Analysis of the Thy-1 pathway of T cell hybridoma activation using 17 rat monoclonal antibodies reactive with distinct Thy-1 epitopes.
Seventeen monoclonal anti-Thy-1 antibodies (mAb) derived from LOU/M rats immunized with mouse T cell clones were used to study the role of Thy-1 in antigen-independent T cell activation. These mAb identified Thy-1.2 or monomorphic determinants and immunoprecipitated a molecule of 25-28 kDa from detergent-solubilized, 125I-labeled T cell surface proteins. Competitive cross-inhibition binding assays demonstrated that these reagents defined 3 epitope groups including either Thy-1.2 (group A) or Thy-1 monomorphic (groups B and C) determinants. Experiments using high titered culture supernatants revealed that all 6 IgG mAb defining the epitope group C, and one IgG2c mAb directed at a determinant in group A were capable of stimulating the terpolymer-L-glutamic acid60-L-alanine33-Ltyrosine10 (GAT) plus I-Ad-reactive BALB/c T cell hybridoma T14-117.9 to produce interleukin 2 (IL2) in the absence of accessory cells. Cross-linking of cell-bound rat mAb by a BALB/c anti-rat kappa chain mAb, or the presence of B cell lymphomas in the culture resulted in an increase of the Thy-1-mediated IL2 responses of this hybridoma. Some mAb from group B required antibody doses exceeding 80 micrograms/ml in order to activate T cells, while others remained nonstimulatory at any dose tested. Striking synergy in mAb-mediated T cell activation was observed when nonmitogenic doses of mAb group groups A and C were mixed in the same culture. Analysis of a panel of GAT plus I-Ad-specific T cell hybridomas revealed that these cells markedly differed in the magnitude of their IL2 responses induced by a given amount of stimulating anti-Thy-1 mAb. Such reagents also stimulated normal thymocytes to express IL2 receptor on their surface. These studies show that the epitopic specificity and the amount of anti-Thy-1 mAb, and the susceptibility of the T cell examined represent important parameters for the triggering of the Thy-1 pathway of T cell activation.
Pont
S
S
Regnier-Vigouroux
A
A
Naquet
P
P
Blanc
D
D
Pierres
A
A
Marchetto
S
S
Pierres
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000276:immunology*; D000939:Epitopes; D006825:Hybridomas / Q000276:immunology; D007376:Interleukin-2 / Q000096:biosynthesis; D008213:Lymphocyte Activation; D051379:Mice; D010455:Peptides / Q000276:immunology; D011108:Polymers; D051381:Rats; D011971:Receptors, Immunologic / Q000502:physiology; D015375:Receptors, Interleukin-2; D013601:T-Lymphocytes / Q000276:immunology*; D018800:Thy-1 Antigens
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D000939:Epitopes; D007376:Interleukin-2; D010455:Peptides; D011108:Polymers; D011971:Receptors, Immunologic; D015375:Receptors, Interleukin-2; D018800:Thy-1 Antigens; C020749:GAT