Yizhou Zou

Identification of MICA as a new polymorphic alloantigen recognized by antibodies in sera of organ transplant recipients

MHC class I-related chain A (MICA) is an HLA-related, polymorphic gene the product of which may be recognized by a subpopulation of intestinal gamma delta T cells and may play a role in the activation of a subpopulation of natural killer cells. Using anti-MICA specific rabbit sera we previously demonstrated that freshly isolated monocytes, keratinocytes, fibroblasts, and endothelial cells express MICA. To analyze whether MICA may be a target for specific antibodies in sera of transplanted patients, we produced three recombinant MICA proteins consisting of the alpha 1, alpha 2, and alpha 3 domains, and used them in an enzyme-linked immunosorbent assay. We found that several patients had specific antibodies against MICA. Most of them were detected in serum samples collected at different times after organ rejection. Although this finding raises the question of how these patients became immunized, the fact that the polymorphic, HLA-like MICA molecule, expressed at the cell surface of endothelial cells, is recognized by specific antibodies in sera of transplanted patients, suggests the MICA may be a target molecule in allograft rejection.

Effect of Human Cytomegalovirus on Expression of MHC Class I-Related Chains A

The MHC-encoded MHC class I-related chains A (MICA) glycoproteins are known to enhance the functions of NK and T cells by ligating the stimulating receptor NKG2D and appear to play an important role in host defense. Human CMV (HCMV) evades the immune response in many different ways, but has not previously been found to down-regulate MICA. We have found that a common form of MICA, which has a nucleotide insertion in exon 5 corresponding to the transmembrane region and no cytoplasmic tail, was increased on the surface of fibroblasts HFS-13 compared with the mock-infected sample of the same cells that had been cultured to confluence. However, an astrocytoma cell line, U373, which has a full-length variant of MICA, showed that the expression of MICA was decreased after HCMV infection. Retroviral transduction of different MICA alleles into fibroblasts HFF-D, which express no MICA of their own, established that full-length MICA was down-regulated by HCMV, and the truncated form was not. Fibroblasts with decreased MICA due to HCMV infection were found to be protected from NK cell killing, whereas in the presence of the truncated form of MICA, the virus-infected cells were destroyed. Thus, the truncated form of MICA, which is the most common, has a mutation that allows it to persist on the surface and hinder efforts of the virus to evade the immune response.

MICA is a target for complement-dependent cytotoxicity with mouse monoclonal antibodies and human alloantibodies

The highly polymorphic major histocompatibility class I related chain A (MICA) gene encodes glycoproteins that have been shown to be expressed in epithelial cells, endothelial cells, keratinocytes, monocytes, and tumor cells. In previous experiments, we have studied MICA antigens using rabbit sera obtained by immunization with MICA peptides. We also found that several transplant recipients had specific antibodies against MICA in an ELISA assay with recombinant of MICA (r-MICA). In the present work we produced monoclonal antibodies by immunization of mice with recombinant MICA*008. Based on the different patterns of reactivity observed in ELISA, Western blot, and flow cytometry, mAbs 1.9C2, 2.4F5, 1.7AD, and 2.3D4 only reacted with denatured MICA and mAb 1.7A8 and 3.2H3 reacted also with native MICA as illustrated by flow cytometry with live cells. These monoclonal antibodies were postulated to bind to different sites of the MICA molecule. In order to investigate whether MICA expressed on the cell surface is able to mediate cell killing, antibody absorption, flow cytometry and complement-dependent cytotoxicity (CDC) were performed. We found that mouse monoclonal antibody 3.2H3 was able to kill 70% of HeLa cells. Absorption of a patient serum with pooled human platelets to remove antibodies against class I HLA resulted in a small shift of fluorescence and reduced killing from 100% to 70-75%. Absorption with the platelets and r-MICA produced a remarkable reduction in fluorescence staining and virtually reduced complement-dependent killing to the level of the negative controls. The results suggested that MICA alloantigens may be more immunogenic than could have been previously suspected.

