The cellular requirements of T cell tolerance induction in the thymus by clonal deletion was investigated by using an in vitro assay: thymocytes from mice expressing a transgenic TcR specific for lymphocytic choriomeningitis virus (LCMV) and H-2Db were co-cultured with various H-2b cell types as antigen-presenting cells in the presence of the antigenic LCMV peptide. The results revealed that all cell lines examined including embryonic and transformed fibroblasts, melanoma cells, cortical thymic epithelial cells, lymphomas and neuronal cells induced an antigen dose-dependent deletion of CD4+8+ thymocytes. Similarly, highly enriched accessory cell populations from thymus and spleen (macrophages, dendritic and cortical epithelial cells, i.e. thymic nurse cells) could induce antigen-specific depletion of immature CD4+8+ thymocytes. Depending on the cell type examined micromolar to picomolar concentration of LCMV peptide were required to induce deletion. The effectiveness of deletion by the different cell types did not correlate with their major histocompatibility class I expression level; it was, however, influenced by the presence of ICAM-1 adhesion molecules.

We have determined the half-life in vivo of antigen/MHC class II complexes in different organ microenvironments. Mice were "pulsed" with myoglobin intravenously and MHC class II-positive antigen-presenting cell (APC) populations from different organs were isolated after various time intervals. Specific antigen/MHC complexes were quantitated by co-cultivation of the APC subsets with myoglobin-specific T-T hybridoma cells in vitro. Half-lives of antigen/MHC complexes differed both between organs and between compartments of the same organ. Half-lives in peripheral organs (spleen and bone marrow) ranged between 3 and 8 h, whereas in the thymus half-lives between 13 h (cortical epithelial cells) and 22 h (medullary dendritic cells) were observed. Half lives in vivo were independent of antigen processing, since intact protein or antigenic peptides yielded similar values. The considerably longer half-life of peptide/MHC complexes in the thymus as compared to peripheral organs may reflect the distinct role which antigen presentation plays in both organs, i.e. induction of tolerance versus induction of immunity.

The CD95 (APO-1/Fas) ligand (CD95L) mediates apoptosis in sensitive target cells, Ca(2+)-independent cytotoxicity of cells from perforin knock-out mice, and peripheral deletion of activated T cells through engagement of its cognate receptor CD95. Double-positive thymocytes show a high constitutive expression of CD95. Therefore, we used a model system and investigated whether negative selection through apoptosis might involve CD95/CD95L. We analyzed whether CD95L may induce antigen-specific deletion of double-positive thymocytes from mice transgenic for a lymphocytic choriomeningitis virus (LCMV)/H2b-specific T cell receptor (TCR). These cells are deleted in vitro upon addition of the LCMV-peptide 33-41 in a major histocompatibility complex-class I-restricted fashion. Deletion was not blocked by soluble mouse and human CD95-Fc receptor decoys. CD95-Fc receptor decoys, however, were effective in blocking apoptosis induced by mouse CD95L-transfected L929 cells in sensitive CD95+ target cells and in thymocytes. These results suggest that TCR-induced deletion of immature thymocytes in vitro is independent of CD95L. Thus, our data argue against a role of CD95L in negative selection of MHC-class I-restricted autoreactive thymocytes.

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