The Expression Of Human Leukocyte Antigen-G In Acute Leukemia

Background and purpose:One kind of the malignant tumors, leukemia, is a serious threat to human life. Acute leukemia is the first rank of death caused by cancer for adults under the age of 35. Recent years, with the wide range of applications of high-resolution multi-parameter flow cytometry and vigorously conduct of malignant blood cell immune phenotype testing, as well as karyotype analysis, FISH, and etc. From the morphology, immunology, cytogenetics and molecular biology level, diagnosis of leukemia and its sub-type and prognosis, the diagnosis and treatment of leukemia are increasingly explicit and accurate. However, specific differentiation antigen and its monoclonal antibody research is not deep enough. Human MHC, also named human leukocyte antigen(HLA), which locates in the short arm of chromosome VI, which is a tightly linked group of highly polymorphic gene cluster. HLA-G is a non-classical HLA-Ⅰclass molecule involved in the formation of maternal-fetal immune tolerance. HLA-G molecule was low restriction polymorphism, evolutionary conservation, limited expression in healthy tissue, with the immune regulatory. Under normal physiological conditions, its product expresses in some areas of immune privilege, such as the interface of maternal-fetal, thymic medulla between the epithelial cells, some of the eye cornea. It can up-regulate in the tissues. In different pathological conditions, such as tumors, the virus infection, skin inflammation, psoriasis, or a well-functioning organ graft case, HLA-G molecule can occur, as well as in renal cell carcinoma, breast cancer, lung cancer, leukemia, neural stromal tumors, early cutaneous Iymphoma, melanoma patients. As a new immunosuppressive molecules, HLA-G have been extensively studied in pregnancy immunization, tumor immune escape, and solid organ transplantation. Preliminary study of its mechanism in the induction of immune tolerance:inhibition of CD8+T cells and natural killer (NK) cell's killing activity, inhibition of the proliferation of CD4+T cells, inhibition of heterologous T cell proliferation reaction cycle, affecting antigen-presenting cells(APC)'s function and maturation process, induction of regulatory T cells(Treg cells), regulation of cytokine secretion, etc. A result of HLA-G and immune tolerance closely related to each other, cellular immunity, other than humoral immunity, plays a more important role in the anti-tumor immune responses. T lymphocytes and NK cells execute the cellular immune function, are the body's important cells of anti-tumor immune activity. By the determination of HLA-G of leukemia patients, this study explores the differences and trends of the HLA-G expression of newly diagnosed leukemia patients and patients with refractory and relapsed leukemia before and after treatment (chemotherapy), and significance of diagnosis and monitoring of the occurrence and development of leukemia with HLA-G concentration.Methods:We resarch 10 healthy people and 50 cases of leukemia patients. Membrane-bound HLA-G(mHLA-G, HLA-G 1) expression before and after treatment of newly diagnosed patients, healthy people, and relapse patients were detected by flow cytometry. Peripheral blood plasma secreted HLA-G(sHLA-G, HLA-G5), transforming growth factorβ(TGF-β) and Interleukin-10(IL-10) expression of newly diagnosed patients before and after treatment, relapse patients and normal human were detected by Enzyme-linked immunosorbent assay (ELISA). Patients' mononuclear cells'HLA-G1, HLA-G5 mRNA were detected by Real-time PCR. Statistical analysis:SPSS13.0 statistical package was used for processing all data, indicated by mean±standard deviation (x±SD). The differences before and after treatment were analyzed with two separate paired-samples t test; When the data was in line with normal distribution and homogeneity of variance, then using single-factor analysis of variance (ONE-WAY ANOVA), further multiple comparisons between groups using LSD method. If the data was not meet the homogeneity of variance, then using Welch's method to correct F values, further multiple comparisons using Dunnett T3 method. A P-value≤0.05 was considered to be statistically significant.Results:1. sHLA-G level:Normal group's sHLA-G plasma level was 5.87±2.07 ng/ml, leukemia patients with newly diagnosed group was 10.05±6.58 ng/ml, refractory and relapse group was 13.37±8.01 ng/ml, the newly diagnosed group's sHLA-G levels were significantly higher than the normal group, the difference was significant (P<0.05). Pre-chemotherapy group and post-chemotherapy group of CR patients was 9.10±4.34 ng/ml,4.74±1.73 ng/ml, respectively; Pre-chemotherapy's sHLA-G levels were significantly higher than post-chemotherapy group, the difference