Dendritic cells in chronic mycobacterial granulomas restrict local anti-bacterial T cell response in a murine model

Abstract

Background: Mycobacterium-induced granulomas are the interface between bacteria and host immune response. During acute infection dendritic cells (DCs) are critical for mycobacterial dissemination and activation of protective T cells. However, their role during chronic infection in the granuloma is poorly understood.

Methodology/principal findings: We report that an inflammatory subset of murine DCs are present in granulomas induced by Mycobacteria bovis strain Bacillus Calmette-guerin (BCG), and both their location in granulomas and costimulatory molecule expression changes throughout infection. By flow cytometric analysis, we found that CD11c(+) cells in chronic granulomas had lower expression of MHCII and co-stimulatory molecules CD40, CD80 and CD86, and higher expression of inhibitory molecules PD-L1 and PD-L2 compared to CD11c(+) cells from acute granulomas. As a consequence of their phenotype, CD11c(+) cells from chronic lesions were unable to support the reactivation of newly-recruited, antigen 85B-specific CD4(+)IFNgamma(+) T cells or induce an IFNgamma response from naïve T cells in vivo and ex vivo. The mechanism of this inhibition involves the PD-1:PD-L signaling pathway, as ex vivo blockade of PD-L1 and PD-L2 restored the ability of isolated CD11c(+) cells from chronic lesions to stimulate a protective IFNgamma T cell response.

Conclusions/significance: Our data suggest that DCs in chronic lesions may facilitate latent infection by down-regulating protective T cell responses, ultimately acting as a shield that promotes mycobacterium survival. This DC shield may explain why mycobacteria are adapted for long-term survival in granulomatous lesions.

Figure 1. Inflammatory DCs present in both acute and chronic mycobacterium-induced granulomas.

C57BL/6 mice were systemically infected intraperitoneally with BCG. Three, six and ten weeks post infection livers were harvested, and granuloma-infiltrating cells were isolated. A, FACS analysis of CD11c and CD11b populations in liver granulomas. Plot obtained from gate of SSC high FSC high population, excluding lymphocytes. B, Phenotypic analysis of CD11c⁺ cells from gating shown in A. Graph shows absolute number of cells per gram of liver tissue of cells positive for designated DC subsets listed below graph. C, Absolute number of CD11c⁺ cells per gram of tissue, population obtained from gate shown in A. Statistical differences among groups shown on graph. FACS plots and graphs generated from 3–7 mice pooled per time point for granuloma preparation and representative of three independent experiments.

Figure 2. Location of DCs in acute and chronic mycobacterium-granulomas is different.

A, Schematic outline of DCs in the center, periphery or outside of the granuloma. B, Distribution of DC location from stained sections. 42, 43 and 17 lesions combined from three mice per time point were observed for CD11c⁺ location for three, six and ten weeks, respectively. C, CD11c-EYFP mice were systemically infected with dsRED BCG for three, six and ten weeks. EYFP expression (green cells) and DAPI nuclear stain (blue). Granulomas are outlined with white dashed lines and shown at 1000× magnification.

Figure 3. A portion of BCG is sustained within CD11c⁺ cells, both in acute and chronic granulomas.

A, Left, Fluorescent images of CD11c-EYFP⁻ (left panels) and CD11c-EYFP⁺ cells (right panels) with dsRED BCG at three and ten weeks. Images magnified from 1000× images. DAPI nuclear stain (blue), dsRED BCG (red rods) and cytoplasmic CD11c-EYFP cells (green). Right, Average number of viable dsRED BCG rods per cell type (CD11c-EYFP+ or non-fluorescent) within the granuloma at three- and ten-weeks after infection. Data are represented as mean +/− SEM, P<0.0001. B, FACS plots generated from CD11c⁺ gate (left plot) and CD11b⁺CD11c⁻ gate (right plot). Boxed gate shows percentage of cells containing GFP-BCG. Gate placement was made from non-fluorescent-BCG infected CD11b+ and CD11c+ cells, not shown. Plots representative of 3- and 10-week time points with 3, 8 and 8 mice per group, respectively. C, Histograms generated from 10 week CD11c⁺ (1.17) and CD11b⁺CD11c⁻ (8.61) GFP BCG+ gates in (B).

Figure 4. Surface expression of activating and inhibitory costimulatory molecules and chemokine receptors is different on CD11c⁺ cells in acute and chronic granulomas.

CD11c⁺ cells from granuloma single cell suspensions obtained from three, six and ten week systemically infected C57BL/6 mice were phenotyped using flow cytometry. A, Histograms represent fluorochrome surface staining with monoclonal antibodies against MHCII, activating costimulatory molecules (CD40, CD80 and CD86) and inhibitory costimulatory molecules (PD-L1 and PD-L2). Black-dashed histograms represent costimulatory molecule expression on naïve splenic CD11c⁺CD11b⁺ cells. Blacks arrows indicate directional shift in MHCII and costimulatory molecule expression compared to 3-week expression levels at time points where substantial change is observed. Histograms representative of 3 independent experiments with 3–7 mice per group. B, Average fold change between three and ten week time points in mean fluorescent intensity of MHCII and costimulatory molecules. Average generated from fold change in three independent experiments.

Figure 5. CD4⁺ T cells maintain contact with CD11c-EYFP⁺ cells in both acute and chronic granulomas.

