Killer Cell Immunoglobin-like Receptor Along With HLA-C Ligand Genes Are Associated With Type 1 Diabetes In Chinese Han Population

Background and ObjectiveType 1 diabetes (T1D) is a chronic disease caused by destruction of pancreaticβcells, resulting in an absolute deficiency of insulin. T1D is considered as a multi-factorial disease with strong contributions from immunogenetic and environmental factors. Genes located in the HLA region (HLA-DR3/DR4) confer most of the genetic susceptibility for T1D, and a small number of genes outside the HLA region also contribute to increased genetic risk.Killer cell immunoglobulin-like receptors (KIRs) gene showed to be associated with autoimmune mediated diabetes in several reports. KIRs are a family of membrane glycoproteins mainly expressed by natural killer (NK) cells. NK cells are population of lymphocytes known as an important component of the innate immune system by participating in early responses against infected or transformed cells (production of cytokines and direct cytotoxicity). NK cells have been reported to play an important role in the pathogenesis of T1D, though their exact role is not clear. The link between the development of diabetes and NK cells was suggested in rodent models of autoimmune T1D. The ligands for KIRs are certain allotypes of HLA class I molecules, which can be classified into several HLA classⅠgroups:HLA-C, HLA-B, HLA-A and HLA-G. Two allotypes of HLA-C molecules showed to have specific binding affinity to KIR:HLA-C2, with a lysine at position 80, and HLA-C1 with an asparagine at position 80. The inhibitory KIR2DL1 has specific affinity to HLA-C2 allotype, while KIR2DL2 and 2DL3 bind specifically to HLA-C1. The activating receptors KIR2DS1 and 2DS2 share sequence similarity in their extracellular domains with their corresponding inhibitory counterparts (KIR2DL1 and 2DL2 respectively), therefore, they are thought to share HLA ligands binding specificities; KIR2DL4 has specificity for HLA-G; KIR3DL1 is known to bind HLA-Bw4 allotypes; and KIR3DL2 interacts with some HLA-A allotypes; KIR2DS4 is thought to interact with HLA-Cw4; KIR3DS1 can also interact with Bw4 allotypes. Ligands for KIR2DL5, KIR2DS3 and KIR2DS5 are yet to be identified. The interaction of KIRs and HLA classⅠmolecules has been explained by several hypothesis such as the "missing self" hypothesis and the "licensing hypothesis"We studied the association of KIR genes and their HLA-C ligands with susceptibility to T1DM in a cohort of patients and controls from China using a PCR based approach. We aimed to test our hypothesis that certain activatory and inhibitory KIRs may, either independently or in combination with their HLA classⅠligands, confer susceptibility to or protection from T1D.Subject and MethodsStudy Subjects:259 T1D patients (Mean age at diagnosis 6.94±3.64 years, Males: 123, Females:136) diagnosed in children's hospital of Fudan University, Shanghai, China were collected as case group.262 healthy children served as controls. The study was approved by Fudan University Pediatric Research Ethics Board. Diabetes was diagnosed according to the criteria established by the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Data on plasma autoantibodies status was available for 128 subjects for GAD65 and 88 subjects for IA2 antibodies. Of thesse subjects, GAD 65 auto-antibody was found positive in 79 subjects, IA2 autoantibody was positive in 59 subjects.KIR genotyping:PCR-sequence specific primers (PCR-SSP) based genotyping was performed to type all KIR genes and HLA-C1 and C2 ligands.ResultsKIR gene frequencies in T1D patients and healthy controls were compared. HLA-C 1 was present in 166 of 259 patients (64.1%) and 122 of 262 controls (46.6%), data showed statistic significance (OR～2.05,95%CI～1.44-2.90, p=0.0013). HLA-C2 gene was present in 127 of 259 patients (49.0%) and 98 of 262 controls (37.4%) in our study, this difference was not significant after correction. However no statistically significant difference was observed in the distribution of KIR genes between T1D patients and healthy controls. The combination of C1+/C2+ was positively (OR=2.11, P=0.0008) and C1-/C2- was negatively (OR=0.46, P=0.0008) associated with T1D. No association with T1D