Mycobacterium bovis bacillus Calmette–Guérin (BCG) immunization still remains the best vaccination strategy available to control the development of active tuberculosis. Protection afforded by BCG vaccination gradually wanes over time and although booster strategies have promise, they remain under development. An alternative approach is to improve BCG efficacy through host-directed therapy. Building upon prior knowledge that blockade of IL-10R1 during early Mycobacterium tuberculosis infection improves and extends control of M. tuberculosis infection in mice, we employed a combined anti–IL-10R1/BCG vaccine strategy. An s.c. single vaccination of BCG/anti–IL10-R1 increased the numbers of CD4+ and CD8+ central memory T cells and reduced Th1 and Th17 cytokine levels in the lung for up to 7 wk postvaccination. Subsequent M. tuberculosis challenge in mice showed both an early (4 wk) and sustained long-term (47 wk) control of infection, which was associated with increased survival. In contrast, protection of BCG/saline-vaccinated mice waned 8 wk after M. tuberculosis infection. Our findings demonstrate that a single and simultaneous vaccination with BCG/anti–IL10-R1 sustains long-term protection, identifying a promising approach to enhance and extend the current BCG-mediated protection against TB.

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Tuberculosis (TB), caused by Mycobacterium tuberculosis, is considered a global public health emergency, as declared by the World Health Organization in 1993. TB is a leading cause of mortality, associated with >10 million new cases and 1.2 million deaths each year (1). The ongoing TB pandemic is worsened by the emergence of multidrug resistance strains, opportunistic coinfections, and limited advancements in chemotherapeutics.

Vaccination is considered the most potent and cost-effective approach for improving public health in both the industrialized and developing world (24). Currently, Mycobacterium bovis bacillus Calmette–Guérin (BCG) is the only available vaccine to provide immune protection against TB (58). The BCG vaccine elicits a robust Th1 response, which is critical toward mitigating TB burden (912). Despite this, BCG has limited protection against pulmonary TB in adults in high TB-endemic countries and is estimated to prevent only 5–15% of all potentially vaccine preventable deaths due to TB (1, 13, 14). Therefore, BCG-based vaccine strategies should aim at inducing long-lasting T cell responses that would contribute to long-term protection.

IL-10 is a correlate of TB disease in mice and humans (1518). We and others have shown that IL-10 negatively regulates the immune response during M. tuberculosis infection in both M. tuberculosis–resistant C57BL/6 and M. tuberculosis–susceptible CBA/J mice (16, 1921). We have also shown that M. tuberculosis infection of CBA/J mice leads to increased IL-10 in the lungs during the later phase of infection and that blockade of IL-10R1 at that time promotes Th1 immunity and stabilizes the bacterial burden (21). Important to this study, IL-10R1 blockade during the first 21 d of M. tuberculosis infection, when IL-10 levels in the lung are negligible, resulted in early recruitment of Th1 cells to the lung and improved long-term control of M. tuberculosis for at least 120 d postinfection. This protection was associated with the formation of mature fibrotic granulomas and extended host survival (22).

Although early IL-10R1 blockade can substantially improve long-term control of M. tuberculosis infection, changes in M. tuberculosis control were not evident until 60 d postinfection (22). We therefore hypothesize that IL-10 negatively influences the initial generation of adaptive immunity required for long-lasting control of M. tuberculosis infection. This concept is supported by previous studies showing that IL-10 can directly or indirectly restrict memory CD4+ and CD8+ T cell differentiation (2327). Studies have also shown that IL-10R1 blockade for 3 wk following BCG vaccination could enhance Ag-specific Th1 and Th17 immune responses in the lungs that subsequently reduced the M. tuberculosis burden for up to 16 wk postinfection (28), showing proof of concept for our hypothesis. In this study, we specifically asked whether a single dose of anti–IL-10R1 Ab, delivered simultaneous with the BCG vaccine, was sufficient to enhance the long-term control of M. tuberculosis infection afforded by BCG. This strategy spatially separates the influence of IL-10 on the generation of protective immunity to BCG from the impact of IL-10 on control of M. tuberculosis infection.

Our results reveal that a single dose of anti–IL-10R1 delivered simultaneously with BCG vaccination stimulated immunity that was capable of maintaining long-term control of M. tuberculosis infection. Long-term protection was sustained for at least 47 wk after M. tuberculosis infection and was associated with extended survival relative to mice receiving BCG alone. Prior to M. tuberculosis challenge, a single anti–IL-10R1/BCG vaccination resulted in the emergence of CD4+ and CD8+ central memory T cells, a reduced proinflammatory cytokine profile in the lungs, and increased M. tuberculosis Ag-specific IFN-γ and IL-17 production. These findings identify IL-10 as an important immunomodulator that impedes the development of long-term BCG-specific memory immunity. Our studies also demonstrate that a single dose of anti–IL-10R1 delivered simultaneous with BCG vaccination is sufficient to reverse the known waning protective efficacy of BCG. Furthermore, our results identify a single-dose anti–IL-10R1 strategy that may be more amendable to implement in humans.