A, Quantitative analysis of CD4⁺ T cell contact with CD11c-EYFP⁺ cells in granulomas from three- and ten-week liver sections. All CD4⁺ T cells per granuloma section were determined to be in contact (green bars), or not in contact (white bars) with CD11c-EYFP⁺ cells. For each time point, liver sections from 3 independent mice were used, and 10–15 lesions per section were analyzed. Data are represented as mean +/− SEM, P<0.0001. B, Fluorescent images demonstrating CD11c-EYFP contact with CD4⁺ cells. Granulomas are outlined with white dashed lines at 1000× magnification and colors shown depict CD11c-EYFP (green cells), anti-CD4 Alexa 647 (red cells), and DAPI nuclear stain (blue).

Figure 6. IFNγ recall response of CD4⁺ T cells is much higher in acute compared to chronic granulomas.

Granuloma cells from three- and ten-week systemic BCG infected animals were isolated and stimulated ex vivo for five hours with anti-CD3. FACS plots show intracellular IFNγ staining and LFA-1 expression from CD4⁺ gated population. Plots representative of five pooled mice per group.

Figure 7. Newly activated Ag85B-specific Th1 cells are recruited at similar levels to acute and chronic granulomas, but produce less IFNγ in chronic granulomas.

A, Experimental schema. Coinciding three- and ten-week dsRED BCG systemically infected CD11c-EYFP mice received s.c. footpad injections of 1.25×10⁶ CFU GFP BCG seven days prior to harvest. The following day 10⁶ CFSE dsRED Ag85B-specifc transgenic CD4⁺ T cells were adoptively transferred via retro-orbital sinus vein injection. Mice were harvested six days later and granuloma cells were isolated. B, Left FACS panel, Presence of transferred P25 dsRED CD4⁺ T cells in both three- and ten-week granulomas. CD4⁺dsRed⁺ co-staining indicates adoptively transferred population. Plots derived from lymphocyte gate on SSC vs. FSC. B, Right FACS panel, CFSE dilution of gated P25 dsRED CD4⁺ T cell population indicates the recent activation of these T cells. B, Lower panels, Fluorescent microscopy reveals contact between CD11c-EYFP cells (green) and P25 dsRED CD4⁺ T cells (red) in granuloma (DAPI-blue). Images taken at 1000× magnification. Right images, Enlarged image of small white box. C, Left FACS panels, ex vivo restimulation of granuloma cells and staining for LFA-1 expression and IFNγ production. Plot was obtained from gated dsRED CD4⁺ T cell population as shown in B. C, Right graph, Average P25 IFNγ production in the granuloma from three independent experiments, displayed as a percent of total CD4⁺dsRED/Vβ11⁺ cells in lymphocyte gate (data are represented as mean +/− SEM). Plots representative of three independent experiments with 2–7 mice per group. These data show that despite CD4⁺IFNγ-producing mycobacterium-specific T cells reaching both acute and chronic granulomas, the IFNγ response is blocked (not reactivated) in chronic lesions.

Figure 8. Naïve CD4⁺ OTII T cell activation by CD11c⁺ cells from acute and chronic granulomas.

A, Experimental schema. B, CD11c⁺ cells from three- and ten-week BCG infected animals were purified and cultured with purified naïve OTII CD4⁺ T cells at 5∶1 ratio of T cell reponders:APCs, respectively. Cells were co-cultured ex vivo for 48 hours, followed by a five hour recall with anti-CD3. Cells were stained for intracellular cytokines IFNγ (left FACS plots) and IL2 (right FACS plots). B right graph, IFNγ ELISA of supernatant following 48 hours ex vivo co-culture. Cells shown were from CD4⁺Vβ5⁺ lymphocyte gate. Granuloma cells from 5–8 mice were pooled for each time point.

Figure 9. Blockade with anti-PD-L1 and anti-PD-L2 mAbs, or PD1-Fc restores 10-week granuloma CD11c⁺ cells' ability to restimulate allogenic CD4⁺ T cells to produce IFNγ.

DCs (2×10⁴) purified from 3- and 10-week granulomas were cocultured ex vivo with purified, primed Ag85B-specifc transgenic CD4⁺ T cells (1×10⁵). Intracellular IFNγ production was measured four hours after DC pretreatment with isotype control or anti-PD-L1 and anti-PD-L2 pretreatment (A), and media or PD1-Fc pretreatment (B). FACS lots were derived from initial lymphocyte gate on SSC vs. FSC, followed by gating of CD4⁺Vβ11⁺ population. Representative FACs plots are shown from groups of 3–7 mice per group. Data are represented as mean +/− SEM.

Figure 10. Changing role of dendritic cells in mycobacterium-induced granulomas over time.

Scheme showing the changing phenotype of granuloma DCs in the transition from acute to chronic infection. Acute lesions contain DCs with high MHCII, and T cell costimulatory molecule (MHCII, CD40, CD80/86) expression. As the infection becomes chronic, DCs within the granuloma have decreased expression of MHCII, activating costimulatory molecules, and increased expression of inhibitory molecules PD-L1 and PD-L2. Expression of these inhibitory molecules results in an inability of local CD11c⁺ cells to reactivate IFNγ-producing CD4⁺ T cells, and therefore, shielding the granuloma-located bacteria from an important anti-bacterial factor.