It is widely reported that Listeria monocytogenes can infect virtually all cell types, however, the degree to which this facultative intracellular pathogen can infect lymphocytes has not been well characterized. Previous studies have shown that a subset of lymphocytes, including activated T cells, are susceptible to apoptosis following exposure to L. monocytogenes, but the ability of the bacteria to replicate in the cytosol of lymphocytes prior to cell death was not examined. In this report, we demonstrate that intracellular L. monocytogenes can survive and multiply in vitro in a variety of transformed cell lines of lymphocytic origin. Intracellular L. monocytogenes were also recovered from splenic B cells, T cells, and NK cells following intravenous infection of mice. In fact, lymphocyte-associated L. monocytogenes comprised a substantial portion of the total bacterial burden in the spleen throughout the course of murine infection and B cell deficient mice had significantly lower titers of bacteria present in the spleen following intravenous infection. These results suggest that lymphocytes can be a reservoir for L. monocytogenes growth in vivo.

Detection of cytotoxic activity by pathogen-specific T cells of unknown antigenic specificity is difficult due to the limitations of using infected cells, instead of peptide-pulsed cells, as targets. We report here that the recently described CD107 mobilization assay readily allowed for the ex vivo detection of cytotoxic T lymphocytes (CTL) with a novel MHC-Ib restriction that specifically recognized Listeria monocytogenes-infected macrophages. The CD107 mobilization assay is likely to be a useful tool for detection of CD8 + T cells that recognize a wide variety of intracellular pathogens.

A subset of CD8؉ T cells can rapidly secrete gamma interferon (IFN-␥) in an antigen-independent and interleukin-12 (IL-12)-and IL-18-dependent manner within 16 h of infection with the intracellular bacterial pathogen Listeria monocytogenes. This rapid IFN-␥ response is robust enough to be detected directly ex vivo and is not observed following infection with intracellular bacterial pathogens that remain sequestered within host cell vacuoles. We demonstrate here that three distinct pathways can lead to rapid secretion of IFN-␥ by CD8 ؉ T cells during L. monocytogenes infection: (i) a direct cytokine-inducing activity encoded by the cholesteroldependent cytolysin (CDC) listeriolysin O (LLO) acts within the infected cell, (ii) the pore-forming activity of LLO promotes cytosolic localization of bacterial products that trigger cytosol-specific signaling pathways, and (iii) the sustained presence of high concentrations of bacterial products can exogenously trigger cytokine production. Although it has been suggested that CDC protein toxins may act as Toll-like receptor 4 (TLR4) agonists to trigger proinflammatory cytokine secretion, we show in this report that TLR4 signaling is not required to induce a maximal rapid IFN-␥ response by CD8 ؉ T cells. The results presented here indicate that multiple mechanisms contribute to the induction of rapid IFN-␥ secretion by CD8 ؉ T cells during Listeria infection and that care must be taken when interpreting the results of in vitro assays, since the contribution of each pathway can vary depending on how the assay is performed.Gamma interferon (IFN-␥) is a multifunctional cytokine that contributes to both innate and adaptive immune responses and is critical for the clearance of intracellular bacterial pathogens such as Listeria monocytogenes. IFN-␥ activates macrophages, which leads to enhanced production of antimicrobial oxygen and nitrogen intermediates and limits bacterial spread during infection (33, 41). IFN-␥ also increases major histocompatibility complex class I expression on macrophages, which promotes recognition of infected cells by CD8 ϩ cytotoxic T lymphocytes (11). Mice that lack IFN-␥ or its receptor are significantly more susceptible to infection with L. monocytogenes (8, 17).It has long been known that T cells are an important source of IFN-␥ during the adaptive phase of the immune response against L. monocytogenes; however, more recently it was demonstrated that a subset of CD8 ϩ T cells can also secrete IFN-␥ during the early, innate phase of the immune response. Berg et al. showed that up to 6% of splenic CD8 ϩ T cells were actively secreting IFN-␥ 16 h after infection of mice, a robust response that could be detected directly ex vivo without any further stimulation of the T cells (2, 3). All of the IFN-␥ ϩ CD8 ϩ T cells had a "memory" phenotype (CD44 hi ); however, the rapid IFN-␥ response was not antigen specific and instead depended only on the presence of the cytokines interleukin-12 (IL-12) and IL-18 (2).In addition to CD8 ϩ T cells, many other cell types ...