Curtis J. Henry

IL-12 Produced by Dendritic Cells Augments CD8+ T Cell Activation through the Production of the Chemokines CCL1 and CCL17

IL-12 family members are an important link between innate and adaptive immunity. IL-12 drives Th1 responses by augmenting IFN-γ production, which is key for clearance of intracellular pathogens. IL-23 promotes the development of IL-17-producing CD4+ T cells that participate in the control of extracellular pathogens and the induction of autoimmunity. However, recent studies have shown that these cytokines can modulate lymphocyte migration and cellular interactions. Therefore, we sought to determine the individual roles of IL-12 and IL-23 in naive CD8+ T cell activation by addressing their ability to influence IFN-γ production and cellular interaction dynamics during priming by Listeria monocytogenes-infected dendritic cells (DC). We found that IL-12 was the major cytokine influencing the level of IFN-γ production by CD8+ T cells while IL-23 had little effect on this response. In addition, we observed that IL-12 promoted longer duration conjugation events between CD8+ T cells and DC. This enhanced cognate interaction time correlated with increased production of the chemokines CCL1 and CCL17 by WT but not IL-12-deficient DC. Neutralization of both chemokines resulted in reduced interaction time and IFN-γ production, demonstrating their importance in priming naive CD8+ T cells. Our study demonstrates a novel mechanism through which IL-12 augments naive CD8+ T cell activation by facilitating chemokine production, thus promoting more stable cognate interactions during priming.

Differential susceptibility of bone marrow-derived dendritic cells and macrophages to productive infection with Listeria monocytogenes.

Dendritic cells (DC) are required for the immune response against Listeria monocytogenes and are permissive for infection in vivo and in vitro. However, it is unclear if DC provide a desirable intracellular niche for bacterial growth. To address this issue, we have compared the behaviour of L. monocytogenes in murine bone marrow‐derived DC and macrophages (BMM). Similar to BMM, bacteria escaped to the cytosol in DC, replicated, and spread to adjacent cells. However, DC infection was less robust in terms of intracellular doubling time and total increase in bacterial numbers. Immunofluorescence analysis using a strain of L. monocytogenes that expresses green fluorescent protein upon bacterial entry into the cytosol suggested that a subpopulation of DC restricted bacteria to vacuoles, a finding that was confirmed by electron microscopy. In unstimulated DC cultures, L. monocytogenes replicated preferentially in phenotypically immature cells. Furthermore, DC that were induced to mature prior to infection were poor hosts for bacterial growth. We conclude that DC provide a suboptimal niche for L. monocytogenes growth, and this is at least in part a function of the DC maturation state. Therefore, the generation of an effective T cell response may be a net effect of both productive and non‐productive infection of DC.

Distinct Responses of Splenic Dendritic Cell Subsets to infection with Listeria monocytogenes: maturation phenotype, level of infection, and T cell priming capacity ex vivo

To determine the relative contributions of DC subsets in the development of protective immunity to Listeria monocytogenes we examined the relationship between maturation, bacterial burden, and T cell priming capacity of four well characterized subsets of splenic DC following infection with Lm. CD8α(+), CD4(+), and CD8α(-)CD4(-) DC and the B220(+) plasmacytoid DC (pDC) were compared for abundance and costimulatory molecule expression at 24, 48, and 72h post i.v. infection. We further determined the bacterial burden associated with each DC subset and their relative capacities to prime CD8(+) T cells at 24hpi. The CD8α(+) DC displayed the highest level of maturation, association with live bacteria, and T cell activation potential. Second, the CD4(+) DC were also mature, yet were associated with fewer bacteria, and stimulated T cell proliferation, but not IFN-γ production. The CD8α(-)CD4(-) DC showed a modest maturation response and were associated with a high number of bacteria, but failed to induce T cell proliferation ex vivo. pDC displayed a strong maturation response, but were not associated with detectable bacteria and also failed to stimulate T cell activation. Finally, we measured the cytokine responses in these subsets and determined that IL-12 was produced predominantly by the CD8(+) DC, correlating with the ability of this subset DC to induce IFN-γ production in T cells. We conclude that Listeria-specific CD8(+) T cell activation in the spleen is most effectively achieved by infection-induced maturation of the CD8α(+) DC subset.

The roles of IL-12 and IL-23 in CD8+ T cell-mediated immunity against Listeria monocytogenes: Insights from a DC vaccination model.

Listeria monocytogenes infection induces a strong inflammatory response characterized by the production of IL-12 and IFN-gamma and protective immunity against this pathogen is dependent on CD8+ T cells (CTL). Recent studies have suggested that these inflammatory cytokines affect the rate of memory CD8+ T cell generation as well as the number of short-lived effector cells generated. The role of the closely related cytokine, IL-23, in this response has not been examined. We hypothesized that IL-12 and IL-23 produced by dendritic cells collectively enhance the generation and function of memory cells. To test this hypothesis, we employed a DC vaccination approach. Mice lacking IL-12 and IL-23 were vaccinated with wild-type (WT), IL-12(-/-), or IL-12/23(-/-) DC and protection to Lm was monitored. Mice vaccinated with WT and IL-12(-/-) DC were resistant to lethal challenge with Lm. Surprisingly, mice vaccinated with IL-12/23(-/-) DC exhibited significantly reduced protection when challenged. Protection correlated with the relative size of the memory pools generated. In summary, these data indicate that IL-23 can partially compensate for the lack of IL-12 in the generation protective immunity against Lm.

Dendritic Cells Inhibit the Progression of Listeria monocytogenes Intracellular Infection by Retaining Bacteria in Major Histocompatibility Complex Class II-Rich Phagosomes and by Limiting Cytosolic Growth

ABSTRACT Dendritic cells (DC) provide a suboptimal niche for the growth of Listeria monocytogenes, a facultative intracellular bacterial pathogen of immunocompromised and pregnant hosts. This is due in part to a failure of large numbers of bacteria to escape to the cytosol, an essential step in the intracellular life cycle that is mediated by listeriolysin O (LLO). Here, we demonstrate that wild-type bacteria that failed to enter the cytosol of bone marrow-derived DC were retained in a LAMP2+ compartment. An isogenic L. monocytogenes strain that produces an LLO protein with reduced pore-forming activity had a severe escape and growth phenotype in DC. Few mutant bacteria entered the cytosol in the first 2 h and were instead found in LAMP2+, major histocompatibility complex class II+ (MHC-II+) H2-DM vesicles characteristic of MHC-II antigen loading compartments (MIIC). In contrast, the mutant had a minor phenotype in bone marrow-derived macrophages (BMM) despite the reduced LLO activity. In the first hour, DC phagosomes acidified to a pH that was, on average, half a point higher than that of BMM phagosomes. Unlike BMM, L. monocytogenes growth in DC was minimal after 5 h, and consequently, DC remained viable and matured late in infection. Taken together, the data are consistent with a model in which phagosomal maturation events associated with the acquisition of MHC-II molecules present a suboptimal environment for L. monocytogenes escape to the DC cytosol, possibly by limiting the activity of LLO. This, in combination with an undefined mechanism that controls bacterial growth late in infection, promotes DC survival during the critical maturation response.