C57BL/6 (B6) mice, originally purchased from Charles River Laboratory (Barcelona, Spain), were bred and maintained at the ICVS animal facility. pMT-10 mice in a B6 background were generated by Drs. António G. Castro and Paulo Vieira, as previously described (25). Briefly, mouse IL-10 cDNA cloned in the p169ZT vector, carrying the sheep metalloprotein 1a promotor, a β-globin splice site, and the SV40 polyadenylation signal, was injected in B6 eggs. The identification of transgenic founders was determined by PCR using sheep metalloprotein and IL-10-specific primers.

To induce IL-10 overexpression, the drinking water of pMT-10 mice was supplemented with a 2% sucrose solution containing 50 mM of zinc sulfate (25). This supplementation induces a rapid increase in the circulating levels of IL-10, which are maintained until zinc sulfate withdraws (25). B6 mice were maintained in the same condition as pMT-10 mice, including drinking water supplemented with 2% sucrose solution containing 50 mM of zinc sulfate.

Both male and female mice between 6 and 12 weeks of age were used for experimental procedures. Mice euthanasia was carried out by controlled CO₂ inhalation.

Mice were subcutaneously vaccinated with 1 × 10⁶ CFU of M. bovis BCG Pasteur and rested for 60 days prior to Mtb aerosol infection.

The H37Rv strain of Mtb used in this study was originally obtained from the Trudeau Institute (Saranac Lake, NY). Mtb H37Rv was first grown to log phase in Middlebrook 7H9 broth supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC), 0.2% glycerol, and 0.05% Tween 80 and then subcultured in Proskauer-Beck medium with 0.05% Tween-80 to mid-log phase before being frozen at -80°C. These frozen stocks were quantified and used to infect mice with a low dose of bacteria (~75 CFUs) using the Glas-Col airborne infection system, as previously described (14).

At different time-points after infection, the lungs were aseptically excised and used to prepare lung single-cell suspensions for flow cytometry analysis or homogenized for CFU counts. Organ homogenates were serially diluted and plated onto Middlebrook 7H11 agar (BD Biosciences, San Jose, CA, USA) for 3 weeks at 37°C, at which point CFUs were counted.

Aseptically excised lungs were sectioned and incubated at 37°C for 30 min with collagenase D (0.7 mg/ml, Sigma, St. Louis, MO, USA). Samples were then disrupted into a single-cell suspension by passage through a 70-μm nylon cell strainer (BD Biosciences). Lung single-cell suspensions were then treated with erythrocyte lysis buffer (0.87% of NH₄Cl) and processed over 40:80% Percoll (GE Healthcare, Chicago, IL, USA). The resulting cell suspension was washed twice and counted.

For intravital flow cytometry analysis, mice received intravenously APC-labeled anti-CD45 antibody 3 min before euthanasia (26). Lung single-cell suspensions prepared as described above were stained with fluorochrome-conjugated antibodies for 30 min on ice. For intracellular cytokine detection, cells were cultured in 5 µg/ml of Ag85b_240-254 or ESAT-6_1-20 peptide for 1.5 h before 10 µg/ml Brefeldin A (Sigma-Aldrich) was added to the culture for an additional 3.5 h. Cells were analyzed by flow cytometry using the following antibodies: CD3-Brilliant Violet 605 (145-2C11, BioLegend, San Diego, CA, USA), CD4-APC-Cy7 (GK1.5, BioLegend), CD44-PercPCy5.5 (IM7, BioLegend), CD45-Brilliant Violet 510 (30-F11, BioLegend), CD45.2-APC (104, BioLegend), and IFNγ-PE-Cy7 (XMG1.2, BioLegend). Data were acquired on a LSRII flow cytometer (BD Biosciences) with Diva Software and analyzed using FlowJo software (BD Biosciences). The total number of cells for each population was calculated by taking into account the percentage of cells determined by flow cytometry and the total number of cells per lung.

