Alloreactive immune responses of transgenic mice expressing a foreign transplantation antigen in a soluble form.

Abstract

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.

Transgenic mice expressing a soluble foreign H-2 class I antigen are tolerant to allogeneic fragments presented by self class I but not to the whole membrane-bound alloantigen.

“…We have recently reported that transgenic mice expressing a foreign class I antigen in soluble form could still react with the same high frequency of CTL precursors to the membranebound alloantigen as normal mice (8). We could, however, not exclude that the lack of tolerance toward the membrane antigen was based on the fact that the concentration of the antigenic protein in the serum (200 ng/ml) might have been insufficient to induce any tolerance.…”

“…Soluble H-2Kk was present in the sera of the transgenic animals (200 ng/ml). In spite of this, the transgenic mice were able to generate H-2Kk-specific CTL responses when stimulated with cell membrane-bound H-2Kk antigens (8). We have now analyzed the CTL responses of these animals to soluble H-2Kk by using either cells or recombinant vaccinia virus expressing soluble H-2Kk for immunization.…”

“…If the number of alloreactive CTLs responding to a processed form of class I were a substantial fraction of the total number of CTLs reacting with class I antigen, this would have major implications for the induction of tolerance to class I antigens and on the modulations of transplant rejections. To stiidy this question in more detail, we have recently designed a transgenic mouse model to test the influence of membrane-bound versus soluble alloantigen on the induction of tolerance and selection of the T-cell repertoire (8).…”

Immunological function of HLA-C antigens in HLA-Cw3 transgenic mice.

“…These results were expected but needed to be verified because in some transgenic systems lack of tolerance was observed. For example, not all mice transgenic for the simian virus 40 large tumor antigen were tolerant due to late expression during ontogeny (33), and transgenic mice expressing soluble H-2 antigens were not tolerant in their respective CTL and antibody compartments (34).…”

Molecular and cellular aspects of immunologic tolerance

“…In such situations, the B cell appears simply to ignore the antigen and no tolerance develops. Examples include transgenic foreign MHC deprived of its transmembrane domain [49], transgenic human insulin in the mouse [SO] and low concentrations of transgenic hen egg lysozyme (HEL) [32]. In those examples no B cell tolerance develops, although T cell tolerance may do so, presumably through passage of the protein concerned into the thymus, followed by local processing and presentation leading to clonal abortion.…”