The products of the I region of the major histocompatibility complex, I region associated (Ia) molecules, are intimately involved with, if not identical to the products of the genes controlling specific immune responses (Ir). I propose to study the way in which Ia molecules on antigen presenting cells (APC) interact with antigen to determine the immune response pattern of antigen-specific T cells by the preparation of specific Ia mutants in functional antigen presenting cells (APC). We have recently prepared somatic cell hybrids between normal heterozygous B cells and a drug-marked variant of a BALB/c B lymphoma cell line. These hybrid cell lines have potent APC ability and express the Ir gene patterns of both the normal B cell and the tumor cell partners. We have recently been successful in producing Ia mutants of one of these cell lines by first mutagenizing the cells with ethylmethane sulfonate, then by immunoselecting with one monoclonal anti-Ia reagent and complement followed by a (single or double) positive selection on the fluorescence activated cell sorter (FACS) with a second monoclonal anti-Ia antibody directed at a distinct Ia determinant. Cells which had lost the expression of the first Ia determinant but which retained the expression of the second one as well as cells which had lost both determinants were obtained. These mutant cell lines were then characterized functionally by correlating the loss of certain Ia determinants with the loss of ability to present specific foreign antigens to antigen-specific Ia-restricted T cell hybridomas. It is hoped that by using a panel of different anti-Ia monoclonal antibodies for both the negative and positive immunoselections a wide range of point mutations may be obtained. In particular, an Ia mutant which has extinguished antigen presenting function for only one antigenic determinant will be sought. The availability of such functional Ia mutant cell lines when used together with a large number of cloned antigen-specific T cell hybridomas and lines, may allow us to assign the presentation of specific antigens to well-defined Ia antigenic determinants. Another complementary approach to this problem will be undertaken in collaboration with Dr. J. Seidman. The genes encoding the murine I-A(R) Alpha and Beta chains will be inserted into both the parent BALB/c tumor line and into some of the already existing I-A(K) mutant lines and the phenotypic and functional characteristics of such transfected cell lines examined in our laboratory as a preliminary step toward the eventual goal of in situ mutagenesis of the cloned I-A gene.
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