow up to March 2015, all patients obtained hematopoietic reconstitution after transplantation and the mean time of neutrophil ≥ 0.5 *109 / L and platelet ≥ 20*109/L were 16.8d and 18.5d respectively. After transplantation, 8 cases of acute GVHD, no chronic GVHD occurred, there were 6 cases of pulmonary infection, 1 case of septicemia, 2 cases of cytomegalovirus viremia, 2 cases of bleeding cystitis, 1 case of oral infection. 1 case died of acute GVHD, 1 case died of pulmonary infection, and the remaining 10 cases were alive. (Figure 1) Figure 1 Survival curves of all cases 2.2 The comparison of T lymphocyte subsets between before and post transplantation Compared to the control group, CD3+ cells of AA children was slightly higher after transplantation, CD4+ cells decreased, CD8+ cells increased, CD4+/CD8+ decreased, CD56+ cells decreased, CD4+CD25high+FOXP3+ cells reduced, and the differences above were statistically significant (P< 0.05). 10 patients existed abnormal immune cells (CD4+/CD8+ inverted ) in 12 cases with AA. (Table 2) Table 2 The comparison of peripheral blood T lymphocyte subsets betweeen AA patients and control group ( x ±SD) Group AA group Cases CD3+ CD4+ 12 (66.79±7.35)% (33.73±7.26)% (35.69±6.78)% (1.23±0.56)% (7.46±2.8)% (3.3±1.5)% (62.74±5.58)% (39.54±3.46)% (25.34±4.36)% (16.73±3.7)% (8.1±1.3)% 0.043 0.037 Control group 12 P CD8+ CD4+/CD8+ CD56+ (1.78±0.34)% 0.000 0.001 CD4+CD25high+FOXP3+ 0.000 0.003 2.3 Changes of T lymphocyte subsets after transplantation This study showed that 1 month after transplantation, the proportion of CD3+ cells decreased, the difference was statistically significant(P< 0.05), CD3+ cells gradually returned to the level of before transplantation on the 3 month after transplantation and CD8+ cells had been restored and even more than the level of before transplantation, CD4+ cells recovery delayed, the ratio of CD4+/ CD8+ sustained inversion more than 1 year; CD56+ cells were significantly lower on 1 month after transplantation, and gradually restored on 3 month. CD4+CD25high+FOXP3+ cells were lower in early stage after transplantation. (Table 3) Table 3 The level of T lymphocyte subsets in AA children before and after transplantation ( x ±SD) Group Pre CD3+ Cases CD4+ CD8+ CD4+/CD8+ CD56+ CD4+CD25high+FOXP3+ 12 (66.79±7.35)% (33.73±7.26)% (35.69±6.78)% (1.23±0.56)% (7.46±2.8)% (3.3±1.5)% Post/+1M 12 (48.0±28.2)%* (20.8±23.7)%* (20.2±23.8)%* (1.5±2.7)%* (5.35±3.2)%* (1.2±1.1)%* (1.1±0.9)% +3M 12 (58.1±27.1)% (10.8±7.7)% (51.3±18.8)% (0.2±0.2)% (12.65±3.6)% +6M 12 (63.9±28.4)% (23.7±7.6)% (37.5±28.3)% (1.0±0.9)% (13.78±2.1)% (1.6±1.7)% (79.1±6.5)% (28.0±2.9)% (47.4±6.1)% (0.6±0.1)% (15.73±4.7)% (1.3±0.6)% +12M 12 Note:* Compared with befor e transplantation,P<0.05 2.4 The correlation between GVHD and CD4+CD25high+FOXP3+ 8 cases developed acute GVHD after transplantation in this group, defined as GVHD positive group, 4 cases with no acute GVHD, defined as GVHD negative group. The difference of CD4+CD25high+FOXP3+ in GVHD positive group and negative GVHD group on+ 1, + 3, + 6 and +12 month after transplantation statistically significant (P < 0.01).(Table 4) Table 4 The comparison of CD4+CD25high+FOXP3+ between GVHD positive group and negative group ( x ±SD) Group CD4+CD25high+FOXP3+ Cases +1M GVHD positive group 8 (0.4±0.6)% +3M +6M (0.7±0.3)% (1.1±0.5)% +12M (1.4±0.3)% GVHD negative P group 4 (1.6±0.7)% (2.7±0.4)% 0.007 0.000 (2.9±0.7)% 0.005 (3.6±0.2)% 0.006 3 Discussion With the continuous improvement of allogeneic hematopoietic stem cell transplantation technology, HSCT has become the most effective for children with SAA[7-8]. The reconstruction of immune function in children requires a long time after transplantation, which means the recovery of various immune effector cells. Hematopoietic stem cells produce lymphatic hematopoietic stem/progenitor cells in the bone marrow after HSCT,then migrate to the thymus, and proliferate, differentiate and maturate[9]. Generally, CD3+ cells gradually returned to normal on +3 month after transplantation, and the reduction of CD4+ cells was more than 1 year, CD8+ cells recovered quickly, and the inversion of CD4+/CD8+ was more than 1 year. The reason may be that the generation of CD4+ cells depend mainly on the thymus, while the formation and maturation of CD8+ cells are not completely dependent on the thymus[10]. Related research[11] showed that, lymphocyte subsets especially the dynamic balance of T lymphocyte subsets was related to immune function closely. Allogeneic hematopoietic