Inhibition by 5-lipoxygenase of the early initiation of an immune response after M. tuberculosis infection. (a,b) Frequency of TB10.4(4–11)-specific CD8⁺ T cells in the thoracic draining lymph nodes (a) and lungs (b) of Ptges^−/−, wild-type (WT) and Alox5^−/− mice 2 weeks after M. tuberculosis infection. Numbers adjacent to outlined areas indicate percent CD8⁺ T cells stained with the TB10.4(4–11)-loaded H-2K^b tetramer (H-2K^b–TB10.4(4–11)). Data are representative of two independent experiments (mean of four to five mice per group). (c) Kinetics of the pulmonary TB10.4-specific CD8⁺ T cell response in Ptges^−/−, wild-type and Alox5^−/− mice. *P < 0.05 (one-way analysis of variance (ANOVA)). Data are representative of two experiments (mean ± s.e.m. of four mice per group).

Transfer of M. tuberculosis–infected Alox5^−/− macrophages into wild-type mice recapitulates the phenotype of intact Alox5^−/− mice. (a) Frequency of TB10.4(4–11)-specific CD8⁺ T cells in the thoracic draining lymph nodes (top) and lungs (bottom) 17 d after intratracheal transfer of M. tuberculosis H37Rv–infected Alox5^−/−, Ptges^−/− or wild-type macrophages into wild-type mice. Numbers adjacent to outlined areas indicate percent CD8⁺ T cells stained with H-2K^b–TB10.4(4–11). Data are representative of four independent experiments (mean and s.e.m. of 16 total mice per group). (b) Enzyme-linked immunospot assay of IFN-γ production showing the frequency of M. tuberculosis–specific CD4⁺ and CD8⁺ T cells 17 d after intratracheal transfer of M. tuberculosis H37Rv–infected Ptges^−/−, wild-type or Alox5^−/− macrophages into wild-type mice; splenic T cells from the wild-type recipient mice were cultured with APCs and Ag85B(241–256), ESAT6(3–15), TB10.4(4–11) or 32c(309–318). Data are representative of three experiments. (c) Kinetics of PLN and lung TB10.4-specific CD8⁺ T cell responses in recipient mice after transfer of infected Ptges^−/−, wild-type or Alox5^−/− macrophages. SFC, spot-forming cells. Data are representative of two experiments (mean ± s.e.m. of four mice per group). (d) Bacterial burden in the lung 28 d after intratracheal transfer of M. tuberculosis H37Rv–infected Alox5^−/−, Ptges^−/− or wild-type macrophages into wild-type mice. Each symbol represents an individual mouse; small horizontal bars indicate the mean. Data are from two independent experiments (n = 4 mice per group). NS, not significant; *P < 0.05, compared with wild-type (one-way ANOVA).

Transfer of infected Alox5^−/− alveolar macrophages generates pulmonary and systemic CD4⁺ and CD8⁺ T cell responses. (a) Frequency of TB10.4(4–11)-specific CD8⁺ T cells in the lungs, PLN and spleen 17 d after intratracheal transfer of alveolar macrophages (MΦ) from wild-type mice (left) or Alox5^−/− mice (right) infected by aerosol with M. tuberculosis (virulent Erdman strain). Numbers adjacent to outlined areas indicate percent CD8⁺ T cells stained with H-2K^b–TB10.4(4–11) or streptavidin-phycoerythrin alone (Control; bottom row). Data are from one experiment. (b) Frequency of H-2K^b–TB10.4(4–11)–stained CD8⁺ T cells in the lungs, PLN and spleens of mice treated as described in a (n = 5 per group). Staining with isotype-matched control antibody (Isotype) is shown for the wild-type group only. *P < 0.05 (one-way ANOVA with the Bonferroni post-test). Data are from one experiment (mean and s.e.m.).

