Construction, expression and recognition of an H—2 molecule lacking its carboxyl terminus

Abstract

A mouse major histocompatibility antigen (H-2) gene, encoding a novel H-2Ld molecule lacking its intracytoplasmic domain, has been constructed and introduced into mouse L-cells. The novel H-2 molecule is found on the surface of the transfected cells at the same level as L-cells transfected with the native H-2Ld gene. Allo- and influenza-specific cytotoxic T lymphocytes can recognize the truncated H-2 gene product nearly as efficiently as the normal H-2Ld gene product. However, vesicular stomatitis virus-specific cytotoxic T lymphocytes recognize the truncated H-2Ld molecule less efficiently than the complete H-2Ld product. The rate of capping of the truncated H-2Ld molecule was investigated and found to be the same as that of the complete H-2Ld gene product.

Recognition of interspecies hybrid class I histocompatibility antigens by antigen-specific cytolytic T lymphocytes.

“…In addition, a variety of reconstructions of the H-2 genes have been made that, when expressed, code for hybrid class I proteins. Results of these studies have (i) confirmed earlier biochemical studies (3)(4)(5) by showing that CTL and most monoclonal antibody (mAb) recognition sites are located in the al and a2 domains (6)(7)(8)(9)(10), (it) suggested that there may be specific conformational interactions between the al and a2 domains required for the stability of antigenic determinants (8,9,11), and (iii) showed that class I proteins that lacked or had altered cytoplasmic domains were normally mobile in the membrane and were recognized by allospecific and most viral-specific CTL (12,13).…”

“…The L-cell transformants expressing the A2 parental and the A21+2/Kb hybrid antigens were assessed for cytolysis by a human HLA-A2,B7-specific long-term human CTL line. As reported (13), neither the A2+ L cell nor the mock-transfected control L cell line was effectively lysed by the human allospecific CTL despite high surface-expression levels of the antigen (Fig. 3A).…”

“…4) may decrease the affinity ofthe T-cell receptor of the CTL for its target cell determinants in the al and a2 domains and (ii) if the a3 domain contains amino acid sequences that act as ligands for CTL accessory molecules, such as T8 or Lyt2, then a diminished level of lysis of target cells bearing the hybrid class I molecule might be due to the lack of recognition by low-affinity CTLs which require T8 or Lyt2 (37,38) to interact with these targets. It seems unlikely that the differences in the intracytoplasmic regions of these proteins would cause this effect, since investigators who have altered or deleted the cytoplasmic region have been unable to demonstrate a defect in allorecognition and have reported only limited effects on H-2-restricted viral recognition (9,12,13). The amino acid differences within the transmembrane region should have little effect on the external polymorphic domains; the conservation of hydrophobicity is necessary for anchoring of the molecule within the membrane (33).…”

H-2Ld antigen encoded by a recombinant retrovirus genome is expressed on the surface of infected cells.

“…An H-2Ld_ containing recombinant MuLV retrovirus, pLTV-11, was constructed by blunt-end ligation of a truncated H-2Ld gene (17) into the XhoI site of the plasmid MSV-neo ( Fig. 1; 15).…”

Biochemical and functional analyses of a secreted H-2Ld molecule.

“…These studies have been extended by the use of recombinant DNA technology to generate altered H-2 genes. H-2 genes altered in vitro have been introduced into L cells and studied for their ability to serve as targets for cytotoxic T lymphocytes (CTLs) (1,2,11,(24)(25)(26)(27)33 To obtain a secreted H-2Ld antigen, we decided to take a similar approach. An H-2Ld gene encoding a secreted H-2 antigen was constructed by using restriction enzymes and BAL 31 exonuclease to delete the transmembrane and cytoplasmic domains, as shown in Fig.…”