stem cell transplantation technique significantly improves the disease free survival rate of the SAA patients. Infection, GVHD, transplantation related complications concern the efficacy after transplantation, and the immune function recovery can reduce transplantation related complications[12]. Therefore, it is very important to monitor the immune reconstitution after transplantation, which has important significance to improve the reconstitution of immune function after transplantation. For the role of T lymphocytes in the pathogenesis of SAA[13], now we consider that the CD4+ and CD8+ T cells regulate hematopoiesis. CD4+ T cells are helper inducer T lymphocyte subset(Th cells) which can stimulate the hematopoiesis, and its increase means the immunologic function enhance. CD8+ T cells are inhibited T lymphocyte subset(Ts cells) which can inhibit the hematopoiesis, and its increase means immunologic function inhibit. The ratio of CD4+/CD8+ indicates the equilibrium state between Th and Ts, and is the most important index of the environmental stability in human immune system,the lower ratio can cause immune function reduced. The imbalance of CD4+ and CD8+ T cells is fundamental cause to inhibit the hematopoietic function in AA children[14]. This study also showed that the T lymphocyte subsets were unbalanced in the peripheral blood among a considerable part of cases. The main performance were that CD8+ cells increased, the CD4+ cells decreased, and the ratio of CD4+ /CD8+ reversed. In addition, the number and activity of natural killer cells in most cases were significantly decreased[15], and the same results were also obtained in this study. The most important reason is that the existence of CD4+CD25+FOXP3+ regulatory T cells (Tregs) inhibits the activation of T cells in the normal state which makes no autoimmune response under normal condition[16]. Immune incompetence and immune suppression of CD4+CD25+FOXP3+ Tregs can be activated to turn into inhibit effector T cells. Foxp3 was discovered in recent years which is the new member of the forkhead/winged-helix transcription factor family, and plays an important role in the Treg differentiation[17]. FOXP3 specifically expressed in the cytoplasm of CD4+CD25+Treg cells, which mediates the development of Treg in the thymus and its expression in the periphery, and maintains its immunologic suppression[18]. Magenau[19] confirmed that + + + CD4 CD25high FOXP3 can be as a predictor of acute GVHD and a valuable biological marker of prognosis. CD4+CD25+FOXP3+ Tregs reduced in most children with AA, this may weaken T cell immune suppression function, to sustain the activation of the effector T cells, eventually led to the imbalance of immune regulation. This study aimed to explore the immune function recovery of the children with SAA after the allogeneic hematopoietic stem cell transplantation through monitoring the T lymphocyte subsets pretransplantation and on +1, +3, +6 and +12 month posttransplantation dynamicly. Our research showed that CD4+CD25high+FOXP3+ increased gradually after transplantation in children withno acute GVHD, and finally reached to the normal level, and CD4+CD25high+FOXP3+ relatively reduced in children with acute GVHD. Compared to 4 cases withno acute GVHD, CD4+CD25high+FOXP3+ lower in 8 cases with acute GVHD (P < 0.05), and no chronic GVHD occurred which related the small sample. In summary, the level of CD4+CD25high+FOXP3+ is closely related to the occurrence of acute GVHD. We can detect and prevent the acute graft-versus-host disease (GVHD) before the appearance of clinical symptoms through monitoring regularly the level of CD4+CD25high+FOXP3+ after transplantation in peripheral blood, so as to improve the survival rate of SAA patients with transplantation. References 1. Jaime-Perez JC, Colunga-Pedraza PR, Gomez-Ramirez CD, et al. Danazol as first-line therapy for aplastic anemia[J]. Ann Hematol 2011,90(5):523-7 2. Szpecht D, Gorczyńska E, Kałwak K, et al. Matched sibling versus matched unrelated allogeneic hematopoietic stem cell transplantation in children with severe acquired aplastic anemia: experience of the polish pediatric group for hematopoietic stem cell transplantation[J]. Arch Immunol Ther Exp (Warsz) 2012,60(3):225-33. 3. Yoshizato T, Dumitriu B, Hosokawa K, et al. Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia[J]. N Engl J Med 2015,373(1):35-47. 4. Dufour C, Pillon M, Sociè G, et al. Outcome of aplastic anaemia in children. 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