Generation and functional characterization of anti-clonotype antibodies to human T-cell receptors

Monoclonal antibodies (mAb) directed against the clonotypic structure of the T-cell receptor (TCR) may be useful reagents in the study and therapy of T-cell-mediated diseases. In contrast to several reports concerning the generation of anti-clonotype mAb to mouse TCR, only very limited numbers of anti-clonotype mAb to human TCR have been described. So far, a suitable method for the generation of anti-clonotype mAb to a given TCR has not been available and in this report we describe a novel strategy for the generation of such mAb. Mice were immunized with intact human T-cells. Then, spleen cell populations were precleared from B-cells reactive to CD3 and the constant region of the TCR by adsorption to TCR/CD3 complexes derived from an irrelevant T-cell clone. Subsequently, clonotype-specific B-cells were selected with TCR/CD3 complexes from the T-cell clone of interest. The small number of B-cells resulting from this selection were clonally expanded in a B-cell culture system and then immortalized by mini-electrofusion. Ten clonotype-specific mAb were generated against a DRBI *0401-restricted T-cell clone recognizing an epitope of the human cartilage glycoprotein 39 (HC gp-39) All mAb immunoprecipitated a heterodimeric 85 kDa protein. Absolute specificity was demonstrated in a T-cell agglutination test with the T-cell clone of interest compared to a set of 16 defined, irrelevant T-cell clones or lines. In functional assays, the mAb were found to inhibit or block antigen-specific T-cell stimulation. In addition, crosslinked mAb were found to stimulate proliferation of the specific clone in the absence of antigen and antigen presenting cells (APC). Such mAb may have clinical relevance in deleting or modulating autoreactive T-cells in a clonotype-specific manner. (C) 1997 Elsevier Science B.V

Generation and functional characterization of anti-clonotype antibodies to human T-cell receptors

Monoclonal antibodies (mAb) directed against the clonotypic structure of the T-cell receptor (TCR) may be useful reagents in the study and therapy of T-cell-mediated diseases. In contrast to several reports concerning the generation of anti-clonotype mAb to mouse TCR, only very limited numbers of anti-clonotype mAb to human TCR have been described. So far, a suitable method for the generation of anti-clonotype mAb to a given TCR has not been available and in this report we describe a novel strategy for the generation of such mAb. Mice were immunized with intact human T-cells. Then, spleen cell populations were precleared from B-cells reactive to CD3 and the constant region of the TCR by adsorption to TCR/CD3 complexes derived from an irrelevant T-cell clone. Subsequently, clonotype-specific B-cells were selected with TCR/CD3 complexes from the T-cell clone of interest. The small number of B-cells resulting from this selection were clonally expanded in a B-cell culture system and then immortalized by mini-electrofusion. Ten clonotype-specific mAb were generated against a DRBI *0401-restricted T-cell clone recognizing an epitope of the human cartilage glycoprotein 39 (HC gp-39) All mAb immunoprecipitated a heterodimeric 85 kDa protein. Absolute specificity was demonstrated in a T-cell agglutination test with the T-cell clone of interest compared to a set of 16 defined, irrelevant T-cell clones or lines. In functional assays, the mAb were found to inhibit or block antigen-specific T-cell stimulation. In addition, crosslinked mAb were found to stimulate proliferation of the specific clone in the absence of antigen and antigen presenting cells (APC). Such mAb may have clinical relevance in deleting or modulating autoreactive T-cells in a clonotype-specific manner. (C) 1997 Elsevier Science B.V.</p

T-cell anergy induced by clonotype-specific antibodies:modulation of an autoreactive human T-cell clone in vitro

Monoclonal antibodies (mAb) specific for the clonotype of an autoreactive T cell may be useful reagents in the modulation of autoimmune disease. We have previously reported the generation of a set of mAb specific for the clonotypic structure of a human T-cell clone recognizing an epitope of human cartilage gp-39. This glycoprotein was recently identified as a candidate autoantigen in rheumatoid arthritis. Here, we demonstrate for the first time that small amounts of immobilized anticlonotype mAb can induce anergy in the autoreactive clone. Following the anergic stimulus, T cells failed to proliferate upon restimulation as a result of a lack of interleukin-2, (IL-2) gene transcription. In addition, a diminished interferon-gamma (IFN-gamma) production was found. Our data indicate that anergy was not a result of T-cell receptor (TCR) downmodulation or the absence of free TCR. The anergic state was induced independent of costimulation or the presence of IL-2 and no protein synthesis was required for the induction of anergy. Anticlonotype mAb-induced anergy was prevented by cyclosporin A, suggesting that active signalling via the calcium/ calcineurin pathway was required for the induction of anergy. In coculture experiments, anergic T cells were found to suppress the response of reactive cells from the same clone. This bystander suppression led to 90% inhibition of peptide-induced proliferation. Together, these findings suggest that mAb to the clonotypic structure of autoreactive T cells may be suitable reagents for the functional inactivation of these T cells in autoimmune diseases

T-cell anergy induced by clonotype-specific antibodies:modulation of an autoreactive human T-cell clone in vitro

Monoclonal antibodies (mAb) specific for the clonotype of an autoreactive T cell may be useful reagents in the modulation of autoimmune disease. We have previously reported the generation of a set of mAb specific for the clonotypic structure of a human T-cell clone recognizing an epitope of human cartilage gp-39. This glycoprotein was recently identified as a candidate autoantigen in rheumatoid arthritis. Here, we demonstrate for the first time that small amounts of immobilized anticlonotype mAb can induce anergy in the autoreactive clone. Following the anergic stimulus, T cells failed to proliferate upon restimulation as a result of a lack of interleukin-2, (IL-2) gene transcription. In addition, a diminished interferon-gamma (IFN-gamma) production was found. Our data indicate that anergy was not a result of T-cell receptor (TCR) downmodulation or the absence of free TCR. The anergic state was induced independent of costimulation or the presence of IL-2 and no protein synthesis was required for the induction of anergy. Anticlonotype mAb-induced anergy was prevented by cyclosporin A, suggesting that active signalling via the calcium/ calcineurin pathway was required for the induction of anergy. In coculture experiments, anergic T cells were found to suppress the response of reactive cells from the same clone. This bystander suppression led to 90% inhibition of peptide-induced proliferation. Together, these findings suggest that mAb to the clonotypic structure of autoreactive T cells may be suitable reagents for the functional inactivation of these T cells in autoimmune diseases.</p