Phenotypic analysis of CD4/10.4 and CD8/10.4 during a persistent infection.

<p>Lung lymphocytes from mice sacrificed at different stages of infection (Early: week 6; late: week 40) were stimulated with (A) TB10.4 _3–11 or (B) TB10.4 _74–88 prior to staining for the surface markers CD44, CD11a and intracellularly for IFN-γ. Samples were then examined by flow cytometry. FACS plots are shown for one mouse, representative of 3–4 mice.</p

Functional characterization of the CD4/10.4 and CD8/10.4 T cells at different stages of infection.

<p>(Early: week 6; Intermediate: week 16, late: week 40) (A and B) Lung cells from infected mice were stimulated in vitro with TB10.4 _3–11 or TB10.4 _74–88 (lower panel) or left non stimulated (“Media”, upper panel) in the presence of αCD107a/b and stained with αIFN-γ and αCD8 (A), or αCD4 (B), antibodies (C) CD107a/b MFI of the IFN-γ positive cells following in vitro stimulation with TB10.4 _3–11 or TB10.4 _74–88. (D) The specific lysis of TB10.4 _3–11 or TB10.4 _74–88 loaded cells was determined in an in vivo cytotoxicity assay. Unloaded splenocytes (CFSE^low) and TB10.4 _3–11 or TB10.4 _74–88 loaded splenocytes (CFSE^high) from naïve mice were transferred into infected mice. The amount of splenocytes killed in vivo by cytotoxic T cells specific for either TB10.4 _3–11 or TB10.4 _74–88 was observed as a reduction in the CFSE^high population and a percent specific lysis was calculated. A value of <i>P</i><0.05 (students paired t-test) was considered significant and is shown by *.</p

The bacterial load in the lungs throughout infection shown as Log10 CFU.

<p>Mice were challenged by the aerosol route with virulent <i>M. tuberculosis</i>. At the indicated timepoints 3–6 mice were killed and the bacterial burden (CFU) was measured in the lung.</p

Changes in cytokine profiles of CD4/10.4 and CD8/10.4 T cells during a chronic infection.

<p>Cells from infected lungs were stimulated with TB10.4 _3–11 or TB10.4 _74–88 prior to staining with anti-CD4, -CD8, -IFN-γ, -TNF-α and –IL-2. (A) Cytokine profiles of CD4/10.4 was determined by first dividing the CD4 T cells into IFN-γ positive (+) or IFN-γ negative (−) cells. Both the IFN-γ+ and IFN-γ- cells were analyzed with respect to the production of TNF-α and IL-2. The numbers in the quadrant gates of the plots denominates each distinct population based on their cytokine production and is color coded as shown. To illustrate the gating sequence two examples from a late infection stage are shown. (B and C). The pie-charts illustrate the relative contribution of the different cytokine T cell populations to the total CD4/10.4 population and data is depicted for both an early and a late time point, for 3 mice at each time point. Background (>0.5%) has been deducted. (D) The proportion of IFN-γ⁺ TNF-α⁺ IL-2⁺ within the CD4/10.4 or CD8/10.4 T cells populations in the early stage compared to the late stage of infection.</p

The kinetics of CD4/10.4 and CD8/10.4 following aerosol infection with <i>M.tb</i>.

<p>(A and B) CB6F1 mice were infected by the aerosol route with virulent <i>M.tb Erdman</i> and lymphocytes were obtained from lungs or blood and then stimulated with TB10.4 _3–11 or TB10.4 _74–88 for assessment of IFN-γ production by FACS analysis. Frequencies represent IFN-γ production out of total T cells. Background staining from cells stimulated with medium alone has been deducted (Background <0.5%). Each time point represents the mean from at least three individual mice±standard error of the mean (SEM). (C) Lung or blood cells were stained directly <i>ex vivo</i> with H2-<i>K^b</i> pentamer loaded with IMYNYPAM. Background staining from naïve mice has been deducted. Each time point consists of data from a pool of 3–6 mice. (**p<0.01, Student's t-test).</p

The TB10.4 CD4 and CD8 T cells epitopes that are recognized by CB6F1 mice (BALB/c×C57BL/6).