Donor-recipient mismatches in MHC class I chain-related gene A in unrelated donor transplantation lead to increased incidence of acute graft-versus-host disease

The polymorphic products of major histocompatibility complex class I-related chain A (MICA) genes are important in solid organ transplantation rejection. MICA expression is limited to gut epithelium and may play a role in triggering acute graft-versus-host disease (aGVHD). A total of 236 recipients of unrelated donor transplantation were studied. Donor-recipient human leukocyte antigen (HLA) match was 10/10 human leukocyte antigen (HLA-A, -B, -C, -DRB1, -DQB1) in 73% and MICA mismatch in 8.4%. Because of physical vicinity of the loci, MICA mismatch was significantly associated with mismatch at HLA-B and HLA-C. A higher rate of grade II-IV aGVHD was seen in MICA-mismatched patients (80% vs 40%, P = .003) irrespective of degree of HLA matching (HLA 10/10 match: 75% vs 39%, P = .02) and HLA any mismatch (83% vs 46%, P = .003). The rate of grade II-IV gastrointestinal aGVHD was also higher in MICA-mismatched patients (35% vs 17%, P = .05). We conclude that MICA may represent novel a transplantation antigen recognized by human allogeneic T cells. This study was registered at ClinicalTrials.gov (Identifier NCT00506922).

Major histocompatibility complex class I-related chain A allele mismatching, antibodies, and rejection in renal transplantation

Even when kidney allografts are well matched for human leukocyte antigen (HLA) and anti-HLA antibodies are not detected, graft rejection can still occur. There is evidence that some patients who lose their graft have antibodies specific for major histocompatibility complex (MHC) class I-related chain A (MICA) antigens. We investigated whether mismatching MICA alleles associates with MICA antibody production and graft rejection or dysfunction. MICA and HLA antibody screening in 442 recipients was performed, and specificities were confirmed in a subgroup of 227 recipients using single-antigen multiplex technology. For assignment of MICA antibody specificity, we used three independent assays. In addition, MICA alleles of 227 recipients and donors were determined by DNA sequencing. In all, 17 patients (7.5%) had MICA antibodies, and 13 patients (6%) developed MICA donor-specific antibodies (DSA). Multivariate analysis revealed MICA mismatching, as an independent significant factor associated with the presence of MICA antibodies (p = 0.009), and 14 mismatched MICA residues significantly correlated with MICA antibody production. MICA and HLA antibodies significantly associated with acute rejection (AR) and MICA DSA and HLA DSA correlated with decreased graft function by univariate and multivariate analysis. We conclude that mismatching for MICA epitopes in renal transplantation is a mechanism leading to production of MICA antibodies that associate with AR and graft dysfunction.

MICA polymorphism in South American Indians

Abstract. We have studied the MICA alleles of 196 unrelated subjects from three South American Indian tribes (Toba, Wichi and Terena). They are members of isolated tribes located in the Gran Chaco area in northeastern Argentina and in Mato Grosso do Sul in South Central Brazil. Of 55 previously known alleles, nine were observed in South American Indians, compared with 16 that were found in North American Caucasians, suggesting a more restricted allelic distribution of MICA in these tribes. In South American Indians, MICA*00201 was the most frequent allele, with a gene frequency of 33% in Toba, 47% in Wichi and 44% in Terena. MICA*00201, MICA*027 (external domain sequence like MICA*008/TM allele A5) and MICA*010 accounted for more than 90% of all the MICA genes in South American Indians. In North American Caucasians, MICA*00801 (*008/A5.1) accounted for 42% of the genes and was the most common allele. We observed a high degree of linkage disequilibrium between certain alleles of MICA and of HLA-B in the South American Indian populations. Phylogenetic trees constructed using gene frequencies of the transmembrane short tandem repeats in the populations reported here, and in other populations taken from published reports, suggest that South American Indians are more closely related to Asians than to Europeans.

Antibodies Against Nucleolin in Recipients of Organ Transplants

Background. Patients who reject allografts frequently make strong antibody responses against donor human leukocyte antigens and autoantigens such as vimentin, collagen V, or alpha-tubulin and it has been postulated that autoantibodies may play a role in allograft failure. Methods. We have used serum from patients who recently rejected an allograft as a source of antibodies in combination with lysates of human umbilical vein endothelial cells as a source of target antigens. Immunoprecipitation and protein identification was performed by mass spectrometry. Recombinant nucleolin was produced and sera were assayed for antibodies by enzyme-linked immunosorbent assay. Results. Immunoprecipitation with serum WW led to the recognition of the protein nucleolin as a target antigen. By enzyme-linked immunosorbent assay, with recombinant nucleolin (r-nucleolin), the frequency of antibodies to nucleolin were found to be 2.0% in normal subjects, 9.1% in patients waiting for a kidney transplant, 25.5% after irreversible rejection of a kidney allograft, 17.1% after a heart transplant, and 43.8% in heart transplant recipients developing transplant-related coronary artery disease. Antibodies against nucleolin from mice or from transplant patients inhibited endothelial cell proliferation and in vitro capillary-like tube formation and caused apoptosis of human umbilical vein endothelial cells. Conclusions. Antibodies against nucleolin seem to inhibit and produce apoptosis of proliferating endothelial cells. These antibodies were found in many transplant patients and seemed to be associated with rejection of kidney allografts and with coronary artery disease in heart transplant recipients.

