Effector T cells do not find the granuloma to be a stimulating environment.

<p>Effector T cells enter the granuloma and only a few exhibit significant migration arrest (dark blue cells) and targeted release of IFNγ, likely when they encounter a high level of cognate antigen on infected phagocytes. As their cognate antigen is reduced, even fewer cells undergo migration arrest, with many more cells continuing to move throughout the granuloma (light blue motile cells). Although these cells do not stop migrating, they do up regulate CD69 in an antigen-specific manner. Cells entering the granuloma may mediate their effector function without the release of IFNγ, and while this activity does require recognition of antigen, it may not need migration arrest.</p

Type 1 immune responses are similar but myeloid cell activation is different between C57BL/6 and AID^−/−µS^−/− Mtb-infected mice.

<p>(A–F) C57BL/6 (filled symbols) and AID^−/−µS^−/− (opened symbols) mice were infected as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a>. (A) Lung cells were cultured with ESAT_1–20 peptide to determine the number of IFN-γ-producing ESAT_1–20-specific cells. The number of CD3⁺CD4⁺IFN-γ⁺ or CD3⁺CD8⁺IFN-γ⁺ was determined by flow cytometry. (B) The presence of mRNA for <i>ifng</i> in the lungs of infected mice was determined by real-time RT-PCR. (B) IFN-γ protein was determined in lung homogenates by Luminex. (C) MHC-II expression on CD11c+ cells (expressed by alveolar macrophages and dendritic cells) derived from the lungs of uninfected (shaded curve) or infected C57BL/6 (solid line) or AID^−/−µS^−/− mice (dashed line) was determined by flow cytometry. (A–C) One experiment representative of 3 independent experiments is shown. <i>n</i> = 4 mice per group. *, p<0.05; **, p<0.01; ***, <i>p</i><0.001 by Student’s <i>t</i> test. (D) At specific time points after infection, the caudal lobe of the lung from each mouse was processed for histologic analysis and stained using H&E. Sections were screened and scored for inflammation, PMN infiltration and necrosis by a pathologist in a blinded manner (0: absent; 1: minimal; 2: mild; 3: moderate; 4: marked). One experiment representative of 2 independent experiments is shown. <i>n</i> = 4 mice per group. (E) At different time points after infection, RNA was extracted from lung tissue and analyzed by real-time PCR for the expression of <i>nos2, irgm1, fizz1, fizz2, arg1 and arg2</i>. (F) At day 60 post-infection, arginase activity was determined in the lungs of C57BL/6 (filled symbols) and AID^−/−µS^−/− (open symbols). One experiment representative of 2 independent experiments is shown. <i>n</i> = 4–5 mice per group. *, p<0.05; **, p<0.01; ***, <i>p</i><0.001 by Student’s <i>t</i> test.</p

Immunization with YopE_69–77 peptide protects mice against <i>Y. pestis</i>.

<p>Wild-type C57BL/6 mice were immunized intranasally with CT adjuvant alone (CT) or CT mixed with YopE_69–77 peptide (YopE) and then challenged intranasally with (A) 20 MLD (2×10⁵ CFU) or (B) 200 MLD (2×10⁶ CFU) <i>Y. pestis</i> strain D27 or (C) 10 MLD (1×10⁴ CFU) <i>Y. pestis</i> strain CO92. In comparison with CT–immunized mice (n = 10–40), YopE_69–77–immunized mice (n = 15–39) exhibited significantly increased survival. Data were pooled from 2–5 independent experiments.</p

TNFα and IFNγ produced by YopE_69–77-specific CD8 T cells have complementary roles during protection against <i>Y. pestis</i>.

<p>(A) Wild-type (WT), TNFα-deficient (TNFaKO) or IFNγ-deficient (IFNgKO) C57BL/6 mice were immunized with CT mixed with YopE_69–77 peptide. WT mice immunized with CT adjuvant only or CT mixed with OVA_257–264 peptide were used as controls. CD8+ splenocytes were then purified and transferred intravenously to naïve WT C57BL/6 mice, which were challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27 the next day. In comparison with mice that received CD8+ T cells from CT-immunized WT mice (n = 50), mice that received CD8+ T cells from YopE_69–77-immunized WT (n = 56), TNFα-deficient (n = 17) and IFNγ-deficient (n = 15) mice were protected against <i>Y. pestis</i> challenge. Notably, mice that received CD8+ T cells from OVA_257–264-immunized WT mice (n = 25) were also protected (p<0.05). Data were pooled from 7 independent experiments. (B) Splenocytes isolated from naïve WT, TNFαKO, IFNγKO, perforin-deficient (PKO) or TNFα/IFNγ-deficient (TNFαIFNγ DKO) mice were transferred to TCRβδ-deficient mice. The mice were then immunized with CT mixed with YopE_69–77. Control mice received naïve WT splenocytes and were immunized with CT mixed with OVA_257–264 peptide. Mice were then challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27. In comparison with control mice (n = 15), YopE_69–77 immunized mice that received WT (n = 22), TNFαKO (n = 25), IFNγKO (n = 20), or PKO (n = 11) splenocytes were protected against <i>Y. pestis</i> challenge. YopE_69–77-immunized mice that received TNFαIFNγ DKO splenocytes were also protected (n = 11) but the survival was significantly lower in comparison with the mice received WT, TNFαKO or IFNγKO splenocytes. Data were pooled from 3 independent experiments.</p

Blocking of IL-10R improves the ability of AID^−/−µS^−/− mice to control Mtb.

