Tolerance induction as a multi-step process.

Tolerant T cells are characterized by their partial or full resistance to activation by antigen. We investigated whether tolerant T cells were still receptive to further tolerogenic signals. T cells expressing a transgenic T cell receptor (TCR) specific for the major histocompatibility complex (MHC) class I molecule Kb were deleted in mice carrying Kb but not in mice expressing the mutant Kb-molecule Kbm1 [TCR (H-2bm1 x k) mice]. These T cells were tolerant in vivo but could be activated in vitro by the Kb antigen. This in vitro reactivity was abolished after the tolerant T cells encountered Kb-positive cells that had been intravenously injected. Furthermore, in TCR (H-2bm1 x k) mice expressing Kb only on hepatocytes, no T lymphocytes bearing the transgenic TCR could be found in the periphery, indicating that the additional contact with Kb on hepatocytes led to deletion of the tolerant T cells. These findings demonstrate that tolerance induction can be a multi-step process.

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.

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.

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

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)F1 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-2Kk antigens. These responses were indistinguishable from those of the nontransgenic littermates. Possible explanations for the observed lack of tolerance are discussed.