Transfection of cells with the H-2Kk gene lacking the transmembrane and cytoplasmic segments resulted in secretion of the H-2Kk protein, as determined by immunoprecipitation with monoclonal anti-H-2Kk antibodies. Transgenic (H-2b x H-2d)Fj mice were established carrying integrated copies of the modified H-2Kk gene. Expression of the soluble H-2Kk antigen in the transgenic mice was demonstrated in cell supernatants of biosynthetically labeled splenic and thymic Con A blasts as well as bone marrow-derived macrophages. Soluble H-2Kk molecules were also present in the sera of the transgenic animals. No cell-surface expression of the H-2Kk antigen could be observed. In spite of the presence of the soluble H-2Kk molecules in the transgenic mice, the animals were able to generate H-2Kk specific cytolytic T cells as well as antibody responses when stimulated with cell-surface-bound H-2K" antigens. These responses were indistinguishable from those of the nontransgenic littermates. Possible explanations for the observed lack of tolerance are discussed.The selection of the T-cell repertoire results in T cells that recognize foreign antigens only in association with the major histocompatibility complex (MHC) antigens (1). These T cells react also with high frequency with allogeneic MHC antigens, possibly due to crossreactivity (2). It is assumed that this selection takes place in the thymus, where T cells with high affinity for self MHC antigens are eliminated, resulting in tolerance for self MHC determinants. The mechanism of these selection processes is not understood. It is known that the presence of MHC molecules in the thymus is mandatory, but it is not clear if these MHC molecules have to be expressed as integral membrane proteins or if soluble MHC antigens are sufficient for induction of tolerance and education of T cells. Transgenic mice expressing a soluble "foreign" transplantation antigen should help to clarify this question.A prerequisite for this study is the conversion of membrane-associated proteins into secreted forms, a process that can be achieved by deletion of gene segments encoding the transmembrane portion of the protein (3). In this paper, we describe such a modification of the H-2Kk gene that leads to secretion of H-2Kk molecules by transfected cells. We also show that transgenic mice carrying the modified gene secrete the soluble H-2Kk protein into their serum. Immunological studies demonstrate that the presence of soluble H-2Kk proteins in these mice does not cause tolerance in cytolytic T lymphocyte (CTL) precursors and B cells specific for cell-surface-bound H-2Kk antigens.

The properties of transmembrane and soluble transplantation antigens were compared with respect to the induction of tolerance and the selection of the T-cell repertoire.For this purpose, transgenic (H-2b X H-2d)F1 mice were constructed that carry integrated copies of a modified H-2Kk gene resulting in the secretion from various cell types including thymocytes of soluble H-2Kk molecules. Despite the presence of H-2Kk antigen, these mice were still able to generate an H-2Kk-specific T-cell response. This response was comparable to that produced by normal littermates when stimulated with cells expressing membrane H-2Kk in a mixed lymphocyte reaction. In contrast, only transgenic mice failed to generate a cytolytic T-cell response to soluble H-2Kk antigen expressed by recombinant vaccinia virus and presented by the H-2Db molecule. These data imply the presence of two populations of alloreactive cytolytic T cells. A small fraction of T cells recognizes alloantigen as antigenic peptide(s) presented by other major histocompatibility complex class I molecules and tolerance can be induced in this population by soluble alloantigen. The majority of T cells, however, require the whole cell membrane-expressed class I molecule for recognition. This population is not affected by tolerance induction to the soluble major histocompatibility complex class I molecule.