| ObjectiveTo explore the role of HLA-DQ in systemic immune response in severe aplastic anemia(SAA),further reveal the immune pathogenesis of SAA and provide theoretical support for the development of new immunotherapy by studying the expression level of human leukocyte antigen(HLA-DQ)in myeloid/conventional dendritic cells(m DCs/c DCs)of SAA patients and its influence on systemic immune downstream effect-T cells.Methods1.HLA-DQ protein and m RNA expression level in peripheral m DCs from 30 SAA patients(15 untreated and 15 recovring patients)and 15 healthy people were detected by flow cytometry,Western blot method(WB)and real-time fluorescence Quantitative Polymerase Chain Reaction(q RT-PCR)methods.Correlation analysis were performed between the levels of HLA-DQ in untreated SSA patients and their clinical index and immune status.HLA global gene sequencing was performed in 12 untreated patients with SAA and 10 healthy subjects by double-end sequencing.2.The expression of HLA-DQm RNA in m DCs of untreated SAA patients was knockdown by small interfering RNA.m DCs proliferation was detected by CCK-8method before and after gene knockdown.The phagocytosis and apoptosis of m DCs and the expression of CD80/CD86 were detected by flow cytometry before and after gene knockdown.3.CD4~+T cells were co-cultured with HLA-DQ-knockdown m DCs of untreated SAA patients.Th1 and Th2 lymphocyte cytokine concentrations and the regulatory T cells(Treg)number were detected by flow cytometry before and after gene knockdown.4.Immune-mediated bone marrow failure mouse model was established.The amount of m DCs and the expression of costimulatory molecules CD40,CD80 and CD86 in peripheral blood of AA and normal control mice were detected by flow cytometry.The expression levels of CD40,CD80 and CD86 on m DCs were detected before and after treatment with cycloporine and levamisole in AA mice.The blood routine and survival rate were observed before and after treatment.Results1.The expression rate of HLA-DQ on peripheral m DCs in untreated SAA patients(SAA)group,recovering SAA patients(RSAA)group(18.44±8.53%,14.88±13.33%)was higher than that of healthy control(HC)group(8.59±6.14%)(p<0.001,p<0.05),it was negatively correlated with peripheral white blood cell count(r=-0.602,p<0.05),absolute neutrophil value(r=-0.530,p<0.05),reticulocyte percentage(r=-0.567,p<0.05),CD4~+T/CD8~+T ratio(r=-0.620,p<0.05),Treg proportion(r=-0.531,p<0.05),the percentage of granulocytoid and erythroid cells in bone marrow(r=-0.627,p<0.05),and was positively correlated with the percentage of lymphocyte in bone marrow(r=0.627)and CD86 expression rate on m DCs(r=-0.586,p<0.05),and was positively correlated with the proportion of m DCs in peripheral blood(r=0.836,p<0.001).The natural logarithm of HLA-DQB1 m RNA relative expression of SAAgroup was(3.33±2.58),which was higher than that RSAA group(-0.31±2.25)and HC group(-1.91±2.60).WB test showed that HLA-DQ expression level in m DCs of SAA patients was higher than that of healthy people.The frequency of HLA-DQB1*06:01:01 in SAA(4.17%)was lower than that of healthy people(30%)(p=0.0353).2.The m DC proliferation(OD value)in HLA-DQ knockdown group(0.40±0.16)was lower than that in control group(0.65±0.20)(p<0.01);the phagocytosis rate(15.99±5.03%)was lower than that of the control group(20.29±4.06%)(p<0.01),the expression rate of CD86 on m DCs(60.99±8.56%)was lower than that of control group(67.29±10.11%)(p<0.05);The percentage of total apoptotic cells in HLA-DQ knockdown group(46.56±18.96%)was higher than that in control group(34.33±15.25%)(p<0.01).3.The concentration of IL-2 in co-culture supernatant of HLA-DQ knockdown group was(171.83±44.92)pg/ml,which was lower than that of control group(236.93±74.79pg/ml)(p<0.05);the concentration of IFN-γ(1515.87±535.87pg/ml)was lower than that of control group(2142.20±714.61pg/ml)(p<0.05);the concentration of TNF-α(162.83±52.46pg/ml)was lower than that of blank control group(264.95±72.70 pg/ml)(p<0.05);there was no significant difference in TNF-αconcentration between HLA-DQ knockdown group and negative control group(p=0.07).There was no significant difference in IL-4,IL-6 and IL-10 concentrations between HLA-DQ knockdown group and control groups.There was no statistical significance in the proportion of Treg cells among the three groups.4.The proportion of m DCs in peripheral blood of AA mice(2.07±0.58%)was higher than that of normal control group(1.30±0.73%)(p<0.05),CD40 expression rate on m DCs in AA mice(74.39±10.30%)was higher than that of normal control group(55.66±9.29%)(p<0.01),CD86 expression rate on m DCs in AA mice(36.53±8.80%)was higher than that of normal control group(27.20±6.93%)(p<0.01),there was no significant difference in CD80 expression on m DCs compared with normal control.The expression rates of CD40 and CD86 in the cyclosporine treatment group decreased,the blood routine gradually recovered,and the survival period was prolonged.There was no statistical significance of the expression of m DC costimulatory molecules in the levamisole intervention group.The blood routine of mice in the levamisole intervention group was significantly lower than that in the other groups at 17 days,but the blood routine cells of all lines of mice survived by levamisole treatment showed a slight slow recovery over time.Conclusion1.The expression level of HLA-DQ in m DCs of untreated SAA patients was higher than that of healthy people,and the pathway protein expressions were also higher.The expression level of HLA-DQ was significantly correlated with the severity of disease and the severity of immune disorders.HLA-DQB1*06:01:01 may be a protective factor for SAA.2.The knockdown of HLA-DQ gene in m DCs in SAA patients resulted in the decrease of m DC proliferation,phagocytosis and expression of costimulatory molecule CD86,and the increase of apoptosis.HLA-DQ plays a positive role in maintaining m DC function and activating downstream immune response.3.The knockdown of HLA-DQ gene in m DCs in SAA patients resulted in the decrease of the secretion levels of the Th1 type of lymphocyte cytokines,and there was no significant effect on the secretion of Th2 lymphocyte cytokines.Treg has no change in the co-culture.4.The number of m DCs in AA mice increased,and the expression of surface costimulatory molecules CD40 and CD86 increased.After cyclosporine treatment,the expression of costimulatory molecules on m DCs in AA mice decreased,blood routine gradually recovered,and life span was prolonged.After levamisole intervention in AA mice,there was no significant difference in the expression of costimulatory molecules on m DCs.In the early stage of levamisole intervention,the disease of mice was more serious,and in the late stage of intervention,the blood routine level of surviving mice tended to recover.5.The role of HLA-DQ in the immune pathogenesis of SAA has great potential research value and may become a breakthrough in the development of new immunotherapy. |
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