Expression And Clinical Significance Of CD8~+CD28~- Regulatory T Cells In Idiopathic Thrombocytopenic Purpura And Acute Leukemia

[Objective] CD8⁺CD28^-Regulatory T cell belongs to an important subset of CD8⁺Treg cells. To be more detail, it is a mature T cell subset, which has the specific suppression of effect antigen of negative regulation, and can control its self-reactive cells. In recent years, CD8⁺CD28^-Treg cell has being an important part of the study about the relationship and balance between the immune response and immune tolerance. It may involve in the incidence of autoimmune diseases and tumor. Through this study is to observe quantity and expression of peripheral blood CD8⁺CD28^-Treg cells and CD8⁺T cells of Idiopathic Thrombocytopenic Purpura(ITP) and Acute Leukemia(AL) patients, discuss them in the relationship of ITP and AL; Through comparing the situation before and after treatment with the normal group, discuss law and clinical significance of the variation of disease incidence in ITP and AL by change of peripheral blood CD8⁺CD28^-Treg cells and CD8⁺T cells; provide the possible ideas and theories for pathogenesis and treatment of immunology.[Objects and Methods]1. Study objects: Collect newly diagnosed ITP and AL patients from Blood Immunology of Yan'an university affiliated hospital in December 2009 February 2011. There are 98 cases. All cases were confirmed according to the third edition "Blood Disease Diagnosis and Treatment Standard"standardization of Zhang Zhinan and others in 2007 by physicians of Blood Immunology, excluded other diseases. Control group 30 cases were normally healthy subjects; the median age was 31.0(5-50) years, male 19 cases, and female11 cases. There was no significant difference with the composition of the experimental group in the age and gender. ITP 35 cases in study have different degree and significant bleeding in the clinic,which were collected from the outpatient and inpatient. The median age was 40.0 (3-80) years, male 23 cases, and female 12 cases, there were 20 cases with platelet for (20-50)×10⁹ / L in the treatment and 15 cases with platelet <20×10⁹ / L. Firstly they received glucocorticoids after the diagnosis. 12 of them received equivalent dose methylprednisolone pulse therapy, 9 of them received the treatment of combination with high dose gamma globulin. After treatment platelets restored, they changed to take orally prednisone. 33 cases obtained efficient treatment. The blood of 2 cases was improved markedly after the treatment by above methods, but they relapsed in the short term when the hormone was diminished a little. In such situation, the spleen was taken off finally. The group detected peripheral blood CD8⁺CD28^-Treg cells and CD8⁺T cells when they were diagnosed and treated to normal platelets after 4 weeks, and compared with the normal group respectively. There were 63 cases AL patients, who were collected from the hospital, among them, male 36 cases, and female 27 cases. The median age was 25.0(2-81) years. All cases were determined by bone marrow cytology,cytochemistry and flow cytometry immunophenotype. While Acute Lymphoblastic Leukemia (ALL) patients also need to be checked by using fluorescent hybridization fusion gene BCR / ABL, M₃ by using PML-RARαand M₂ by using AML1-ETO. Among these 63 cases, 33 cases were Diagnosed ALL, 1 case mixed type, 29 cases Acute Non-Lymphocytic Leukemia(ANLL), 3 cases M₁, 5 cases M₂, 8 cases M₃, 7 cases M₄, 4 cases M₅, 2 cases M₆. ALL were released by using the chemotherapies of VMP,VDLP,CEA respectively. 1 case fusion gene BCR-ABL (+) patient added to take imatinibmesylate; M₃ PML-RARα(+) patients were done chemotherapy , taking ATRA and As₂O₃. Other ANLL patients were released by using the chemotherapies of MA (DA or HA),MEA (or DEA). After the second course, 39 cases were released complete remission (CR) and 21 cases partial remission (PR). 1 case died during the first course of chemotherapy, 1 case gave up and 1 case died after the second course of chemotherapy. 60 cases with complete data were drawn peripheral blood CD8⁺CD28^-Treg cells and CD8⁺T cells to compare with normal group when diagnosed, done chemotherapy released PR, released CR respectively.2. Methods: Take the fasting blood 7ml with empty stomach in the morning,â‘ Detect quantity and expression of peripheral blood CD8⁺CD28^-Treg cells,CD8⁺T cells of ITP,AL patients before and after treatment compared with normal group by micro flow cytometry of labeled doors with fluorescence CD3;â‘¡Use the Cellquest software to analyze data, record the percentage of cells in each group, diminish non-specificity control group and carry out comprehensive analysis;â‘¢Detect platelet(PTL) and white blood cell (WBC)parameters of ITP and AL patients by resistance-type blood cell analyzer provided by the German COULTER JENS company. Operations were strictly carried out in accordance with instructions;â‘£Statistical analysis, use the SPSSl6.0 statistical software , the comparison of distribution that meet the measurement data was checked by independent sample T ; the comparison of distribution that didn't meet the measurement data was checked by rank sum . Use non-parametric Spearman rank correlation test to straight line correlation analysis. P = 0.05 was the standard.