Six- to 8-wk-old, specific pathogen–free male or female wild-type (WT) CBA/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were acclimatized for at least 1 wk before any experimental manipulation. IL-10−/− mice on the CBA/J background were developed and bred in-house (stock now available at The Jackson Laboratory; strain 036145, CBA.129P2(B6)-Il10tm1Cgn/TrnrJ) and age and sex matched with WT mice. Mice were housed in ventilated micro-isolator cages in ABSL-2 or ABSL-3 animal facilities and maintained with sterilized water and food ad libitum. Mice were euthanized at predetermined time points by CO2 asphyxiation. The Ohio State University (2009A0226) or Texas Biomedical Research Institute (1617 MU) Institutional Laboratory Animal Care and Use Committees approved animal protocols.

BCG Pasteur (ATCC 35734) and M. tuberculosis Erdman (ATCC 35801) were grown in supplemented Proskauer–Beck medium as previously described (29). Mice were immunized by the s.c. route with 1 × 105 CFU of BCG prepared in normal saline (0.9% NaCl) or with normal saline alone (sham). To coimmunize mice, 1 × 105 CFU of BCG and 1.6 mg of anti–IL-10R1 Ab (BE0050, clone 1B1.3A; Bio X Cell) or its isotype IgG1 control Ab (BE0088, clone HRPN; Bio X Cell) were admixed prior to vaccination. Anti–IL-10R1 and IgG1 dosing was based on methods in Cyktor et al. (22). All vaccine and control formulations were given in a 200-μl final volume. IL-10 knockout (IL-10−/−) mice were immunized with 1 × 105 CFU of BCG in normal saline (0.9% NaCl) or normal saline alone (sham) by the s.c. route.

Seven weeks after BCG immunization, mice were infected with a low-dose aerosol of M. tuberculosis Erdman using an inhalation exposure system (Glas-Col, Terre Haute, IN) calibrated to deliver 50–100 CFU to each individual mouse (29). Mice were euthanized at specific time points postinfection and lungs were aseptically harvested and homogenized. Lung homogenates were serially diluted and plated onto 7H11 agar plates enriched with OADC (oleic acid, albumin, dextrose, and catalase, Sigma-Aldrich, St. Louis, MO) and incubated at 37°C for 3 wk. CFU were counted to determine the burden in each organ (30). Mice allocated to survival studies were monitored over a period of 50 wk, and any surviving mice were euthanized at the study endpoint. Mice were euthanized when they reached a body condition score of ≤2 (31). Scores were determined by weekly visual and hands-on examination of each animal.

Mice were euthanized at predetermined time points after vaccination or M. tuberculosis challenge, and lungs were perfused with 10 ml of PBS containing 50 U/ml heparin via the right ventricle of the heart. Lung lobes were extracted and placed in 4 ml of enriched complete DMEM (DMEM containing l-glutamine [500 ml, Life Sciences, Tewksbury, MA]), supplemented with a sterile-filtered mixture of 5 ml of HEPES buffer (1 M; Sigma-Aldrich), 10 ml of MEM nonessential amino acid solution (100×; Sigma-Aldrich), 660 µl of 2-ME (50 mM; Sigma-Aldrich), and 45 ml of heat-inactivated FBS (Atlas Biologicals, Ft. Collins, CO). Lungs were dissociated using a gentleMACS dissociator, in the presence of collagenase A (type XI; 0.7 mg/ml obtained from Clostridium histolyticum; Sigma-Aldrich) and type IV bovine pancreatic DNase (30 μg/ml; Sigma-Aldrich), and incubated for 30 min at 37°C, 5% CO2. The enzymatic reaction was stopped by adding complete DMEM. Single-cell suspensions were achieved by passing the digested lung tissue through 70-µm cell strainers. Cells were treated with 2 ml of Gey’s solution (8 mM NH4Cl, 5 mM KHCO3 in water) to lyse residual RBCs and suspended in complete DMEM. Live cells were counted by using a trypan blue live-dead exclusion method or using a Cellometer K2 (Nexcelom Bioscience, Lawrence, MA) with acridine orange/propidium iodide stain (29).