<p>C57BL/6 and AID^−/−µS<i>^−/−</i> mice were infected as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a>. (A) The concentration of IL-10, IL-6 and G-CSF in lung homogenates of Mtb-infected mice was determined by Luminex. One experiment representative of 2 independent experiments is shown (<i>n</i> = 4). *, p<0.05; **, p<0.01 ***, <i>p</i><0.001 by Student’s <i>t</i> test. (B) Lung sections from mice infected for 60 days as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a> were stained for B220 (red) and IL-10 (green), white dashed line represents edge of B cell follicle area (bar represents 50 microns). Representative sections shown for C57BL/6 (left) and AID^−/−µS^−/− (right panel), reproduced in five other mice and in 2 separate experiments. (C) C57BL/6 (filled symbols) and AID^−/−µS^−/− (open symbols) mice were infected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a>. At day 0 and once a week thereafter, mice were treated with anti-IL-10R antibody (squares) or with the same concentration of an isotype control antibody (circles). Lung and spleen bacterial burden was determined at day 60 post-infection. One experiment representative of two independent experiments is shown (<i>n</i> = 5). *, p<0.05; ***, <i>p</i><0.001 by one-way ANOVA with Tukey’s multiple comparisons post-test.</p

B cell depletion in Mtb-infected AID^−/−µS^−/− mice allows for improved control of bacterial burdens in the spleen, but not in the lung.

<p>C57BL/6 (closed symbols) and AID^−/−µS^−/− (open symbols) mice were infected as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a> and then treated every other week starting either day 0 or day 30 with an isotype control antibody (circles) or with an anti-mouse CD20 antibody (αCD20, squares) and (A) flow cytometry was used to determine depletion of CD19⁺CD3⁻ cells at day 30 (depletion was maintained through day 60 - not shown). (B) The bacterial burden was determined by plating the lung and spleen on agar and counting viable colony forming units. (C) Radiation bone marrow chimeras were made with bone marrow from C57BL/6 (closed circles) AID^−/−µS^−/− (open circles) or a mixture of the two (half filled circles) and then infected as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a> and the bacterial burden in the spleen measured. (D) C57BL/6 (closed symbols) and AID^−/−µS^−/− (open symbols) mice were infected as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061681#pone-0061681-g001" target="_blank">Figure 1</a> and then treated every three days starting at day 15 with normal mouse serum (triangles) or were left untreated (circles). Bacterial burden was assessed at day 60. Data from two separate experiments showing the same results independently have been combined in A–D (<i>n</i> = 8–10 mice per group). **, p<0.01; ***, <i>p</i><0.001 by ANOVA with Tukey’s multiple comparisons post-test.</p

AID^−/−µS^−/− mice have an altered B cell population in the lung and are more susceptible to aerosol infection with Mtb.

<p>C57BL/6 (filled circles) and AID^−/−µS^−/− mice (opened circles) were infected with Mtb H37Rv via the aerosol route. (A–C) At day 30 of infection, B cells (gated on live lymphocytes and CD19 expression) were analyzed for the expression of IgM and IgD by flow cytometry. The frequency (A) and total number of CD19⁺ cells, CD19⁺IgD⁻IgM⁺ cells and CD19⁺IgD⁺IgM⁺ cells in the lung (B) and spleen (C) was calculated. (D) The bacterial burden was determined in lungs and spleen over time (<i>n</i> = 4). (E) Survival of infected mice was determined over the course of the experimental infection (<i>n</i> = 5). (A–E) One experiment representative of at least three independent experiments is shown *, p<0.05**, p<0.01; ***, <i>p</i><0.001 by Student’s <i>t</i> test. (F–H) C57BL/6 (filled circles), AID<i>^−/−</i> (F), µS^−/− (G) and µMT (H) mice (opened circles) were infected, and the bacterial burden was determined in the lungs and spleen over time (<i>n</i> = 4–8). Data sets from a total of two experiments were combined. *, p<0.05; ***, p<0.001 by Student’s <i>t</i> test.</p

Perforin is dispensable for YopE_69–77–specific CD8 T cell-mediated protection against <i>Y. pestis</i> and <i>Y. pseudotuberculosis</i>.

