Activated Human T Lymphocytes Express Mhc Class I Heavy Chains Not Associated with 02

Class I MHC molecules associated with R 2-microglobulin (a2m) are peptide-binding proteins on the surface of nearly all nucleated cells and present antigens to CTL (1). HLA class I molecules may also associate with other surface proteins (2-4) and exert a signal-transducing function in these aggregates. The genes coding for the MHC molecules are the most polymorphic loci known in higher vertebrates. It is generally held that the noncovalent association between the monomorphic light chain 02m is essential for the transport of the class I molecule to the surface and that surface expressed HLA molecules are linked to 02M (1, 5). Here we report that in vitro or in vivo activated, but not resting T lymphocytes, express a considerable number of surface HLA class I heavy chain molecules not associated with a2m. The conformational properties of such class I molecules un-linked to 02m (5, 6) are known to be very different from peptide binding, 02M-associated heavy chain molecules. It is therefore likely that the loss of the 02m and the conformational alteration of this class I protein upon T cell activation have some important functional implications. The CD25 (clone 3G10) and LA45 antibodies were raised against HUT102 cells in our laboratory ; the CD3 antibody OKT3 was purchased from Ortho Pharmaceuticals , Raritan, NJ .The antibody W6/32 (7) reacts with a monomorphic determinant of human HLA-A,B,C molecules and was purchased form Sera-lab, Sussex, UK, and the anti-02m antibody (clone SV393) was from Behring, Marburg, FRG. HC10 was a gift of H. Ploegh (Netherlands Cancer Institute, Amsterdam) (8). Stimulation and Fluorescence Staining ofLymphocytes. Mononuclear cells (MNC) from healthy donors and patients were isolated by Ficoll Hypaque (Pharmacia, Uppsala, Sweden) gradient centrifugation and either freshly analyzed or cultured for 3 d with 2 mg/ml PHA (Wellcome, Beckenham, UK) in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with l0olo FCS (Flow Laboratories, Beckenham, UK) or 10% human AB serum (gave no difference), L-glutamine, and antibiotics. Cells were double stained with antibodies CD3-PE, LA45-FITC,