Abstract
This thesis describes a novel approach for the generation of human or murine MAb producing hybridomas. The approach is based on expansion of B cells in the presence of human T-cell supernatant and irradiated murine thymoma cells, followed by immortalization of individual cultures of expanded B cells in a mini-electrofusion procedure (B-cell culture/mini-electrofusion approach). Following such a protocol, a very high immortalization frequency of B cells was achieved. Approximately 50% of murine B cells and 30% of human B cells could be immortalized. This is at least 1000-fold more efficient than a standard electrofusion procedure, and more than 100,000-fold more efficient than the classical PEG-fusion technique. The high frequency of the B-celculture/mini-electrofusion approach enables the processing and immortalization of small numbers of B cells obtained from antigen-specific selection procedures. Thus, many antigen-specific hybridomas can be generated without laborious screening of large numbers of irrelevant hybridomas. Furthermore, it has been possible to immortalize a small number of B cells obtained from human donor material, murine blood samples or murine lymph nodes. The B-cell culture/mini-electrofusion approach has been the basis for generation of antibodies to the clonotypic structure of human T cells. A set of ten clonotype-specific MAb were generated against a human autoreactive T-cell clone that recognizes an epitope of the human cartilage glycoprotein 39 (HC gp-39). This protein was recently found to be candidate antigen involved in rheumatoid arthritis. In vitro functional assays have shown that these MAb can inhibit or block antigen-specific T-cell stimulation. Furthermore, small amounts of immobilized MAb can modulate the autoreactive T-cell clone by the induction of anergy. These findings suggest that such MAb may have clinical relevance in deleting or modulating autoreactive T cells in a clonotype-specific manner.
