IL-10 receptor blockade during T cell priming prevents the suppressive effects of <i>L. monocytogenes</i> within a phagosome.

<p>Mice were infected with ActA-Lm–OVA, LLO-Lm-OVA, or the combination of both strains in combination with αIL-10R antibody. (A) 30 days post infection mice were challenged with a lethal dose of wt <i>L. monocytogenes</i>-OVA. Spleens were harvested 3 days later and CFU per spleen determined. Each bar represents the mean and standard error of 5 mice per group. (B) B6.MyD88−/− mice were infected with 1×10⁵ CFU ActA-Lm-OVA, 1×10⁸ CFU LLO-Lm-OVA, or the combination of both strains. 16 weeks later, mice were challenged with 1×10³ CFU wt <i>L. monocytogenes</i>-OVA. Spleens and livers were harvested 3 days later and CFU per organ determined. Each bar represents the mean and standard error of 3–5 mice per group. Data are from one representative experiment of two.</p

Suppression of the primary T cell response is antigen-independent.

<p>(A) Mice were infected with the indicated combinations of 1×10⁵ CFU ActA-Lm, 1×10⁵ CFU ActA-Lm–OVA, 1×10⁸ CFU LLO-Lm, and 1×10⁸ CFU LLO-Lm-OVA. 7 days later, the frequency of OVA_257–264 -specific CD8 and LLO_190–201 -specific CD4 T cells was determined by IFN-γ intracellular cytokine staining. (B) ActA-Lm and LLO-Lm <i>L. monocytogenes</i> were engineered to express 4 defined epitopes from vaccinia virus. Mice were infected intravenously with the indicated combinations of ActA-Lm-QuadVacc and LLO-Lm-QuadVacc. 7 days later, spleens were harvested and the frequency of CD8 T cells specific for each epitope was determined by IFN-γ intracellular cytokine staining. Total splenocyte number and absolute CD8 T cells per spleen were consistent between all groups. Values in each plot represent the mean±SEM of IFN-γ+ cells within the CD4 or CD8 population from 5 animals per group.</p

<i>L. monocytogenes</i> within a phagosome impairs protective immunity.

<p>Mice were infected with 1×10⁵ CFU ActA-Lm–OVA alone, or in combination with increasing doses of phagosome-confined LLO-Lm-OVA. (A) Mice were challenged 60 days later with a lethal dose of wt <i>L. monocytogenes</i>-OVA. Spleens were harvested 3 days later and CFU per spleen determined. (B) Mice were infected with 1×10⁵ CFU ActA-Lm-Erm^R alone, or in combination with 1×10⁸ CFU LLO-Lm. Erythromycin-resistant colonies were enumerated from the spleen and liver over 96 hours. Each data point represents the mean and standard error of 5 mice per group. (C) Mice were infected with 1×10⁵ CFU ActA-Lm-Erm^R alone, or in combination with 1×10⁸ CFU LLO-Lm. Erythromycin-resistant colonies were enumerated from the spleen and liver at 1 and 6 hours post infection. Each data point represents the mean and standard error of 5 mice per group from one representative experiment of two. (D) Bone marrow-derived macrophages were infected with ActA-Lm-Erm^R alone (at 1∶10,000), or in combination with 1×10⁸ CFU LLO-Lm (at 1∶200). Erythromycin-resistant colonies were enumerated at the indicated timepoints. Each data point represents the mean and standard error of 3 independent coverslips per timepoint from one representative experiment of two. (E) Mice were infected with the indicated combinations of 1×10⁵ CFU ActA-Lm–OVA and 1×10⁸ heat-killed ActA-Lm–OVA. 30 days later, mice were challenged with 1×10⁵ CFU of wt <i>L. monocytogenes</i>-OVA. Spleens were harvested 3 days later and CFU per spleen determined. (F) Mice were infected with 1×10⁵ CFU ActA-Lm, 1×10⁵ CFU <i>B. subtilis</i>, or the combination of both strains. 30 days post infection, mice were challenged and CFU determined. (G) Mice were infected with the indicated combinations of 1×10³ CFU wild-type and 1×10⁶ CFU LLO-Lm. 58 days later, mice were challenged with 1×10⁵ CFU of wild-type <i>L. monocytognes</i>. Spleens were harvested 3 days later and CFU per spleen determined. In all panels, each point represents a single animal with † indicating animals that died before CFU were determined. Lines indicate the median of each group.</p

<i>L. monocytogenes</i> within a phagosome suppress the host inflammatory response to bacteria within the cytosol.

<p>(A) Mice were infected with 1×10⁵ CFU ActA-Lm–OVA alone, or in combination with increasing doses of LLO-Lm-OVA. Serum was collected 24 hours later and assayed for IFN-γ, IL-12p70, IL-6, and MCP-1. (B) C57Bl/6 and B6.MyD88−/− mice were infected with 1×10⁵ CFU ActA-Lm–OVA, 1×10⁸ CFU LLO-Lm-OVA, or the combination of both strains. Serum was collected 4 hours later and assayed for IL-10 and IL-12p40. Bars represent the mean and standard error of 5 mice per group.</p

<i>L. monocytogenes</i> within a phagosome impairs the primary T cell response.

<p>Mice were infected with 1×10⁵ CFU ActA-Lm–OVA alone, or in combination with increasing doses of LLO-Lm-OVA. 7 days later, the frequency of OVA_257–264-specific CD8 T cells and LLO_190–201-specific CD4 T cells was determined by pentamer and IFN-γ intracellular cytokine staining. Total splenocyte number and absolute CD8 T cells per spleen were consistent between all groups. Values in each plot represent the mean±SEM of antigen-specific cells within the CD4 or CD8 population from 5 animals per group.</p