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.

The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membraneperforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly + rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C-deficient hly + rBCG (∆ureC hly + rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. ∆ureC hly + rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of ∆ureC hly + rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell-mediated immunity.

Rapid removal of pathogens from the circulation by secondary lymphoid organs is prerequisite for successful control of infection. Blood-borne Ags are trapped mainly in the splenic marginal zone. To identify the cell populations responsible for Ag trapping in the marginal zone, mice were selectively depleted of marginal zone macrophages and marginal metallophilic macrophages. In the absence of these cells, trapping of microspheres and Listeria monocytogenes organisms was lost, and early control of infection was impaired. Depletion of marginal zone macrophages and marginal metallophilic macrophages, however, did not limit Ag presentation because Listeria-specific protective T cell immunity was induced. Therefore, marginal zone macrophages and marginal metallophilic macrophages are crucial for trapping of particulate Ag but dispensable for Ag presentation.

In vitro, T cell-dependent cytotoxicity is mediated by two distinct mechanisms, one being perforin-, the other Fas-dependent. The contribution of both of these mechanisms to clearance of viral infections was investigated in mice for the non-cytopathic lymphocytic choriomeningitis virus (LCMV) and the cytopathic vaccinia, vesicular stomatitis (VSV) and Semliki forest (SFV) viruses. Clearance of an acute LCMV infection was mediated by the perforin-dependent mechanism without measurable involvement of the Fas-dependent pathway. For the resolution of vaccinia virus infection and for resistance against VSV and SFV, however, neither of the two pathways was required. These data suggest that perforin-dependent cytotoxicity mediated by T cells is crucial for protection against non-cytopathic viruses, whereas infections with cytopathic viruses are controlled by nonlytic T cell-dependent soluble mediators such as cytokines (IFN-gamma against vaccinia virus) and neutralizing antibodies (against VSV and SFV).

Among the first cells to invade a site of infection, polymorphonuclear neutrophils (PMN) play an important role in the control of numerous infections. While PMN are considered critical for control of acute infections, their role in chronic infections remains less well understood. Here we report that PMN are essential for accurate early granuloma formation during chronic M. tuberculosis infection without influencing mycobacterial growth restriction. The PMNmediated regulation of granuloma formation depended on chemokines signaling through CXCR3, in particular MIG, as indicated by immune histochemical analysis of lung sections from C57BL/6 wild-type and CXCR3 -/-mutant mice and supported by microarray transcriptome analysis. Hence, PMN play a central role in regulating the focal granulomatous response in the lung, and this early granuloma formation can be segregated from long-term protection against pulmonary M. tuberculosis infection.

To investigate the role of T cell–mediated, perforin-dependent cytotoxicity in autoimmune diabetes, perforin-deficient mice were backcrossed with the nonobese diabetes mouse strain. It was found that the incidence of spontaneous diabetes over a 1 yr period was reduced from 77% in perforin +/+ control to 16% in perforin-deficient mice. Also, the disease onset was markedly delayed (median onset of 39.5 versus 19 wk) in the latter. Insulitis with infiltration of CD4+ and CD8+ T cells occurred similarly in both groups of animals. Lower incidence and delayed disease onset were also evident in perforin-deficient mice when diabetes was induced by cyclophosphamide injection. Thus, perforin-dependent cytotoxicity is a crucial effector mechanism for β cell elimination by cytotoxic T cells in autoimmune diabetes. However, in the absence of perforin chronic inflammation of the islets can lead to diabetogenic β cell loss by less efficient secondary effector mechanisms.

Macrophages play a key role in the immune defense against pathogens. They control early invasion by antigen-unspecific phagocytosis of pathogens and act as professional antigen-presenting cells to induce antigen-specific T cell responses. To investigate the involvement of particular subsets of the splenic macrophages in an antiviral immune response, we selectively depleted mice of splenic marginal zone macrophages (MZM) and marginal zone metallophils (MM) using the clodronate liposome depletion technique. MZM- and MM-depleted mice were not able to control an infection with lymphocytic choriomeningitis virus (LCMV). In these mice, LCMV spread from the spleen to peripheral organs at an early phase of infection. The virus-specific cytotoxic T lymphocyte (CTL) response was induced initially, yet was exhausted in parallel with the overwhelming virus replication. These findings suggest that MZM and MM play a crucial role in the early control of a LCMV infection by preventing immediate virus spread to peripheral organs, but are not essential for the induction of the LCMV-specific CTL response.

A potent Th1 immune response is critical to the control of tuberculosis. The impact of an additive Th2 response on the course of disease has so far been insufficiently characterized, despite increased morbidity after co-infection with Mycobacterium tuberculosis and Th2-eliciting helminths and possible involvement of Th2 polarization in reactivation of latent tuberculosis. Here, we describe the gene expression profile of murine bone marrow-derived macrophages alternatively activated by IL-4 in response to infection with M. tuberculosis. Comparison of transcriptional profiles of infected IL-4-and IFN-c-activated macrophages revealed delayed and partially diminished responses to intracellular bacteria in alternatively activated macrophages, characterized by reduced exposure to nitrosative stress and increased iron availability, respectively. Alternative activation of host macrophages correlated with elevated expression of the M. tuberculosis iron storage protein bacterioferritin as well as reduced expression of the mycobactin synthesis genes mbtI and mbtJ. The extracellular matrix-remodeling enzyme matrix metalloproteinase (MMP)-12 was induced in alternatively activated macrophages in vitro, and MMP-12-expressing macrophages were abundant at late, but not early, stages of tuberculosis in murine lungs. Our findings emphasize that alternative activation deprives macrophages of control mechanisms that limit mycobacterial growth in vivo, thus supporting intracellular persistence of M. tuberculosis.