<p>Wild-type (WT) and perforin-deficient (PKO) C57BL/6 mice were immunized intranasally with CT adjuvant alone, or CT mixed with YopE_69–77 or OVA_257–264 peptides and then challenged with (A and B) 20 MLD (2×10⁵ CFU) <i>Y. pestis</i> strain D27 intranasally, (C) 10 MLD (5×10⁹ CFU) <i>Y. pseudotuberculosis</i> strain 32777 intragastrically or (D) 10 MLD (1.2×10² CFU) <i>Y. pseudotuberculosis</i> strain 32777 intravenously. (A) <i>Y. pestis</i> survival data pooled from 3 independent experiments (n = 9–30 mice/group). (B) Day 4 bacterial burden in lung and liver tissues after <i>Y. pestis</i> challenge (Kruskal-Wallis test, compared with CT- or OVA_257–264–immunized PKO or WT mice). Data are pooled from 2 independent experiments (n = 9–11 mice/group). Solid bar depicts median; broken line depicts the limit of detection. (C and D) <i>Y. pseudotuberculosis</i> survival data (n = 6–7 mice/group for CT, n = 10–11 mice/group for YopE). Data were pooled from 2 independent experiments.</p

Mice immunized with YopE_69–77 exhibit perforin-dependent cytotoxic activity.

<p>Wild-type (WT) and perforin-deficient (PKO) C57BL/6 mice were immunized with CT adjuvant alone or CT mixed with 1 or 10 µg of YopE_69–77 or OVA_257–264 peptides. Splenocytes from naïve congenic WT mice (CD45.1+) that were either pulsed with YopE_69–77 peptide and labeled with 10 µM of CFSE or pulsed with OVA_257–264 peptide and labeled with 1 µM of CFSE were mixed together at a 1∶1 ratio and injected into the immunized recipient mice. Splenocytes of recipient mice were then collected 20–22 h later and stained for congenic marker. The target cells (CD45.1+CD45.2−) were gated and the proportion of each CFSE-labeled population was analyzed by flow cytometry. The percent specific lysis was then calculated as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004142#s4" target="_blank">Materials and Methods</a>. (A) Representative plots of the flow cytometry analysis of naïve or immunized WT recipient mice. The numbers on the lower right corner of the plots depict the percentage of specific lysis of YopE_69–77-pulsed target cells or OVA_257–264-pulsed target cells. (B and C) The percentages of specific lysis of YopE_69–77-pulsed target cells by YopE_69–77-immunized mice (B) or OVA_257–264-pulsed target cells by OVA_257–264-immunized mice (C). In comparison with WT mice, PKO mice displayed significantly decreased cytotoxicity (one-way ANOVA). Data shown are pooled from 5 independent experiments. (D and E) The percentages of PBL staining positive for CD8 and K^bYopE_69–77 (D) or K^bOVA_257–264 (E) tetramers two days before the cytotoxicity assay. In comparison with WT mice, PKO mice immunized with the same amount of peptide have comparable Ag-specific CD8 T cell frequencies (one-way ANOVA). Data shown are pooled from 5 independent experiments.</p

YopE_69–77-specific CD8 T cells lacking the capacity to produce TNFα and IFNγ fail to protect mice and control bacterial burden.

<p>TCRβδ-deficient (TCRbdKO) mice were lethally irradiated and reconstituted with 75% TCRβδKO bone marrow cells and 25% of either WT, TNFαKO, IFNγKO, PKO, or TNFαIFNγ DKO bone marrow cells. Six weeks later they were immunized with CT mixed with YopE_69–77 or OVA_257–264 and then challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27. (A) Survival. In comparison with OVA_257–264-immunized mice reconstituted with WT T cells (n = 20), the YopE_69–77-immunized chimeric mice reconstituted with WT (n = 25), TNFαKO (n = 17), IFNγKO (n = 19), PKO (n = 8) or TNFαIFNγ DKO (n = 33) T cells all showed significant protection. (B) The percentage of CD8+ T cells that stained positive for MHC class I tetramer K^bYopE_69–77 in PBL on the day before challenge. Solid bar depicts the mean. All groups of chimeric mice that were immunized with YopE_69–77 had significantly increased frequency of K^bYopE_69–77+CD8+ T cells in compared with the chimeric mice immunized with OVA_257–264 (p<0.001). YopE_69–77-immunized chimeric mice reconstituted with TNFαKO T cells had significantly higher frequency of K^bYopE_69–77+CD8+ T cells in compared with YopE_69–77-immunized chimeric mice reconstituted with WT T cells (p<0.01). Data for (A) and (B) are pooled from 6 independent experiments. (C and D) Bacterial burden in lung (C) and liver (D) tissues was measured at day 4 after challenge (Kruskal-Wallis test). Data are pooled from 3 independent experiments. Solid bar depicts median; broken line depicts the limit of detection.</p