The right upper lobe of each lung was inflated with 4% paraformaldehyde (PFA) and processed routinely for light microscopy with hematoxylin and eosin stain. Morphometric analysis was performed in a blinded manner using ImageJ software (version 1.50e; NIH, Bethesda, MD, USA). The percentage of the lung inflamed for each sample was calculated by dividing the cumulative area of inflammation by the total lung surface area. The percentage of B-cell aggregates in the granuloma was calculated by dividing the area of lung tissue stained positive for B220 by the area of inflammation in the lung.

Immunofluorescence was performed on formalin-fixed lung sections, as described previously (14). Sections were stained with either purified rabbit polyclonal anti-CD3e (ab185811; Abcam, Cambridge, MA, USA) or CD45R (RA3-6B2; Abcam) followed by streptavidin–Alexa Fluor 488 (Invitrogen). SlowFade Gold antifade with DAPI (Invitrogen) was used to counterstain tissues and to reveal nuclei. Representative images were obtained with an Olympus BX61 microscope and were recorded with the DP70 digital camera using the cell^P software.

Total RNA from infected lungs was extracted using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. cDNA was generated from 1 μg of total RNA using the GRS cDNA Synthesis Master Mix (GRiSP, Porto, Portugal) following the manufacturer’s instructions. The resultant cDNA template was used to quantify the expression of target genes by real-time (RT)-PCR (Bio‐Rad CFX96 Real‐Time System with C1000 Thermal Cycler) using the following protocol: one cycle of 95°C for 3 min, followed by 40 cycles of a two-stage temperature profile of 95°C for 3 s and 60°C for 30 s. Gene expression was normalized to ubiquitin mRNA levels using the ΔCt method. Target gene mRNA expression was quantified using SYBR Green (Thermo Scientific, Waltham, MA, USA) and specific oligonucleotides ( Table 1 ; Invitrogen, Carlsbad, CA).

Differences between groups were determined using one-way ANOVA. Differences were considered significant for p ≤ 0.05.

Our previous data demonstrated that, in an IL-10-enriched environment CD4⁺ T cells differentiate into a vasculature-associated phenotype with reduced ability to migrate into the parenchyma and infiltrate the Mtb lung lesion (14). This migratory deficit prevented the control of Mtb, resulting in exacerbated bacterial proliferation and lung pathology (14). As early protection conferred by BCG vaccination has been shown to depend on lung-resident memory CD4⁺ T cells (16, 27), we used our pMT-10 mouse model to test the impact of IL-10 in vaccinated hosts. To do this, we vaccinated B6 and pMT-10 mice with BCG through the subcutaneous route. Two months after vaccination, mice were infected with Mtb through the aerosol route, and the expression of IL-10 in pMT-10 mice initiated by supplementing the drinking water with 50 mM of zinc sulfate, as previously described (14, 25). Vaccinated and unvaccinated B6 mice maintained in the same conditions as pMT-10 mice were used as controls.

We began by analyzing bacterial burdens in the lung at days 15, 20, and 30 postinfection ( Figure 1A ). As expected, BCG-vaccinated B6 mice began controlling Mtb growth at day 15 postinfection (16, 28) and, by day 30 postinfection, they had 1.4 Log₁₀ less bacterial burden than unvaccinated mice ( Figure 1A ). In line with our previous study (14), pMT-10 mice exhibited exacerbated lung bacterial burdens when compared with B6 mice ( Figure 1A ). Interestingly, however, BCG-vaccinated pMT-10 mice displayed significantly lower lung bacterial burdens than control pMT-10 mice. Indeed, bacterial burdens in the lungs of vaccinated pMT-10 mice were similar to control B6 mice throughout the infection period analyzed ( Figure 1A ). In accordance with these data, we found that both B6- and pMT-10-vaccinated mice had fewer areas of lesioned lung tissue than the respective control animals ( Figure 1B ). Together, these data show that BCG vaccination prevents the acute susceptibility to Mtb infection induced by IL-10 overexpression, allowing for improved control of bacterial proliferation and reduced pathology.

