Analysis of the Thy‐1 pathway of T cell hybridoma activation using 17 rat monoclonal antibodies reactive with distinct Thy‐1 epitopes

Abstract

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-sol- ubilized, 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 IgG_2c mAb directed at a determinant in group A were capable of stimulating the terpolymer-L-glutamic acid⁶⁰-L-alanine³³-Ltyrosine¹⁰(GAT) plus I-A^d-reactive BALB/c T cell hybridoma T14–117.9 to produce inter-leukin 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 üg/ml in order to activate T cells, while others remained nonstimulatory at any dose tested. Striking synergy in rnAb-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-A^d-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.