The long-term objective of this project is to understand how antigens can have antithetical effects on the immune system, foreign antigens usually provoking immunity, but self antigens usually inducing the state of immunologic tolerance. The particular focus will be the B lymphocyte and thus antibody formation. Freshly deaggregated antigens will be introduced into adult mice as surrogate self antigens to determine the mechanisms which prevent B cells fortuitously hypermutating to acquire anti-self reactivity, thus potentially posing the threat of autoimmunity. Insights gained into immunogenic versus toleragenic activity of antigen will be relevant to the design of better vaccines and to the control of autoimmune diseases and organ transplant rejection. Within this broad program, the first investigation will study the details of germinal center formation in C57Bl/6 mice following T-dependent immunization with the hapten 4-hydroxy- 3-nitrophenylacetyl (NP) coupled to protein carriers and administered with adjuvants. Splenocytes will be fractionated by 6-parameter flow cytometry to exclude cells with light-scattering characteristics atypical of lymphocytes, and all cells binding anti-IgM, anti-granulocyte and anti- macrophage antibodies, but to isolate cells which are hapten-binding of the IgG1 isotype and also phenotyped for one of three other characteristics. These are lambda light chains at the surface, capacity or otherwise to bind peanut agglutinin (positivity meaning germinal center status), or expression of syndecan at the surface (positive means antibody-secreting status). Isolated cells will be (a) analyzed singly ex vivo by polymerase chain reaction (pcr) techniques for IgH V gene mutations, and (b) cultured singly in specialized T-dependent or T- independent microcultures for the capacity for anti-NP antibody formation. Results will illuminate the earliest stages of germinal center mutation and selection processes, and the differences between germinal center and extra-follicular B cell differentiation pathways. Then the role of soluble, deaggregated toleragens in disturbing the germinal center reaction will be investigated both by the above techniques and by immunohistology, with special reference to effects on CD4+ T helper cells. T cell receptor transgenic mice will be used to differentiate between clonal deletion, clonal anergy or other possible mechanisms of tolerance among CD4+ T cells. The effects of transgenic overexpression of the bcl-2 gene or of deletion of the fas gene on germinal center and memory B cells will be determined.
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