was significant (P<0.05). Pre-chemotherapy group and post-chemotherapy group of NR patients was 10.10±7.72 ng/ml,9.29±0.56 ng/ml, respectively; the difference was not significant (P>0.05). NR patients'sHLA-G expression in post-chemotherapy group was significantly higher than that of CR patients, the difference was significant (P<0.05).2. mHLA-G level:Normal group's mHLA-G level was 0.29%±0.20%, leukemia patients with newly diagnosed group was 0.60±0.44%, refractory and relapse group was 0.67±0.21%; the newly diagnosed group and refractory and relapse group's mHLA-G levels were significantly higher than the normal group, the difference was statistically significant (P<0.05); Pre-chemotherapy group and post-chemotherapy group of CR patients was 1.03±0.50%,0.31±0.17%, respectively; pre-chemotherapy's mHLA-G levels were significantly higher than post-chemotherapy group, the difference was statistically significant (P<0.05). Pre-chemotherapy group and post-chemotherapy group of NR patients was 0.81±0.23%,0.88±0.27%, respectively; the difference was not significant (P>0.05). NR patients' mHLA-G expression in post-chemotherapy group was significantly higher than that of CR patients, the difference was significant (P<0.01).3. HLA-G mRNA expression:Newly diagnosed patients'HLA-G5 mRNA expression level was significantly higher than the healthy control group, the difference was significant (P<0.05); CR patients' HLA-G5 mRNA in pre-chemotherapy group was significantly higher than that in post-chemotherapy group, the difference was significant (P<0.05). The difference was not significant between newly diagnosed patients'HLA-G1 mRNA and healthy people'(P>0.05). The difference of HLA-G1 mRNA of pre-chemotherapy group and post-chemotherapy group of CR,NR patients was not significant (P>0.05). the same result can be obversed between NR patients and CR patients in the post-chemotherapy group(P>0.05). But NR patients'HLA-G5 mRNA expression was significantly higher than that of CR patients, the difference was significant (P<0.01). 4. HLA-G and FAB type:Patients with various leukemia type had no significant difference among the mHLA-G, sHLA-G and HLA-G mRNA expression. (P>0.05) 5. Cytokines level:The TGF-βlevels of newly diagnosed leukemia patient group were significantly lower than that of normal group, the difference was significant (P <0.01); The IL-10 levels of newly diagnosed leukemia patient group were significantly higher than that of normal group, the difference was significant (P <0.05). The TGF-β, IL-10 levels of pre-chemotherapy group of CR patients were significantly higher than that of post-chemotherapy group, the difference was significant (P<0.01). The difference of TGF-β, IL-10 of pre-chemotherapy group and post-chemotherapy group of NR patients was not significant (P>0.05). NR patients' TGF-P, IL-10 levels in post-chemotherapy group was significantly higher than that of CR patients, the difference was significant (P<0.01). The sHLA-G levels and TGF-P levels in the peripheral blood in patients with newly diagnosed acute leukemia had a significant negative correlation, but not in relapse patients. The sHLA-G levels and TGF-P levels in the peripheral blood between the CR and NR patients in the pre-chemotherapy group and post-chemotherapy group showed no significant correlation; The sHLA-G expression and IL-10 levels in the peripheral blood in patients with newly diagnosed acute leukemia had a significant positive correlation, but not in relapse patients. The sHLA-G levels and IL-10 levels in the peripheral blood between the CR and NR patients in the pre-chemotherapy group and post-chemotherapy group showed no significant correlation.Conclusions:Whether patients are acute leukemia in newly diagnosisd or refractory relapse status, the HLA-G expression in peripheral blood was significantly increase, suggesting that HLA-G may inhibit tumor immune in the state of leukemia, also may be involved in the development of acute leukemia. The CR patients's HLA-G, mHLA-G expression and HLA-G5 mRNA expression after treatment were significantly lower than that before treatment, while the NR patients'sHLA-G and mHLA-G expression did not change after treatment, and it maintained the high status as that before treatment, suggesting that monitoring of sHLA-G and mHLA-G expression in acute leukemia play an important role in monitoring and evaluating the state of acute leukemia. The sHLA-G expression and TGF-p level in newly diagnosed acute leukemia patients had a significantly negative correlation, while the sHLA-G expression and IL-10 had a significant positive correlation, suggesting that cytokines like TGF-P and IL-10 in newly diagnosed acute leukemia patients had the interactions with the HLA-G involved in the pathogenesis of acute leukemia.