Tolerance induction by thymic epithelium induces a state of so-called "split tolerance," characterized in vivo by tolerance and in vitro by reactivity to a given thymically expressed antigen. Using a model major histocompatibility complex class I antigen, H-2Kb (Kb), three mechanisms of thymic epithelium-induced tolerance were tested: induction of tolerance of tissue-specific antigens exclusively, selective inactivation of T helper cell-independent cytotoxic T lymphocytes, and deletion of high-avidity T cells. To this end, thymic anlagen from Kb-transgenic embryonic day 10 mouse embryos, taken before colonization by cells of hemopoietic origin, were grafted to nude mice. Tolerance by thymic epithelium was not tissue-specific, since Kb-bearing skin and spleen grafts were maintained indefinitely. Only strong priming in vivo could partially overcome the tolerant state and induce rejection of some skin grafts overexpressing transgenic Kb. Furthermore, the hypothesis that thymic epithelium selectively inactivates those T cells that reject skin grafts in a T helperindependent fashion could not be supported. Thus, when T-cell help was provided by a second skin graft bearing an additional major histocompatibility complex class II disparity, tolerance to the Kb skin graft was not broken. Finally, direct evidence could be obtained for the avidity model of thymic epithelium-induced negative selection, using Kbspecific T-cell receptor (TCR) transgenic mice. Thymic epithelium-grafted TCR transgenic mice showed a selective deletion of tkpse CD8+ T cells with the highest density of the clonotyph 'CR. These cells presumably represent the T cells with the $ghest avidity for Kb. We conclude that split tolerance induced by thymic epithelium was mediated by the deletion of those CD8+ T lymphocytes that have the highest avidity for antigen.

In continuation of previous work on macrophage activation by a Mycoplasma fermentans-derived product, originally named "mycoplasma-derived high mol. wt. material" (MDHM), we have investigated whether MDHM was capable of inducing synthesis of the reactive nitrogen intermediate nitric oxide (NO), thus rendering macrophages cytocidal. Mycoplasmas were first delipidated with acetone, and MDHM activity was then extracted with 50 mM 1-O-octyl-beta-D-glucopyranoside to yield a particularly active new preparation of MDHM which we have named MDHM-D (D for detergent). In combination with IFN-gamma, MDHM-D activated macrophages to produce reactive nitrogen intermediates and kill P815 mastocytoma cells in co-culture. P815 target cells were chosen because they are TNF-resistant. Macrophages from the LPS-low responder strain C3H/HeJ were used to minimize interference from possible LPS contamination. MDHM-D activity in this system was strictly IFN-gamma-dependent. In the presence of 25 U/ml IFN-gamma MDHM-D gave a half maximal response at a dilution of 1/100,000, showing a parallel concentration dependency for nitrite production and cytocidal activity.

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