GENERATION AND CHARACTERIZATION OF I-E(D) MUTANTS FROM AN ANTIGEN PRESENTING MACROPHAGE CELL LINE (SOMAHI GENETICS, IA MOLECULES)

Abstract

Ia molecules on antigen presenting cells function as restriction elements in the recognition of antigen by T lymphocytes. In order to learn more about structure-function relationships of Ia antigens, structural I-E('d) variants have been generated from a murine macrophage cell line, BAC1.2. This line, obtained by SV40 transfection of splenic adherent cells from a (BALB/cxA.CA) F(,1) mouse, has properties of primary macrophages, including Fc receptors, Fc mediated phagocytosis, and lysozyme secretion. Because the BAC1.2 line is pseudo-diploid, and the A.CA strain expresses only an I-A molecule, making neither an E(alpha) nor E(beta) chain, the BAC1.2 cell line is haploid with respect to I-E expression. This facilitates selection of I-E('d) mutants. The BAC1.2 cell line is capable of presenting antigen to both T cell hydridomas and antigen-primed lymph node cells. Variants of the BAC1.2 cell line in I-E('d) that no longer interact with an antigen-specific I-E('d) restricted T cell hybridoma have been isolated. A T cell hybridoma 3D026.1, specific for I-E('d) and ovalbumin, was pretreated with tubercidin, a lethal adenosine analog. These tubercidin-containing T cells were then added to mutagenized, ovalbumin pulsed BAC1.2 cells. BAC1.2 cells with wild-type I-E('d) molecules bind and phagocytize a small number of tubercidin treated T cells, and so die. Variant BAC1.2 cells with either reduced levels of I-E('d) expression, or with mutations in the I-E('d) molecule such that they do not bind the T cell hybridomas, survive the selection. The variants obtained have lost the ability to present ovalbumin to the I-E('d) and ovalbumin specific T cell line used in the selections, but retain the ability to stimulate an I-A('d) and ovalbumin restricted T cell line, and T cell line reactive with I-E('d) alone. The variant progeny obtained appear not to differ with respect to I-E expression or ability to process antigen, but to represent structural variants. These mutants should aid in the elucidation of structual features of the I-E('d) molecule required for interactions with T helper cells.