title: MICA is a target for complement-dependent cytotoxicity with mouse monoclonal antibodies and human alloantibodies.
abstract: 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.
authors.affiliation: University of Texas Southwestern Medical Center, Dallas 75390-8886, USA.
authors.forename: Yizhou
authors.initials: Y
authors.lastname: Zou
authors.forename: Fariba
authors.initials: F
authors.lastname: Mirbaha
authors.forename: Ana
authors.initials: A
authors.lastname: Lazaro
authors.forename: Yanzheng
authors.initials: Y
authors.lastname: Zhang
authors.forename: Bhavna
authors.initials: B
authors.lastname: Lavingia
authors.forename: Peter
authors.initials: P
authors.lastname: Stastny
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003165:Complement System Proteins / Q000378:metabolism*; D003602:Cytotoxicity, Immunologic; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007518:Isoantibodies; D051379:Mice; D011817:Rabbits
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D015395:Histocompatibility Antigens Class I; D007518:Isoantibodies; C099187:MHC class I-related chain A; D003165:Complement System Proteins