The objectives of this proposal are to study the CH series B cell lymphomas which arose in B10.H-2aH-4bp/Wts mice and the normal Ly-1+ B cells from which they are derived. The CH lymphomas will be employed as models to study B cell differentiation. We will use tissue-culture adapted subclones of lymphomas, whose cells bear membrane IgM reactive with known antigens. We will concentrate on a group of tumors reactive with membrane phospholipids such as phosphatidyl choline, recognizable by the tumor IgM on sheep erythrocytes and bromelain treated mouse erythrocytes. We will study the influence of specific antigen, mitogens, lymphokines and MHC restricted T helper cells on the differentiation of the B cells. Differentiation will be detected as secretion of IgM, by localized hemolysis, or as a change in the immunoglobulin isotype produced. The latter will be detected by flow cytometry and by isotype specific ELISA of the supernatant from cultures grown to high density. We will sort and clone variant cells producing the changed isotypes and will compare the specificity and idiotype of the Ig produced with that of the IgM produced by the parent tumor. We will determine whether the same VH and VL genes are used for the parental IgM and the switched isotype. We will use restriction digest analysis of DNA, sandwich hybridization of RNA and nucleotide sequencing to elucidate the genetic mechanisms responsible for iusotype switching. We will use tissue culture, flow cytometry and ELISA to investigate the nature of signals which control isotype switching. We will also compare the specificity and idiotype of immunoglobulins produced by hybridomas derived from normal Ly- 1+B cells with those expressed by 27 previously characterized CH lymphomas. The objectives is to determine whether the CH lymphomas are derived from a random selection of the normal Ly- 1+B cells of syngeneic mice and to determine whether the Ig produced by normal Ly-1+B cells is as limited in specificity and idiotype as is that of the CH lymphomas. We will undertake exploratory experiments to test the importance of idiotype/antiidotype regulation in controlling the normal population of Ly-1+B cells.
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