Association Of Killer Cell Immunoglobulin-Like Receptor Gene Diversity With Graves Disease

Background:Graves disease (Graves diseases, GD) is a kind of organ-specific autoimmune diseases, which is associated with high secretion of Thyroid hormone (TH), and characterized by high metabolic syndrome, diffuse enlargement of the thyroid gland, Graves ophthalmopathy (GO) and pretibial myxedema. Graves disease, whose incidence keeps increasing, is the most common cause of hyperthyroidism, affecting between2%and4%of women and up to1%of men. At the same time, GD has the youth oriented tendency, which severely threatens our people’s health.The pathogenesis of Graves disease has not been fully elucidated, but it is well-established that GD takes genetic susceptibility as a background, starts with factors such as infection, toxins, and medicines, finally leads to immune dysfunction in the body. Researches suggest that Ts cells function deficiencies weaken the inhibitory effects of Th cells, who assist B lymphocytes in generating TSH receptor antibodies (TRAb). These antibodies, which combined with TSH receptor extracellular domain structure, lead to the occurrence of hyperthyroidism, by causing hyperplasia of the thyroid gland, resulting in secreting and releasing a large amount of thyroid hormone. Acting as immune modulators, immune effector molecules, or other factors involving in this procedure, T lymphocytes, NK cells and a variety of cytokines play a significant role in the occurrence, development and prognosis of GD.Natural killer cells (NK cells), which are a class of lymphocytes distinct from T cells and B cells, have direct killing effect of target cells, and function as anti-tumor, anti-infection, and immune regulation, they also participate in transplantation rejection and autoimmune disease. NK cells function mainly depends on the synthesis of activating and inhibitory receptors on the cells surface, and the balance of these two receptors will affect the function of NK cells. In recent years, the role of NK cells in Graves disease has been under the spotlight. And study proves that the GD patiences have abnormalities in NK cells counts, activity, and secretion of cytokines.Killer cell immunoglobulin receptors (KIRs) expressed by natural killer cells and subsets of T lymphocytes, belong to immunoglobulin superfamily (Ig-SF), and modulate its function upon recognition of MHC class I molecules. According to the function, KIRs can be parted to inhibitory and activating receptors. In humans, the KIR locus, containing a family of polymorphic and highly homologous genes, maps to chromosome19q13.4within the leukocyte receptor complex (LRC). So far,14expressed KIR genes and2pesudogenes have been identified. Besides allelic polymorphism, haplotype diversity also consists of human KIR genes diversity. The high polymorphism of KIR genes formed the basis of disease susceptibility and different immune responses between different individuals and racials. Our previous research found that GD patients KIR2DS4gene frequency was significantly lower than the control group, so we speculated KIR2DS4might be the protective gene of GD.Objective:To investigate the association of KIR-HLA gene combination diversity with Graves disease; To analysis the expression of KIR2DS4on NK cells and its expression level in correlation with Graves disease. To determine the proportion and killing activity of NK cells, preliminary discussing the immune mechanism of NK cells in Graves disease.Method:1. Select100Graves disease patients and86healthy controls matched for age, sex, and use the method of PCR/SSP to augment their KIR genes (KIR2DL1, KIR2DS1, KIR2DL2, KIR2DS2, KIR2DL4) and HLA-Cw01-08genes. Two different primer sets were designed for each KIR gene except KIR2DL4. According to the HLA and KIR receptor ligands combination rule, We analyzed association of HLA-C/KIR combinations with GD.2.95Graves disease patients and83age, sex matched healthy controls were enrolled to detect the expression of protective gene KIR2DS4(CD158i) on GD using the method of flow cytometry. Whereafter, the relevance of the KIR2DS4(CD158i) expression level to GD was studied. 3. Gather clinical dates of T lymphocyte subsets distribution and proportion of NK cells in GD group and control group peripheral blood, to discuss T cell subsets and NK cells’function in GD mechanism.4. We detected people’s peripheral blood mononuclear cell surface molecules, labeled with specific antibodies including CD3, CD56and CD69, who took part in method2, so as to measure the portion and activity of NK cells and analysis their roles in GD.Result:1. The comparison of HLA gene frequencies between the patients with GD and the control group:GD patients had a higher proportion of HLA-Cw01(p<0.05), and a lower proportion of HLA-Cw03and HLA-Cw06(p<0.01).2. The comparison of KIR-2D/HLA-Cw combinations between GD group and the control group:KIR2DL1(+)HLA-CwLys(+)(p<0.05) and KIR2DS1(+)HLA-CwLys(+)(p<0.05) frequencies were lower in the GD patients than in the control group, and the difference was significant. However, the results showed no reveal statistically significant difference in KIR2DL1/S1/HLA-CwLys combinations.3. In GD patients, the expression of KIR2DS4(CD158i) on NK cells was not significantly lower than that in heathy people.4. Clinical dates showed that the GD patients had a lower proportion of CD8+T cells and NK cells, and higher ratio of CD4/8, with p<0.05.5. GD patients had a lower proportion and activity of NK cells compare to the control group. And preliminary analysis showed no correlation between NK cells activity and the expression of KIR2DS4. Conclusion:1. The frequencies of inhibitory KIR2DL1(+)HLA-CwLys(+) and active KIR2DS1(+)HLA-CwLys(+) were significantly lower in GD group than that in the control group, which illustrated there was a correlation between KIR-HLA combinations and GD.2. The expression of KIR2DS4on NK cells was lower in the GD patient group than control group, but did not reveal statistically significant difference, which stated the discrepancy between genetic and expression level disappeared in expression level, but the specific factors that influence the expression are unclear. 3. The lower proportion of CD8+T cells and high CD4/CD8ratio both suggest that CD4+T cells predominate in the GD patient group, and this may be related to TRAb production. Higher ratio of CD4/CD8, namely imbalance of helper and inhibitory T lymphocytes, weakens inhibitory T lymphocytes’ role upon antigen specific T lymphocytes Th cell and B cell, which are thought to be important condition for GD start-up and development.4. The experimental results show that the percentage of NK cells and activity are lower in GD group than control group. NK cells play roles in inhibiting or killing active B cell and its antigen-presenting function of dendritic cells, also, inhibiting the generation of immunoglobulin and cytotoxic T cells. So the decrease in the number of NK cells and/or their activities may allow the proliferation of T and B cells in thyroid gland, thereby, promote the production of antibodies.