CD8⁺ T cell activation induced by M. tuberculosis–infected Alox5^−/− macrophages requires cross-presentation by CD11c⁺ DCs. (a,b) CD8⁺ T cell proliferation (a) and population expansion of cells positive for the H-2K^b–SIINFEKL pentamer (b) 5 d after intravenous transfer of CFSE-labeled Thy-1.2⁺ splenic OT-I CD8⁺ T cells into Thy-1.1⁺ B6.PL mice, followed within 24 h by M. tuberculosis H37Rv–infected wild-type or Alox5^−/− macrophages pulsed with SIINFEKL. Numbers above bracketed lines (a) indicate percent CFSE⁻ cells (left) or CFSE⁺ cells (right); numbers adjacent to outlined areas (b) indicate percent CD8⁺ T cells stained with H-2K^b–SIINFEKL. (c) Frequency of SIINFEKL-specific CD8⁺ T cells in the draining lymph nodes of CD11c-DTR–transgenic mice (CD11c-DTR) and nontransgenic littermate control mice (Non-TG) given intravenous injection of OT-I CD8⁺ T cells, followed by subcutaneous transfer of M. tuberculosis H37Rv–infected, SIINFEKL-pulsed Alox5^−/− macrophages and treatment with diphtheria toxin. No Mϕ (right), control mice that did not receive macrophages. Numbers adjacent to outlined areas indicate percent CD8⁺ T cells stained with H-2K^b–SIINFEKL after 5 d. Data are representative of three experiments (mean and s.e.m. of four to five mice per group). (d) OVA-specific OT-I CD8⁺ T cells in draining lymph nodes of β₂-microglobulin-deficient (B2m^−/−), TAP-1-deficient (Tap1^−/−) or Thy-1.1⁺ recipient mice 5 d after the transfer of CFSE-labeled Thy-1.2⁺ splenic OT-I CD8⁺ T cells and M. tuberculosis H37Rv–infected, SIINFEKL-pulsed wild-type or Alox5^−/− macrophages. Data are representative of two to three independent experiments (mean ± s.e.m. of three to five mice per group). *P < 0.05 (one-way ANOVA).

Caspase-dependent apoptosis of M. tuberculosis-infected Alox5^−/− macrophages is required for the early initiation of T cell immunity. (a) Apoptosis of Alox5^−/− macrophages infected with M. tuberculosis H37Rv (multiplicity of infection, ~2), assessed 3 d after treatment for 2 h with a negative control peptide (Negative control) or with an inhibitor of caspase-8 or caspase-9 or both (0.5 μM each), presented relative to that of uninfected Alox5^−/− macrophages (Untreated). *P < 0.05, compared with control group (one-way ANOVA with Dunnett’s multiple-comparison test). Data are representative of three experiments (mean and s.e.m.). (b) Frequency of TB10.4(4–11)-specific CD8⁺ T cells in the lungs of mice 17 d after the intratracheal transfer of M. tuberculosis H37Rv–infected Alox5^−/− macrophages treated with inhibitors of caspase-8 and caspase-9 (Both inhibitors) or a negative control peptide (Negative control); results (right) are normalized by division of the absorbance by the absorbance of cultures of uninfected macrophages. Numbers adjacent to outlined areas indicate percent CD8⁺ T cells stained with H-2K^b–TB10.4(4–11). *P < 0.05 (Mann-Whitney test). Data are representative of two independent experiments (mean and s.e.m. of four mice per group).
Supplementary Figure 1. The immune response is similar in Ptges^−/−, wild type, and Alox5^−/− mice five weeks after Mtb infection. The frequency of TB10.4(4-11)-specific CD8⁺ T cells was determined five weeks after aerosol Mtb infection (~100 CFU) in the lungs (a) and the spleens (b) of Ptges^−/−, wild type, and Alox5^−/− mice. The percentage of CD8⁺ T cells that stained with the H-2 K^b–TB10.4(4-11) tetramer is indicated in each representative FACS plot. Each bar is the mean ± SE, of 5 individual mice. (c) The pulmonary bacterial burden in Ptges^−/−, wild type, and Alox5^−/− mice (n=5/group) five weeks after infection with aerosolized Mtb. Results are representative of two independent experiments. *, p<0.05 by a one-way ANOVA compared to wild type group, or †, p<0.05 Alox5^−/− vs. Ptges^−/−.
Supplementary Figure 2. The intrinsic ability of T cells from Ptges^−/−, wild type, and Alox5^−/− mice to provide protection against pulmonary Mtb infection is similar. (a) Sublethally irradiated wild type mice were used as recipients for splenic T cells (CD3⁺) from Mtb infected Ptges^−/−, wild type, or Alox5^−/− mice. Recipient mice were infected with Mtb by the aerosol route within 24 hrs after transfer of purified T cells. Three weeks after infection, the bacterial burden in the lung and spleen were determined. Each point represents data from an individual mouse, and the bars represent the mean (n=4-5 mice per group). The differences in lung CFU between Ptges^−/− and wild type, or wild type and Alox5^−/− mice were not significant (as determined by a one-way ANOVA). (b) Control experiment done under the same conditions as (a). Recipient mice were irradiated and purified splenic T cells from Mtb infected syngeneic mice were transferred intravenously (WT). These were compared to control mice that did not receive T cells (No Tx). These two groups were infected as above and analyzed after three weeks. The difference in lung CFU was very significant as determined using a t-test (p = 0.0002).