Mtb burdens are similar in both BCG-vaccinated and unvaccinated mice before day 15 post-challenge (28). After this time-point, control of Mtb in BCG-vaccinated mice has been shown to depend on lung-resident CD4⁺ T cells induced by BCG vaccination (16, 29). Therefore, to determine if the increased protection of vaccinated mice overexpressing IL-10 correlated with an accelerated CD4⁺ T-cell response, we first analyzed the accumulation of CD4⁺ T cells expressing the activation marker CD44 in the lungs at days 15 and 30 postinfection.

We found an increased accumulation of CD4⁺CD44⁺ T cells in the lungs of vaccinated mice at day 15 postinfection as compared to unvaccinated mice ( Figure 2A ). However, by day 30 postinfection, the frequency and number of CD4⁺CD44⁺ T cells were similar across all groups of mice ( Figure 2A ). To determine further the impact of BCG vaccination in the development of the Ag-specific response in the IL-10-enriched environment, we evaluated the frequency and number of Ag85b-specific CD4⁺ T cells and their ability to produce IFN-γ. To do this, we restimulated lung cells with Ag85b and quantified IFN-γ-producing CD4⁺ T cells at 15 and 30 days after Mtb infection ( Figure 2B ). Our data show that both groups of vaccinated mice present an increased frequency and number of Ag85b-specific IFN-γ-producing CD4⁺ T cells at day 15 postinfection when compared to control mice ( Figure 2B ). Importantly, the accumulation of Ag85b-specific IFN-γ-producing CD4⁺ T cells in the lungs of vaccinated pMT-10 mice appears to occur in a similar proportion as in vaccinated B6 mice, suggesting a minor role for IL-10 in the development of this response after BCG vaccination ( Figure 2B ). By day 30 postinfection, the frequency and number of CD4⁺ T cells producing IFN-γ in response to Ag85b were similar across all groups of mice ( Figure 2B ). These results support previous evidence that BCG-vaccinated mice exhibit an accelerated Ag85b-specific CD4⁺ T-cell response when compared to unvaccinated mice (28). Additionally, these data demonstrate that IL-10 overexpression during Mtb infection does not influence the rapid accumulation of Ag85b-specific CD4⁺ T cells in BCG-vaccinated mice.

Our previous study revealed that IL-10 delays the onset of the ESAT-6-specific CD4⁺ T-cell response. Therefore, we decided to explore the impact of IL-10 expression on the development of ESAT-6-specific CD4⁺ T-cell response in BCG-vaccinated mice. To do this, we analyzed the frequency and number of IFN-γ-producing CD4⁺ T cells in response to ESAT-6 restimulation at days 15 and 30 postinfection. We found a similar frequency and number of ESAT-6-specific IFN-γ-producing CD4⁺ T cells in the lungs of all experimental groups by day 15 postinfection. These data suggest that the increased expansion of Ag85b-specific CD4+ T cells at day 15 ( Figure 2B ) are lung-resident memory cells induced by BCG vaccination. However, at day 30 after infection, the frequency of ESAT-6-specific IFN-γ-producing CD4⁺ T cells in the lungs of both unvaccinated B6 and pMT-10 mice were higher than in vaccinated B6 and pMT-10 mice ( Figure 2C ). This decreased expansion is likely a consequence of the lower lung bacterial burdens and the stronger Ag85b-specific CD4⁺ T-cell response observed in these mice at day 15 postinfection ( Figure 2B ).

Altogether, these data demonstrate that the onset of the BCG-specific CD4⁺ T-cell response is similar in both B6- and pMT-10-vaccinated mice, thus supporting the hypothesis that IL-10 overexpression during infection does not significantly impair BCG-mediated immunity.

