Studies of T- and B-cells for the generation of human antigen specific antibodies
Abstract: Human antibodies generated in vitro may be highly valuable for diagnostic and therapeutic purposes. For a long time most human antibodies produced in vitro have been of the IgM isotype with rather low affinities. In this thesis a method for production of IgG switched antigen-specific human antibodies is presented. This is a two step protocol, in which the first step is a selection procedure of donors with sufficient T-cell reactivity against tetanus toxoid (TT). The culture consists of a primary and a secondary immunization, of peripheral blood cells, where the secondary step includes CD40 activation together with antigen and preactivated T cells. The antigen is a heterotope with one B- and one T-cell (TT) epitope. In the model system 60 % of the donors produced anti-V3 specific IgG antibodies. The immunization system was used to study the humoral response against the tumor related antigen Mucin 1. Cells from the immunization were used for preparation of an Ab gene library, which was screened by using phage display. The selected Fab fragments were of low affinity with high dissociation rate constants. Mucin 1 gave a lower response, in this immunization system, compared to V3 probably due to the repetitive nature of the antigen. The in vitro immunization system somewhat mimics the germinal center reaction in vivo. In the germinal center B cells mutate their antibody genes, are selected by their ability to bind, take up and process antigen, and differentiate into plasma cells or memory cells. The germinal center reaction is dependent on T cells and a fraction of the CD4+ T cells in the germinal center coexpresses CD57. We have studied this population and a population in peripheral blood which also coexpresses CD4 and CD57. The GC derived population was able to stimulate B cells to Ig production when IL-2 was added while the blood derived population could not. The GC population was also able to promote survival of GC B cells, in vitro, to the same extent as CD57- Th cells. Furthermore the GC population expressed the costimulatory molecule, CD28 while the blood population did not. Neither GC nor blood derived CD57+ cells expressed any detectable amounts of mRNA specific for IL-2, IL-4 or IFN-g.
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