Supplementary Figure 3. Adoptively transferred macrophages traffic into the lung. Uninfected CD45.2⁺ macrophages were transferred into CD45.1 recipient mice via the intratracheal route. The presence of donor CD45.2⁺ macrophages was evaluated in the air space (by bronchoalveolar lavage) and lung tissue (after collagenase digestion) 6h, 24h, 48h, and 7 days after adoptive transfer.
Supplementary Figure 4. The frequency of TB10.4(4-11) specific CD8⁺ T cells in the lungs and spleens of wild type mice is similar 28 days after the intratracheal transfer of H37Rv-infected Ptges^−/−, wild type, or Alox5^−/− macrophages. Representative of FACS plots showing the H-2 K^b–TB10.4(4-11) tetramer staining of CD8⁺ T cells from the lung (top row) or spleen (bottom row) of recipient mice 4 weeks after infected macrophages transfer. The frequency of TB10.4(4-11)-specific CD8⁺ T cells is indicated in each plot and in the bar graph. Bars, mean ± SE bar graph of 4 mice per group.
Supplementary Figure 5. The frequency of Mtb-specific CD4⁺ and CD8⁺ T cells following adoptive transfer of Mtb infected alveolar macrophages from wild-type or Alox5−/− mice into naïve wild type mice. Seventeen days after the adoptive transfer of Mtb infected alveolar macrophages from wild type or Alox5^−/− mice into wild type recipient mice, the frequency of Mtb-specific CD4⁺ and CD8⁺ T cells was enumerated using an IFN-γ elispot to measure the response to Ag85B(247-256), ESAT6(3-15), TB10.4(4-11), or 32c(309-318) synthetic peptides in the lungs, pulmonary lymph nodes (PLN), and spleens. Mice that received Alox5^−/− alveolar macrophages had larger IFN-γ responses to ESAT6 (CD4⁺ T cell epitope) and TB10.4 (CD8⁺ T cell epitope) in the PLN and spleen, respectively, at this early time point (p<0.05, t-test). Statistical analysis of the other elispot responses is difficult because many of the mice that received wild type alveolar macrophages had no detectable response, and the Alox5^−/− alveolar macrophages group showed significant variability in their responses. To compare the magnitude of the response, recipient mice were categorized as responders or non-responders based on detection of IFN-γ secreting T cells. When all the possible responses were analyzed in aggregate, it was clear that transfer of infected Alox5^−/− alveolar macrophages induced a better response than elicited by infected wild type alveolar macrophages (p < 0.0001 by Fischer exact test).
Supplementary Figure 6. Ptges^−/−, wild type, and Alox5^−/− macrophages have a similar ability to process and present antigen. Ptges^−/−, wild type, and Alox5^−/− macrophages were pulsed with different concentration of sonicated Mtb, Mtb-culture filtrate proteins, Ag85 protein, or Ag85B(241-256) peptide, for 6 hrs. BB7 hybidoma cells were added to macrophages and IL-2 production was measured in the supernatants 24 hrs later. The ratio of the macrophages to T cells was 1:1. Values represent mean ± SE of triplicate samples.
Supplementary Figure 7. Wild type and Alox5^−/− macrophages have a similar capacity to enhance OVA-antigen specific T cell response in the absence of Mtb infection. (a) CD8⁺ T cell proliferation 5 days after IV transfer of CFSE-labeled Thy1.2⁺ splenic OT-I CD8⁺ T cells into Thy1.1⁺ B6.PL mice followed within 24 hrs by uninfected wild type or Alox5^−/− macrophages pulsed with SIINFEKL. (b) Purified CD45.1⁺ OT-1 CD8⁺ T cells were injected IV into CD45.2⁺ C57BL/6 mice (n=2-5/group). Within 24 hours, uninfected wild type or Alox5^−/− macrophages cultured with SIINFEKL (pOVA) and treated with LPS (100 ng/ml) or staurosporine (1 μM) for 6 hrs. OT-1 cells were identified as CD45.1⁺CD8⁺Vβ5⁺Vα2⁺ cells. The frequency of OT-1 cells in each draining LN is shown. Bars represent mean ± SE.
Supplementary Figure 8. In vivo depletion of CD11c⁺ cells. Three different doses of diphtheria toxin (DT) (25, 50, 100 ng/mouse) were administered by the IP route to CD11c–DTR TG and non-TG littermate mice. The frequency of CD11c⁺ cells were measured in the spleens 24 hours later. The majority of CD11c⁺ cells were depleted following 100 ng of DT compared to their littermate non-tg controls.