[Results]1. The analysis of peripheral blood CD8⁺T cells and CD8⁺CD28^-T cells immunophenotype:1.1 Quantity of peripheral blood CD8⁺T cells and CD8⁺CD28^-T cells of ITP patients before treatment was compared with normal group: CD8⁺T cells were increased, while CD8⁺CD28^-T cells were decreased significantly, and the differences possessed statistical significance (P +T cells and CD8⁺CD28^-T cells were compared with those before treatment, CD8 + T cells were decreased. While CD8⁺CD28^-T cells were increased, the differences also possessed statistical significance (P +T cells and CD8⁺CD28^-T cells became normal when they compared with normal group, and there was no significant difference between the two groups. The blood of 2 cases was improved markedly after the treatment by above methods, but they relapsed in the short term when the hormone was diminished a little. In such situation, the spleen was taken off finally. And peripheral blood CD8⁺CD28^-T cells were detected repeatedly, which did not change significantly before and after treatment.1.2 Quantity of peripheral blood CD8⁺T cells and CD8⁺CD28^-T cell of AL patients before treatment was higher than the normal group, the differences possessed statistical significance (P +CD28^-T cells of PR and CR patients was lower than those before treatment, CD8⁺CD28^-T cells of CR patients was especially decreased more significantly than PR, the differences of them possessed statistical significance(P +CD28^-/CD8⁺T cells immunophenotype:Before treatment CD8⁺CD28^-/CD8⁺T cell of ITP patients was lower than the normal group, while AL patients was higher than the normal group, the differences possessed statistical significance (P +T cells and CD8⁺CD28^-Treg cells of ITP, AL patients:Though checking between peripheral blood CD8⁺T cells of ITP, AL patients and CD8⁺CD28^-Treg cells as Spearman rank correlation test showed that: there were no correlation relationship between peripheral blood CD8⁺ T cells and CD8⁺CD28^-Treg cells in ITP patients, (r = 0.151, P = 0.386); and there was positive correlation relationship in AL patients, (r = 0.857, P = 0.000).4. The correlation analysis of peripheral blood CD8 +T cells, CD8⁺CD28^-Treg cells and quantity of PTLand WBC:Use non-parametric Spearman rank correlation coefficient test result showed that: peripheral blood CD8⁺T cells, CD8⁺CD28^-Treg cells and quantity of PTL of ITP patients were no correlation relationship; peripheral blood CD8⁺T cells, CD8⁺CD28^-Treg cells and quantity of WBC of AL were also no correlation relationship, namely, (r =- 0.116, P = 0.507), (r =- -0.082, P = 0.638), (r =- 0.070, P = 0.6051 ), (R =- 0.051, P = 0.704).[Conclusion]1. Expression levels of peripheral blood CD8⁺CD28^-Treg and CD8⁺T cells may be closely related to the occurrence and development of ITP, and its height may reflect the degree of cellular immune dysfunction and situation of disease activity coercion in ITP, and CD8⁺CD28^-Treg cells may be one of important cell populations that involved in its pathogenesis in the cellular immunity of ITP patients.2. Peripheral blood CD8⁺CD28^-Treg cells and CD8⁺T cells have some relevance in the pathogenesis of AL. Monitoring the expressive level of its ratio mayhave certain value of clinical guidance in the therapy and prognosis of AL. After treatment the quantity of peripheral blood CD8⁺CD28^-Treg cells in CR patients were lower than PR patients, and tended to be normal. It also showed that cellular immunity was suppressed severely in AL patients.3. Expression levels of peripheral blood CD8⁺CD28^-Treg cells in ITP patients was lower than normal, CD8⁺T cells was higher than normal; While expression levels of peripheral blood CD8⁺CD28^-Treg cells and CD8⁺T cells in AL patients was even higher than normal.4. Expressive levels of peripheral blood CD8⁺CD28^-Treg cells in ITP patients was no correlation with CD8⁺T cells; While in AL patients CD8⁺CD28^-Treg cells were positive correlation with CD8⁺T cells; Expression levels of peripheral blood CD8⁺CD28^-Treg cells in ITP and AL patients were respectively no correlation with quantity of PTL and WBC. Immune dysfunction was easier to correct in ITP patients, AL followed.