Perforin-deficient mice have been generated by homologous recombination to determine whether the effects of CD8+ cytolytic T cells and natural killer cells are mediated by pore formation involving perforin. These mice are viable and fertile and have normal numbers of CD8+ T cells and natural killer cells which do not lyse virus-infected or allogeneic fibroblasts or natural killer target cells in vitro. The mice fail to clear lymphocytic choriomeningitis virus and they eliminate fibrosarcoma tumour cells with reduced efficiency. Perforin is therefore a key effector molecule for T-cell- and natural killer-cell-mediated cytolysis.

Two molecular mechanisms of T cell-mediated cytotoxicity, one perforin-based, the other Fas-based, have been demonstrated. To determine the extent of their contribution to T cell-mediated cytotoxicity, a range of effector cells from normal control or perforin-deficient mice were tested against a panel of target cells with various levels of Fas expression. All cytotoxicity observed was due to either of these mechanisms, and no third mechanism was detected. Thus, the perforin- and Fas-based mechanisms may account for all T cell-mediated cytotoxicity in short-term in vitro assays.

The immunoglobulin A (IgA) is produced to defend mucosal surfaces from environmental organisms, but host defenses against the very heavy load of intestinal commensal microorganisms are poorly understood. The IgA against intestinal commensal bacterial antigens was analyzed; it was not simply "natural antibody" but was specifically induced and responded to antigenic changes within an established gut flora. In contrast to IgA responses against exotoxins, a significant proportion of this specific anti-commensal IgA induction was through a pathway that was independent of T cell help and of follicular lymphoid tissue organization, which may reflect an evolutionarily primitive form of specific immune defense.

The crucial role of the thymus in immunological tolerance has been demonstrated by establishing that T cells are positively selected to express a specificity for self major histocompatibility complex (MHC), and that those T cells bearing receptors potentially reactive to self antigen fragments, presumably presented by thymic MHC, are selected against. The precise mechanism by which tolerance is induced and the stage of T-cell development at which it occurs are not known. We have now studied T-cell tolerance in transgenic mice expressing a T-cell receptor with double specificities for lymphocytic choriomeningitis virus (LCMV)-H-2Db and for the mixed-lymphocyte stimulatory (MIsa) antigen. We report that alpha beta TCR transgenic mice tolerant to LCMV have drastically reduced numbers of CD4+CD8+ thymocytes and of peripheral T cells carrying the CD8 antigen. By contrast, tolerance to MIsa antigen in the same alpha beta TCR transgenic MIsa mice leads to deletion of only mature thymocytes and peripheral T cells and does not affect CD4+CD8+ thymocytes. Thus the same transgenic TCR-expressing T cells may be tolerized at different stages of their maturation and at different locations in the thymus depending on the antigen involved.

Natural antibodies are often dismissed from immunological analysis as "background," but they may play an important role in conferring immunity against infections. In antibody-free mice infected with various viruses or with Listeria monocytogenes, viral or bacterial titers in peripheral organs, including the kidney and brain, were 10 to 100 times greater than in antibody-competent mice (and enhanced their susceptibility to some infections), and titers in secondary lymphoid organs were 10 to 100 times lower than in antibody-competent mice. Thus, natural antibodies play a crucial role by preventing pathogen dissemination to vital organs and by improving immunogenicity through enhanced antigen-trapping in secondary lymphoid organs.