Our previous study revealed that IL-10 antagonized the control of Mtb infection by promoting the generation of vasculature-associated CD4⁺ T cells with reduced ability to migrate into the lung parenchyma and induce control of infection (14). In the present study, we showed that BCG vaccination prevents the antagonistic effects of IL-10 likely because of memory lymphocytes that are retained in the lung and rapidly respond following Mtb challenge (29). However, as BCG expresses Ag85b but not ESAT-6 we wanted to determine if BCG vaccination would overcome the impaired migration of ESAT-6-specific CD4⁺ T cells to the lung parenchyma observed in unvaccinated pMT-10 mice overexpressing IL-10 (14). Our histological analysis showed that T cells from B6 mice infiltrated the granuloma whereas in pMT-10 mice it accumulated in perivascular cuffs ( Figure 3A ). To confirm this observation and determine the location of CD4⁺ T cells in the lungs of B6 and pMT-10 mice, we performed intravital flow cytometry at day 30 postinfection. To do this, we intravenously injected mice with a fluorochrome-labeled anti-CD45.2 antibody and sacrificed them 3 min later. Lungs were then collected and processed for flow cytometry analysis, as previously described (14). Our results revealed that vaccinated B6 mice had a reduced frequency of intravascular CD4⁺CD44⁺ T cells comparatively to control B6 mice ( Figure 3B ). This finding supports previous studies, showing that the presence of parenchymal CD4⁺ T cells in the lungs of infected mice correlates with control of infection (26, 30). We also found that the frequency of intravascular CD4⁺ T cells was similar in the lungs of pMT-10 mice irrespective of their vaccination status ( Figure 3B ). Importantly, pMT-10 mice had a higher frequency of CD4⁺CD44⁺ T cells than vaccinated B6 mice, and even unvaccinated B6 mice ( Figure 3B ). The same data were found for intravascular ESAT-6-specific IFN-γ-producing CD4⁺ T cells ( Figure 3C ). Together, these data show that BCG vaccination does not overcome the IL-10-mediated migration impairment of newly activated CD4⁺ T cells to the lung parenchyma.

It has been previously shown that the formation of perivascular cuffs in Mtb-infected lungs associates with defective formation of tertiary lymphoid follicles, as determined by the accumulation of B cells within the granuloma (17). Since our data revealed that BCG vaccination did not improve the accumulation of newly activated CD4⁺ T cells in the lung parenchyma of pMT-10 mice, we investigated the impact of BCG vaccination in the formation of tertiary lymphoid follicles. Our histological examination of lung sections of Mtb-infected lungs 30 days after infection revealed a decreased accumulation of B cells (marked with B220) in the granuloma of vaccinated and unvaccinated pMT-10 mice as compared to their B6 counterparts ( Figure 4A ). Specifically, vaccinated pMT-10 mice show smaller B-cell areas within granulomas than vaccinated B6 mice, suggesting that BCG vaccination does not overcome the IL-10-dependent impaired development of lymphoid follicles during Mtb infection.

To explore whether the impaired development of these structures was associated with reduced expression of homeostatic cytokines, we assessed the expression of CCL19, CCL21, and CXCL13 in the lungs of vaccinated and unvaccinated B6 and pMT-10 mice ( Figure 4B ). We found no significant differences in the expression of these chemokines across all groups of mice. Interestingly, however, we found that vaccinated pMT-10 mice expressed reduced levels of CCR7 and CXCR5 mRNA in the lungs when compared to vaccinated B6 mice ( Figure 4B ). These findings suggest that IL-10 expression during Mtb infection does not impair the expression of homeostatic chemokines, but it reduces the expression of chemokine receptors essential for the development of tertiary lymphoid follicles in the lungs of vaccinated mice.

In all, our data show that the presence of lung-resident Ag85b-specific T cells prior to Mtb infection is sufficient to overcome the antagonistic effects of IL-10 in the control of Mtb infection. Despite this, BCG vaccination does not appear to overcome the migration deficiency of CD4⁺ T cells primed in an IL-10-enriched environment, as is the case of ESAT-6-specific T cells, which may have a detrimental impact in the long-lasting control of infection.