Major histocompatibility complex (MHC) class II molecules are heterodimeric glycoproteins with one a and one ,B polypeptide chain of similar molecular size. In this report, we describe the binding of an acetylated N-terminal peptide of myelin basic protein, , to purified individual a and 13 chains of murine I-Ak molecules. Purified complexes of isolated single chains and antigenic peptide bind to cloned T cells restricted by I-Ak and tetradecapeptide. The binding is blocked by a/13 anti-T-cell receptor (TCR) monoclonal antibody. Cell triggering as measured by an increase in extracellular acidfication rate is observed when cloned T cells are exposed to purified complexes of isolated chains and antigenic peptide. This increase in the extracellular acidification rate is antigen specific and MHCrestricted, as chains alone or irrelevant chain-peptide complexes do not trigger an increase in the metabolic acidification rate. These results together demonstrate that in vitro cloned T cells are triggered by complexes of specific antigenic peptides and isolated individual chains of their cognate MHC proteins. MATERIALS AND METHODS Purification of Murine I-Ak and I-As. I-Ak and I-As were purified from Nonidet P-40 extracts of membrane prepared from cultured CH27 cells and SJL/J mouse spleen cells, respectively, by using an affinity support prepared with monoclonal antibody 10-2.16 (specific for I-Ak and I-AS) coupled to Sepharose 4B beads by the standard cyanogen bromide coupling method. Briefly, a membrane fraction from high-speed centrifugation (100,000 x g) was detergentextracted in a buffer containing 10 mM Tris HCI (pH 8.3), 0.5% Nonidet P-40, 0.1 M NaCl, 5 mM EDTA, 0.02% sodium azide, and 1 mM phenylmethylsulfonyl fluoride (PMSF), and the lysate was recycled over the preequilibrated antibody column at 4TC for 16 hr. The column was washed with 10 bed volumes of deoxycholate buffer containing 10 mM Tris HCl (pH 8.3), 0.5% deoxycholate, 0.1 M NaCl, 5 mM EDTA, 0.02% sodium azide, and 1 mM PMSF followed by 5 bed volumes of phosphate-buffered saline (PBS) containing 1% n-octyl-,f-D-glucopyranoside (OG) buffer. Finally, the Ia molecules were eluted with 20 mM phosphate buffer (pH 11) containing 0.1 M NaCl, 1% OG, 0.02% sodium azide, and 1 mM PMSF. Each fraction was neutralized with 1 M acetic acid to a final concentration of 12 mM, and the MHC class II molecules were concentrated by using an Amicon Centriprep-10 concentrator. Affinity-purified I-Ak and I-AS molecules were characterized by 12% one-dimensional SDS/ polyacrylamide gel electrophoresis.Isolation of a and 13 Chains of MHC Class I Molecules. Purified I-Ak was concentrated to 1 mg/ml with Amicon concentrators and applied onto a 12% preparative (16 cm x 18 cm) slab gel unit. The electrophoresis was conducted under nonreducing conditions for 16 hr at room temperature at constant volts (100 V). One lane was excised from the center ofthe gel and developed by silver staining. The stained gel lane was used as a guide to excise bands ofa and 13 chains, and th...
