The Role Of Major Histocompatibility Complex Class I Chain-related Gene A In Organ Transplantation

1. Aims of Research(1) MICA (MHC class I related Chain A) are polymorphic glycoproteins expressed on cell surface. In order to explore the role of polymorphic MICA molecules on organ transplantation, we analyzed the expression of MICA in freshly isolated human umbilical vein endothelial cells (HUVEC) and other cell lines.(2) Patients who lose their graft have been found to have specific allo-antibodies against MICA antigens. To understand the development of MICA antibodies and their role in organ transplantation, we defined the serologic types of MICA antibodies from recipients with allografts.(3) The patients with late liver diseases were found to have higher levels of serum sMICA than the healthy population. In order to observe the effect of sMICA on liver transplantation, we investigated whether high levels serum sMICA are associated with incidence of biliary cast sysdrome (BCS) following liver transplantation.2. Methods and Materials(1) Establishment of MICA allele-level typing method.One PCR amplification was performed to obtain templates of 2.2 kb including exons 2,3,4, and 5 of MICA to be sequenced with two forward and two reverse primers. Overlay of nucleotide sequencing signals resulting from presence of different GCT repeats in exon 5 from two different MICA alleles can be identified by a computer-based analysis.(2) Isolation and culture of HUVEC.Human umbilical vein endothelial cells were isolated from umbilical cord veins and cultured within 4-6 passages. RT-PCR was used to analyze MICA mRNA that expressed on 11 HUEVCs and 7 culture cell lines. Surface MICA expression was examined by flow cytometry with monoclonal antibody staining. Total MICA proteins were accessed by Western blot.(3)Establishment of MICA stable transfected cells.The cDNA containing full-length MICA was cloned into plasmid pEGFP-N1. Then the constructs were transfected into human B cell line Hmy.C1R. After G418 selection, stable transfected cells were monitored by fluorescence microscopy and sorted by flow cytometry. Suface MICA expression was demonstrated by flow cytometry with the monoclonal antibodies 6B3 and 3.2H3.(4) Production of MICA allele recombinants using a Bac-to-Bac Baculovirus Expression system.11 MICA alleles were cloned into the plasmid pFastBac-1.2 hybrids and 2 mutants MICA molecules were made from two normal MICA alleles. The baculovirus with MICA gene was obtained from transformation of DH10Bac (E. coli) with the constructed-vectors. MICA recombinant proteins were produced in insect cells (Sf9) which were infected with baculovirus. Soluble MICA recombinant proteins fused with a His-tag (6 histidines) were purified by nickel-affinity agarose followed by mAb 6B3-affinity agarose. Using such materials, we have established the Luminex Flow Cytometry assay to identify the specificity of MICA antibodies. MICA serologic patterns of reactivity were determined with single MICA antigens which were bound to Luminex beads.(5) Quantitation of serum sMICA and patients with liver transplantation.Serum sMICA was retrospectively evaluated in pre-and post-transplant sera from 133 consecutive primary liver transplant patients and in sera from 88 healthy volunteers using sandwich ELISA. Normal distribution of serum sMICA was described by the data obtained from healthy population and sMICA concentration that was greater than the upper bound 95% normal range was considered as high levels of sMICA. Patient records were reviewed to identify patients who developed BCS.3. Results(1) MICA was expressed on the surface of human endothelial cells.The results show that all of tested cells were found to have MICA mRNA expression by RT-PCR. HUEVCs have surface MICA expressed in high level, but MICA protein expression in T and B lymphocytes was not detected in flow cytometry and Western blot with MICA specifice monoclonal antibodies 6B3 and 1.7AD.(2) Determination of the MICA epitopes that are recognized by human antibodies against MICA.Human sera selected in this study were found to recognize up to 14 distinct MICA epitopes. Among these, nine epitopes correlated with a single unique amino acid:one shared two signature amino acids, one shared three signature amino acids in close proximity, and three epitopes involved multiple amino acids in a nonlinear sequence. Two groups of public epitopes (MICA-G1 and MICA-G2) were characterized and their epitopes were speculated to relate six amino acids.(3) High level serum sMICA are associated with BCS following liver transplantation.We show that 37.6% of patients with end-stage liver diseases had significantly higher pre-transplant serum sMICA than in healthy population.34.4% of recipients with post-transplant high levels of sMICA developed BCS. In contrast,17.3% of patients with post-transplant normal levels of sMICA developed BCS. The risk of BCS development is significantly associated with the presence of post-transplant high levels of sMICA (P=0.0365). Further analysis disclosed that patients with decreased post-transplant sMICA following liver transplantation had a lower incidence rate of BCS than those with remained high levels of sMICA after transplantation (10.5% vs.38.7%, P=0.0302). Furthermore, log-rank test showed that BCS occurrence was significantly associated with dynamic changes of sMICA among different groups (P=0.0188).4. Conclusion(1) The expression of MICA on the surface of endothelial cells makes this polymorphic molecule a possible target during the immune response of graft rejection in organ transplantation.(2) We first reported 14 MICA epitopes that were indentified by human MICA antibodies. Those epitopes may help to understand the development of MICA antibodies and to identify the suitable donors for the sensitized transplant recipients.(3) We found that biliary cast syndrome is more likely to develop in recipients who have post-transplant high levels of sMICA. The data suggested that sMICA might have some immunologic effect on BCS development following liver transplantation. Monitoring of serum sMICA could have a prognostic value in assessment of patients with liver transplantation.