Production and characterization of an Mls-1-specific monoclonal antibody.
Superantigens (SAGs) represent a new class of antigens, characterized as T cell receptor (TCR) V beta-reactive elements. Bacterial toxins constitute the major group of exogenous SAGs, while the mouse mammary tumor virus (MMTV)-encoded Mls molecules represent the endogenous SAGs. Mls-1 is the prototype of the latter SAGs, because it elicits a very potent T cell stimulatory response in vitro in unprimed T cells expressing the TCR V beta 6 or 8.1 chains. In vivo, Mls-1 causes deletion of immature T cells bearing the V beta 6, 7, 8.1, or 9 chains. Although Mls-1 was functionally discovered > 20 yr ago, it has not been possible to raise antibodies against this molecule. We have previously cloned and sequenced the Mtv-7 sag gene, which encodes Mls-1. Sequence comparisons with other MMTV sag genes suggested that the polymorphic 3' end encodes the TCR V beta specificity of these SAGs. We have, therefore, immunized hamsters with a 14-amino acid peptide from the deduced COOH-terminal sequence of the Mtv-7 sag gene. We describe here the production of a monoclonal antibody (mAb), 3B12, which is peptide specific and reacts with a recombinant baculovirus product of Mtv-7 sag. This mAb blocks Mls-1-specific T cell recognition and detects the Mls-1 protein on the surface of the B cell hybridoma LBB.A, but not on LBB.11, which is an Mtv-7 loss variant of LBB.A. Transfection of the Mtv-7 sag gene into LBB.11 renders this cell functionally Mls-1+ as well as positive for 3B12 binding, confirming the specificity of this mAb. It is well documented that B cells and CD8+ T cells express T cell stimulatory Mls-1 determinants, and we show here that this functional profile correlates with the expression of MMTV-specific mRNA. However, primary lymphocytes derived from Mls-1+ mice do not stain with 3B12, even after in vitro activation with mitogens or phorbol ester.
Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts 02111.
N
N
Mohan
D
D
Mottershead
M
M
Subramanyam
U
U
Beutner
B T
BT
Huber
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology*; D001483:Base Sequence; D006224:Cricetinae; D015870:Gene Expression; D005814:Genes, Viral; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008811:Mice, Inbred DBA; D016747:Minor Lymphocyte Stimulatory Antigens / Q000276:immunology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D012367:RNA, Viral / Q000235:genetics; D011994:Recombinant Proteins / Q000276:immunology; D012091:Repetitive Sequences, Nucleic Acid; D015678:Viral Structural Proteins / Q000235:genetics
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D016747:Minor Lymphocyte Stimulatory Antigens; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D012367:RNA, Viral; D011994:Recombinant Proteins; D015678:Viral Structural Proteins