Polymorphisms of MICA recognized by human alloantibodies

MICA antigens are polymorphic glycoproteins expressed on the surface of human endothelial cells and other cells. Antibodies against MICA have been found in transplant recipients and were found to be associated with decreased survival of kidney allografts. In the present work, we investigated the polymorphisms that are recognized by antibodies against MICA. Soluble MICA recombinant proteins representing 11 common alleles, two hybrid alleles, and two single amino acid mutated alleles were produced. Patterns of reactivity were determined with MICA bound to Luminex beads. In some studies, sera containing antibodies against MICA were absorbed by cell lines transfected with MICA*001, MICA*002, MICA*008, and MICA*009 or with untransfected cells, followed by testing of antibody reactivity against MICA proteins bound to beads. The monoclonal antibodies and sera used in this study were found to recognize up to 14 distinct MICA epitopes as demonstrated by their differential absorption/reactivity patterns. 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. MICA shared epitopes were determined by reactivity loss in single MICA antigen bead assays by absorption with MICA transfectants. Since these epitopes may be targets for antibody binding and possibly antibody-mediated allograft rejection, epitope identification may help understand the development of MICA antibodies and to identify suitable donors for sensitized transplant recipients.

The emerging issue of MICA antibodies: antibodies to MICA and other antigens of endothelial cells.

The major histocompatibility complex (MHC) encodes the HLA class I antigens expressed on the surface of most nucleated cells and the HLA class II antigens which are expressed mostly in B lymphocytes, monocytes and dendritic cells. Mismatched HLA antigens are the main source of the immune response that leads to the rejection of allografts. In some patients however, rejection may occur without a detectable response to donor HLA antigens. We have been interested in characterizing antibodies that develop in transplant recipients who do not appear to have antibodies against HLA. For this purpose, we focused our attention to antigens which are expressed on the surface of endothelial cells and are not found on peripheral blood lymphocytes. These include the MICA and MICB antigens, which are encoded by loci in the MHC; certain autoantigens expressed on the endothelium; and a family of polymorphic antigens expressed on endothelial cells which are distinct from HLA and elicit production of antibodies that appear also to be associated with graft failure. Antibodies against MICA have been associated with allograft rejection. MICB antibodies are only rarely found. The autoantibodies and the endothelial specific alloantibodies are being characterized in ongoing studies.

Identification of endothelial cell surface antigens encoded by genes other than HLA. A combined immunoprecipitation and proteomic approach for the identification of antigens recognized by antibodies against endothelial cells in transplant recipients

It has been known for some time that transplant recipients may have antibodies to endothelial cells which are not detected on lymphocytes. However, little progress has been made in the analysis of these endothelial antigens. In the present experiments we have attempted to characterize endothelial cell surface antigens to which antibodies were produced during graft rejection. We have used a panel of endothelial cells from umbilical cord veins and found that antibodies with a polymorphic pattern in the panel appeared to correlate with transplant failure of kidney allografts and with the development of transplant-related coronary artery disease (TCAD) in heart transplant recipients. Among 39 patients with kidney allografts, 21 were negative for antibodies to endothelial cells and did well and 18 were positive and had frequent transplant loss (p=0.001). In 18 patients with TCAD and 20 patients of a comparator group without TCAD, association of coronary disease with endothelial cell antibodies was observed (p<0.02). To characterize the endothelial antigens responsible for these serologic reactions we performed immunoprecipitation of reactive antibodies with the corresponding endothelial cell surface antigens, followed by protein identification of the target antigens. Nine proteins were identified in these experiments, 5 were non-polymorphic and appeared to represent autoantigens. Four of the isolated proteins appeared to be polymorphic. They were the Human Major Histocompatibility Complex class I chain-related gene A (MICA), already known to be associated with antibody production and graft failure, human keratin 1, a protein known to be polymorphic and expressed on the surface of endothelial cells, eukaryotic translation initiation factor (EIF) 2A and ErbB3-binding protein 1. The possible role of keratin 1 and the other antigens in allograft rejection requires further investigation.