Human Major Histocompatibility Molecules Have the Intrinsic Ability to Form Homotypic Associations Kathy Triantafilou, Martha Triantafilou, Keith M. Wilson, and Nelson Fernandez ABSTRACT: We have investigated the homotypic asso- ciations of major histocompatibilty, class II and class I molecules using immunoprecipitation from detergent solubilised cell extracts. A 120-kDa structure correspond- ing to an HLA-DR dimer of dimers was immunoprecipi- tated by the HLA-DR specific mAb L243 from both biotinylated cell-surface and metabolically labeled B cells and transfectant fibroblasts. The thermostability of this structure in SDS was examined. It was detected at 4°C, 22°C, and 37°C, but not at 50°C or 100°C. Experiments performed with L243 Fab fragments and with purified HLA-DR molecules, indicated the presence of HLA-DR dimers of dimers and single heterodimers on B cells. HLA-DQ was also found to form SDS-stable dimers of dimers and single heterodimers on the cell surface of B cells, demonstrating that HLA class II isotypes, other than HLA-DR, also form homotypic associations. Similar ex- periments performed with HLA class I specific mAb, W632, revealed the existence of a 90 kDa and a 135-kDa structure corresponding to a MHC class I multimers. Under the same conditions, non-MHC molecules such as CD14 were found not to self-associate. These findings indicate that major histocompatibility molecules have the intrinsic ability to form homotypic associations at the cell surface of antigen presenting cells. Human Immunology 61, 585–598 (2000). © American Society for Histocom- patibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: dimers of dimers; HLA-DR; HLA-DQ; HLA class I ABBREVIATIONS ATCC American type culture collection CHAPS (3-[(cholamidopropyl)-dimethylammonio]- 1-propanesulfonate NEPHGE non-equilibrium pH gradient gel electrophoresis PBS-T phosphate buffered saline 0.1% Tween-20 HRP horseradish peroxidase INTRODUCTION The major histocompatibility complex (MHC) loci en- codes two major subsets of histocompatibility products, MHC class II and MHC class I. MHC Class II molecules consist of two non-covalently associated glycoprotein polypeptides, the alpha and the beta chains, and are expressed on antigen presenting cells. MHC Class I molecules consist of a non-covalently associated trimo- lecular complex consisting of the class I MHC heavy chain, the  2 -microglobulin ( 2 m) light chain, and a peptide bound to the peptide binding groove [1]. An important structural property of MHC molecules is that both polypeptide chains in the case of Class II, and the heavy chain of Class I are anchored to the lipid bilayer of the cell via a transmembrane domain followed by a short cytoplasmic domain. It is, therefore, possible that the functional properties of MHC molecules on antigen pre- senting or target cells are related to the fluid properties of the lipid bilayer. Thus, MHC molecules could form From the Department of Biological Sciences, University of Essex, Colches- ter, United Kingdom (K.T., M.T., N.F.), and Biotechnology Analytical Laboratories, GlaxoWellcome Research and Development, Beckenham, United Kingdom (K.M.W., present address). Address reprint requests to: Kathy Triantafilou, Ph.D., Department of Biological Sciences, Central Campus, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ; Fax: (+44) 01206-872592; E-mail: ktrian@essex. ac.uk. Received November 19, 1999; revised February 2, 2000; accepted Feb- ruary 11, 2000. Human Immunology 61, 585–598 (2000) 0198-8859/00/$–see front matter © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc. PII S0198-8859(00)00112-9