Stimulation of T cells by antigenic peptide complexed with isolated chains of major histocompatibility complex class II molecules.

Abstract

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...

Endothelial cell-binding activity of anti-U1-ribonucleoprotein antibodies in patients with connective tissue diseases

“…A membrane fraction was prepared from HPAECs according to methods reported previously, with modifications [36]. Briefly, 5 × 10 7 HPAECs were washed three times with PBS.…”

Antigen‐Specific apoptosis in immortalized T cells by soluble MHC class II‐peptide complexes

“…In a recent study, human T cell clones derived from a myasthenic patient showed similar tendencies to undergo programmed cell deaths upon exposure to human DR4.AChRa peptide complexes. '•* Similarly, apoptosis was observed in a murine T cell clone restricted for IA** and rat MBP (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) peptide (B. Nag, unpubl.…”

Rationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activity

“…Each subunit consists of a short cytoplasmic tail, a single membrane‐spanning sequence, and two extracellular domains. X‐ray crystallographic studies have demonstrated that peptides from processed antigen bind to MHC molecules in the membrane distal pocket formed by the β1 and α1 domains 13, 14. Moreover, the β2 domain contains a CD4 binding region that co‐ligates CD4 when the α1 and β1 domains with associated antigenic peptide interact with the TCR αβ heterodimer, whereas the α2 domain appears to contribute to ordered oligomerization in T cell activation 15.…”

“…Complexes of MHC/antigen have been purified as detergent extracts of lymphocyte membranes,16 and as associated recombinant proteins using baculovirus and bacterial expression systems 17–21. These two‐chain, four‐domain molecular complexes, after loading with selected peptide epitopes, have been demonstrated to interact with T cells in an antigen‐specific manner 13, 14, 19, 22–24. In some cases, in the absence of co‐stimulation, these complexes induced antigen‐specific apoptosis rather than anergy 20.…”