Lung mononuclear cells (250,000) were cultured with medium or 10 μg/ml M. tuberculosis culture filtrate proteins (CFPs) for 48 h at 37°C, 5% CO2 (29). Culture supernatants were collected and stored at −80°C.

Clarified lung homogenates and cell culture supernatants were thawed and analyzed for IFN-γ, IL-12p70, TNF-α, IL-17, and IL-10 by ELISA following the manufacturer’s instructions (BD Biosciences, San Jose, CA).

Lung mononuclear cells were suspended in incomplete RPMI 1640 medium (Sigma-Aldrich) containing 0.1% sodium azide. Surface marker staining was performed as described (22). Specific Abs for surface marker staining were purchased from BioLegend (PerCP anti-CD4 [clone GK1.5], allophycocyanin/Cyanine7 anti-CD8 [clone 53-6.7], PE/Cy7 anti-CD62L [clone MEL-14], allophycocyanin anti-CD44 [clone IM7], and Brilliant Violet 421 anti-CD197 [CCR7; clone 4B12]). Briefly, cells were blocked with mouse Fc Block (clone 2.4G2; BD Biosciences) for 10 min followed by staining with fluorescent dye–conjugated Abs specific to surface markers for 20 min at 4°C in the dark. Cells were fixed and samples were acquired using a BD FACSCanto or Beckman Coulter CyAn ADP flow cytometer, and results were analyzed using FlowJo software versions 10.5 and 10.6 (Tree Star, Ashland, OR).

The right caudal lung lobe was isolated from individual mice and inflated with and stored in an excess of 10% neutral-buffered formalin as described (32). Lung tissue was processed, sectioned, and stained with H&E for light microscopy with lobe orientation designed to allow for maximum surface area of each lobe to be seen. Sections were examined in a blinded manner by a board-certified veterinary pathologist without prior knowledge of the experimental groups and evaluated according to the percent affected tissue area, granuloma distribution, granuloma character, granuloma border and cellular composition. The percent affected area was microscopically quantified by calculating the total area of the involved tissue over the total area of the lobe for each individual mouse and graded as 1, 2, 3, 4, and 5, corresponding to <10%, 10–25%, 25–50%, 50–75%, and >75% affected tissue, respectively.

Statistics were performed using Prism version 7 software (GraphPad Software, San Diego, CA). An unpaired, two-tailed Student t test was used for two group comparisons. A log-rank test was used to determine statistical significance of survival experiments. Statistical significance was reported as *p < 0.05, **p < 0.01, ***p < 0.005, or ****p < 0.001.

WT mice were immunized with BCG (or saline control) by the s.c. route, and IL-10 levels were measured in the lung at weeks 1, 2, 4, 5, and 7 postimmunization. BCG immunization resulted in significantly higher IL-10 levels at each time point tested. IL-10 could be detected in the lung as early as 1 wk after BCG vaccination and remained high for up to 7 wk postimmunization compared with the saline-vaccinated group (Fig. 1A). These data demonstrate that s.c. BCG vaccination can stimulate IL-10 production in the lung.

FIGURE 1.

IL-10 in the lung in response to BCG or BCG/anti–IL-10-R1 administration. (A and B) WT mice were immunized with saline or BCG by the s.c. route and euthanized at predetermined time points (A), or immunized with BCG/anti–IL-10R1 or BCG/IgG1 and euthanized at 1 or 7 wk postimmunization (B). IL-10 was determined in lung homogenate by ELISA. Data represent the mean ± SE of one of two independent experiments with three to five mice in each group at each time point. A Student t test was performed to determine statistical significance between the saline- and BCG-immunized groups (A) or between the BCG/IgG1 and BCG/anti–IL-10R1 groups (B). *p < 0.05.

FIGURE 1.

IL-10 in the lung in response to BCG or BCG/anti–IL-10-R1 administration. (A and B) WT mice were immunized with saline or BCG by the s.c. route and euthanized at predetermined time points (A), or immunized with BCG/anti–IL-10R1 or BCG/IgG1 and euthanized at 1 or 7 wk postimmunization (B). IL-10 was determined in lung homogenate by ELISA. Data represent the mean ± SE of one of two independent experiments with three to five mice in each group at each time point. A Student t test was performed to determine statistical significance between the saline- and BCG-immunized groups (A) or between the BCG/IgG1 and BCG/anti–IL-10R1 groups (B). *p < 0.05.