was found in other two HLA-C combinations (C1+/C2-C1-/C2+).Stratification analyses were performed using all possible combinations of KIR 2D genes and their HLA-C ligands. After correction of P value, our results showed that the combinations 2DL2-/HLA-C1+; 2DL3+/HLA-C1+; 2DS2-/HLAC1+ were positively associated with T1D. The combinations 2DL1+/HLA-C2-; 2DL2-/HLA-C1-; 2DL3+/HLA-C1-; 2DS2-/HLAC1- were found to be negatively associated with T1D.To identify the strongest genetic association, we performed statistical analyses as described by Svejgaard and Ryder. Results are:1) HLA-C2 was positively associated with T1D in 2DL1+ ones; 2) HLA-C1 was positively associated with T1D in 2DL2-ones; 3) HLA-C1 was positively associated with T1D in 2DL3+ ones; 4) HLA-C1 was positively associated with T1D in 2DS2+ ones; 5) HLA-C1 and C2 combination confers association, and this association is stronger than C1 alone.To test whether this association is due to interaction between KIR and HLA-C ligand genes, we performed relative excess risk due to interaction (RERI) analyses. However we did not detect any significant association.T1D patients were divided into two groups based in the presence or absence of autoantibodies against GAD65 and IA-2. The rationale for doing this was to find our whether the KIR association to T1D was modified by autoantibody positivity. The frequencies of KIRs 2DL1,2DS2 HLA-C1 were higher in T1D patients positive for GAD autoantibody; frequency of KIRs 2DS4 is higher in T1D patients positive for IA-2 autoantibody. However the association was not statistically significant after applying Bonferroni correction for multiple testing.ConclusionOur results showed no direct association of KIR genes with T1D in a Chinese Han population after correction of p value, HLA-C ligands (a combination of HLA-C1 and C2) showed positive association with T1D. Different combinations of HLA-C and KIR showed positive and negative association with T1D. Objectives:Transplantation of islets of Langerhans represents a potential cure for type 1 diabetes, but the success of it is hampered by destruction of the islets and loss of beta cell to apoptosis. A20 has a dual antiapoptosis and anti-inflammatory function in primary endothelial cells. Our aim was to evaluate the protective effect of A20 gene on rejection in islet transplantation.Methods:In this study, C57BL/6J Mice islet cells were isolated, purified and cultured, the recombinant expression Lentivirus vector, A20 was constructed and prepared to transfect islet cells which were isolated and purified from C57BL/6J mouse pancreases. The effects of islet cells modified with A20 before transplantation on the rejection were investigated. Then Sprague-Dawley (SD) rats were administered with STZ through peritoneal injection to make the rats model of diabetes. Then the islet cells were transplanted under the left kidney capsule of the diabetes rats.The rat recipients were divided into 3 groups randomly, experiment group, control group and sham-operated group. In the experiment group, the graft islet cells were infected with Lentivirus vector expressing A20; in the control group, the islet cells weren't infected; in the sham-operated group, the rats were transplanted without islets, but phosphate buffer solution (PBS). After transplantation the blood glucose were measured to evaluate the effect.Results:On the first day after transplant the blood glucose began decreasing obviously in both experiment group and control group. In the experiment group, the effective existing time of islet cells was 3 to 11 days, the average was 6±1 days. Compare with the experiment group, the time of the control group was 2 to 5 days, the average was 3±1 days.The results verify that A20 modified islet transplant can prolong the survival of grafts significantly. This study supplies a new experimental proof for using A20 gene modified islet.Conclusions:The data showed that A20 gene could prolong the existing time of rodent pancreas xenograft islet cells. A20 might be a relevant gene for protection of beta cells against the autoimmunity in islets xenotransplant. Therefore, the intervention measures based on gene therapy may have significant theoretic and applicative values in the treatment of diseases.