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-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.

Coaggregation of the T-cell receptor with CD4 and other T-cell surface molecules enhances T-cell activation.

“…Some anti-Thy-1.2 antibodies have been reported to activate T cells (20). However, (T-CD3)-T-cell variants are refractory to anti-Thy-1 activation (21,22), suggesting TCR involvement.…”

Accessory molecules and T cell activation I. Antigen receptor avidity differentially influences T cell sensitivity to inhibition by monoclonal antibodies to LFA‐1 and L3T4

“…The use of rat mAb has permitted delineation of several topographic domains that are differentially involved in the function(s) of this molecule [30]. By testing LFA-1 a chainspecific mAb reactive with epitopes (A, C, D) close to the functional site of this molecule, a clear relationship was observed between the degree of inhibition of IL2 responses and the avidity of the T hybridoma cells examined (Fig.…”

“…The IL 2-producing, GAT plus Ad-reactive T hybridoma cells used in these experiments were established according to procedures described elsewhere [24], from in vivo primed and in vitro restimulated BALBlc lymph node T cells fused with the AKR thymoma BW5147. T hybridoma cells were selected for their capacity to produce I L 2 in response to GAT and the antigen presenting BALB/c B lymphoma cells M12.4.1 [25].…”

A novel anti-Thy-1 (CD90) monoclonal antibody induces apoptosis in mouse malignant T-lymphoma cells in spite of inducing bcl-2 expression

“…These special antiThy-1 MAbs may have a capacity to aggregate by themselves because the F(ab')z fragment of KT16 induces T-cell growth only in the presence of a second antibody (Millrain et al, 1992). These anti-Thy-1 MAbs are known to recognize both Thy-1.1 and Thy-1.2 (Millrain et aL, 1992), and the recognized epitope is designated Thy-1A (Pont et aL, 1985). We therefore examined 9 of our established anti-Thy-1 MAbs for their recognition capability.…”

“…2a), indicating that MCS-34 recognized both the Thy-1.1 and Thy-1.2 allotypes, like G7 and KT16 (Millrain et al, 1992). We further determined the epitope recognized by MCS-34 on Thy-1 glycoprotein by cross-blocking analysis (Pont et al, 1985) using FACScan. Pre-incubation of the thymocytes of BALB/c mice with anti-CD45 MAb MCSJ did not affect the recognition of PE-labeled anti-Thy-1.2 MAbs [30H-12 (Fig.…”