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We subsequently performed a separate experiment to determine the impact of simultaneous immunization of BCG with anti–IL-10R1 on the production of IL-10 in the lung. Prior to conducting the study presented, we performed a pilot study to confirm that BCG/IgG1 was the appropriate control (data not shown). Mice were immunized with a combined dose of BCG/anti–IL-10R1 or BCG/IgG1 control Ab via the s.c. route. BCG/anti–IL-10R1 significantly reduced IL-10 in the lung at weeks 1 and 7 postimmunization (Fig. 1B). These results indicate that a transient blockade of IL-10R1 delivered simultaneous with BCG vaccination has a long-term impact on local IL-10 production in the lung. IL-10 has a known deleterious impact on the control of M. tuberculosis infection (15), and therefore the IL-10–inducing properties of BCG vaccination, or any other lung insult that drives IL-10 production, could inadvertently have a negative consequence on M. tuberculosis infection control.

BCG/anti–IL-10R1 vaccination generated a relatively low proinflammatory environment in the lung compared with BCG/IgG1 vaccination. This is contrary to expectations from prior studies with M. tuberculosis (16, 22, 28). Indeed, we detected a significant reduction in IFN-γ, TNF-α, IL-17, and IL-12p70 in the BCG/anti–IL-10R1 group compared with BCG/IgG1 at both time points studied (Fig. 2). Cytokine levels in mice receiving BCG/anti–IL-10R1 were marginally elevated relative to mice receiving saline at week 1 postimmunization (Fig. 2).

FIGURE 2.

Proinflammatory cytokines in the lung in response to BCG/anti–IL-10R1 administration. WT mice were immunized with BCG/anti–IL-10R1 or BCG/IgG1 Ab s.c. Immunized mice were euthanized at 1 and 7 wk postimmunization. Lungs were homogenized and centrifuged to obtain clarified homogenate. (AD) IFN-γ (A), TNF-α (B), IL-17 (C), and IL-12p70 (D) were measured by ELISA. Data represent the mean ± SE of one of two independent experiments with three to five mice in each group at each time point. The saline group is pooled data of mice (n = 4) from 1 and 7 wk postimmunization. A Student t test was performed to determine statistical significance between the BCG/IgG1 and BCG/anti–IL-10R1 experimental groups at 1 and 7 wk postvaccination. *p < 0.05, **p < 0.01.

FIGURE 2.

Proinflammatory cytokines in the lung in response to BCG/anti–IL-10R1 administration. WT mice were immunized with BCG/anti–IL-10R1 or BCG/IgG1 Ab s.c. Immunized mice were euthanized at 1 and 7 wk postimmunization. Lungs were homogenized and centrifuged to obtain clarified homogenate. (AD) IFN-γ (A), TNF-α (B), IL-17 (C), and IL-12p70 (D) were measured by ELISA. Data represent the mean ± SE of one of two independent experiments with three to five mice in each group at each time point. The saline group is pooled data of mice (n = 4) from 1 and 7 wk postimmunization. A Student t test was performed to determine statistical significance between the BCG/IgG1 and BCG/anti–IL-10R1 experimental groups at 1 and 7 wk postvaccination. *p < 0.05, **p < 0.01.

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IL-10 is known to interfere with the generation of memory T cells (2326). We therefore determined whether BCG/anti–IL-10R1 vaccination produced a phenotypically different memory T cell pool in the lung. The total numbers of CD4+ and CD8+ T cells were modestly increased at 7 wk postimmunization in mice receiving BCG/anti–IL-10R1, although this did not reach significance (Fig. 3A, 3E). BCG/anti–IL-10R1 vaccination led to a consistent increase in the total number of memory (CD44hi), central memory (CD4+CD44hiCD62L+CCR7+), and effector memory (CD4+CD44hiCD62LCCR7) cells in both CD4+ and CD8+ T cell subsets, but again this did not reach significance (Fig. 3B–(3D and (3F–(3H). Corresponding cellular frequencies were also shown (Fig. 3I–P); cellular frequencies of CD4+ T or CD8+ T cell subsets were comparable between BCG/IgG1 and BCG/anti–IL-10R1 groups at 4 and 8 wk postvaccination. These results suggest that short-term IL-10R1 blockade during BCG vaccination results in a modest accumulation of central and effector memory cells at 7 wk postvaccination in the lung.

FIGURE 3.

BCG/anti–IL-10R1 administration causes accumulation of central memory cells in lung. (AP) WT mice were s.c. immunized with BCG/anti–IL-10R1 or BCG/IgG1. Immunized mice were euthanized at 7 wk postimmunization, and lung mononuclear cells were harvested and stained with fluorescent dye–tagged Abs specific for CD4 and CD8 in combination with CD44, CD62L, and CCR7 and acquired by flow cytometry and analyzed by FlowJo software. Absolute number (A–H) and frequency (I–P) of (A and I) CD4+, (B and J) CD4+CD44hi, (C and K) CD4+CD44hiCD62L+CCR7+ central memory cells, (D and L) CD4+CD44hiCD62LCCR7 effector memory cells, (E and M) CD8+, (F and N) CD8+CD44hi, (G and O) CD8+CD44hiCD62L+CCR7+ central memory cells, and (H and P) CD8+CD44hiCD62LCCR7 effector memory cells are shown. Data represent the mean ± SE of one of two independent experiment with three to five mice in each group. A Student t test was performed to determine the statistical significance between BCG/IgG1 and BCG/anti–IL-10R1 experimental groups. No statistically significant differences were found.