<p>(A) the amino acid sequence of the TB10.4 protein with the CD4 and CD8 T cell epitopes underlined. (B) Lung lymphocytes from mice infected at week six after aerosol infection were stimulated in vitro with either the TB10.4 _3–11 (QIMYNYPAM) or the TB10.4 _74–88 (THEANTMAMMARDT) for 6 hours before being assessed for IFN-γ production by flow cytometry. A gating sequence was applied to the data to determine the frequencies of IFN-γ positive CD4 and CD8 T cells. The rationale for the FSC-H vs. FSC-A gating is to capture only singlet particles and eliminate doublets that may occur as a result of e.g. cells sticking together.</p

MyD88 and IL-6, IL-10, G-CSF-dependent pathway genes are significantly enriched in HN878 infected vs. non-infected macrophages

<p>. BMDM were stimulated with IL-4/IL-13 or left untreated. After 24 hours of stimulation, cells were infected with HN878. Total RNA was extracted at 4, 12 and 48 hours PI for microarray and GSEA analysis. Enrichment plots and heat maps for (A) MyD88, (B) IL-6, IL-10, G-CSF and (C) IL-4Rα pathway are shown. Enrichment analysis compared log2-fold changes in <i>Mtb</i>-infected samples vs. non-infected samples. The rows in heat maps are listed according to pre-ranking metric scores. Replicates shown are from two independent experiments.</p

IL-4Rα-Dependent Alternative Activation of Macrophages Is Not Decisive for <i>Mycobacterium tuberculosis</i> Pathology and Bacterial Burden in Mice

<div><p>Classical activation of macrophages (caMph or M1) is crucial for host protection against <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) infection. Evidence suggests that IL-4/IL-13 alternatively activated macrophages (aaMph or M2) are exploited by <i>Mtb</i> to divert microbicidal functions of caMph. To define the functions of M2 macrophages during tuberculosis (TB), we infected mice deficient for IL-4 receptor α on macrophages (LysM^creIL-4Rα^-/lox) with <i>Mtb</i>. We show that absence of IL-4Rα on macrophages does not play a major role during infection with <i>Mtb</i> H37Rv, or the clinical Beijing strain HN878. This was demonstrated by similar mortality, bacterial burden, histopathology and T cell proliferation between infected wild-type (WT) and LysM^creIL-4Rα^-/lox mice. Interestingly, we observed no differences in the lung expression of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg1), well-established markers for M1/M2 macrophages among the <i>Mtb</i>-infected groups. Kinetic expression studies of IL-4/IL-13 activated bone marrow-derived macrophages (BMDM) infected with HN878, followed by gene set enrichment analysis, revealed that the MyD88 and IL-6, IL-10, G-CSF pathways are significantly enriched, but not the IL-4Rα driven pathway. Together, these results suggest that IL-4Rα-macrophages do not play a central role in TB disease progression.</p></div

No major differences in expression of iNOS, Arg1, lung immune cell populations and T cell proliferation between wild-type and macrophage cell-specific IL-4Rα deficient mice following low-dose <i>Mtb</i> H37Rv infection (100 CFU/mouse).

<p>(A) iNOS and Arg1 staining (brown colour) from lung sections collected at indicated times PI, original magnification: 40X. Lung sections from 5 mice/group were quantified. N.D. = not detectable. (B) iNOS and Arg1 expression on various immune cells were analysed by flow cytometry at 18 weeks PI (6–7 mice/group, *<i>P</i> < 0.05). (C) T cell proliferation with co-cultured CD11c-sorted macrophages from the lung tissue of naïve non-infected and mice infected with H37Rv (100 CFU/mouse) by aerosol at 4 and 18 weeks. Data shown in A is representative of two independent experiments and results obtained in B and C are from one experiment.</p

Similar mortality, inflammation, bacterial burden, Arg1/iNOS expression and T cell proliferation in wild-type and LysM^creIL-4Rα^-/lox mice following high dose infection with <i>Mtb</i> H37Rv

<p>. Wild-type (BALB/c) and IL-4Rα macrophage cell-specific deficient mice (LysM^creIL-4Rα^-/lox) were infected intranasally with high dose of 10⁴ CFU/mouse of <i>Mtb</i> H37Rv (n = 20/group). (A) Survival of infected mice was recorded weekly. (B) Individual bacterial titers (CFU/organ) with group medians are shown (*<i>P</i> < 0.05). (C) iNOS and Arg1 staining (brown colour) from lung sections collected at 3 and 10 weeks PI. Lung sections from 5 mice/group were quantified. Original magnification: 40X. N.D. = not detectable. (D) T cell proliferation with co-cultured CD11c-sorted macrophages from the lung tissue of naïve non-infected and 3 weeks infected mice. All data shown is representative of two independent experiments except for B which is representative of three independent experiments.</p