FIGURE 3.

BCG/anti–IL-10R1 administration causes accumulation of central memory cells in lung. (AP) WT mice were s.c. immunized with BCG/anti–IL-10R1 or BCG/IgG1. Immunized mice were euthanized at 7 wk postimmunization, and lung mononuclear cells were harvested and stained with fluorescent dye–tagged Abs specific for CD4 and CD8 in combination with CD44, CD62L, and CCR7 and acquired by flow cytometry and analyzed by FlowJo software. Absolute number (A–H) and frequency (I–P) of (A and I) CD4+, (B and J) CD4+CD44hi, (C and K) CD4+CD44hiCD62L+CCR7+ central memory cells, (D and L) CD4+CD44hiCD62LCCR7 effector memory cells, (E and M) CD8+, (F and N) CD8+CD44hi, (G and O) CD8+CD44hiCD62L+CCR7+ central memory cells, and (H and P) CD8+CD44hiCD62LCCR7 effector memory cells are shown. Data represent the mean ± SE of one of two independent experiment with three to five mice in each group. A Student t test was performed to determine the statistical significance between BCG/IgG1 and BCG/anti–IL-10R1 experimental groups. No statistically significant differences were found.

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IL-10 suppresses Th1 and Th17 immune responses necessary to control M. tuberculosis infection (28, 3337). Additionally, BCG/anti–IL-10R1 vaccination reduced IL-10 levels (Fig. 1B) and stimulated a moderate accumulation of memory T cells in the lung (Fig. 3C, 3G). We therefore determined whether BCG/anti–IL-10R1 vaccination could enhance Ag-specific Th1 and Th17 cytokine production. Lung cells from WT mice receiving BCG/anti–IL-10R1 secreted significantly more Ag-specific IFN-γ, TNF-α, and IL-17 (Fig. 4A–C) than did those receiving BCG/IgG1. Therefore, IL-10 receptor blockade at the time of BCG vaccination promoted the generation of functional Th1 and Th17 Ag-specific T cells in the lungs.

FIGURE 4.

BCG/anti–IL-10R1 administration increases Ag-specific Th1 and Th17 cytokine responses. (AC) WT mice were immunized with BCG/anti–IL-10R1 or BCG/IgG1. (DF) IL-10 −/− CBA/J mice were vaccinated with saline or BCG. Mice were euthanized at 7 wk postvaccination and lung mononuclear cells ex vivo–stimulated without (medium) or with CFPs for 48 h. Culture supernatants were analyzed for the production of IFN-γ (A and D), TNF-α (B and E), and IL-17 (C and F), respectively. Data represent the mean ± SEM of three to five mice in each group. A Student t test was performed to determine statistical significance between the response to CFPs of BCG/IgG1 and BCG/anti–IL-10R1 experimental groups (A–C) or IL-10 −/− saline and BCG experimental groups (D–F). *p < 0.05.

FIGURE 4.

BCG/anti–IL-10R1 administration increases Ag-specific Th1 and Th17 cytokine responses. (AC) WT mice were immunized with BCG/anti–IL-10R1 or BCG/IgG1. (DF) IL-10 −/− CBA/J mice were vaccinated with saline or BCG. Mice were euthanized at 7 wk postvaccination and lung mononuclear cells ex vivo–stimulated without (medium) or with CFPs for 48 h. Culture supernatants were analyzed for the production of IFN-γ (A and D), TNF-α (B and E), and IL-17 (C and F), respectively. Data represent the mean ± SEM of three to five mice in each group. A Student t test was performed to determine statistical significance between the response to CFPs of BCG/IgG1 and BCG/anti–IL-10R1 experimental groups (A–C) or IL-10 −/− saline and BCG experimental groups (D–F). *p < 0.05.

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To further establish that the absence of IL-10 signaling during BCG vaccination increases Ag-specific Th1 and Th17 cytokine secretion, we immunized IL-10−/− mice with BCG or saline (control) and assessed Ag-specific cytokine responses by lung cells at 7 wk postvaccination. Similar to WT mice vaccinated with BCG/anti–IL-10R1, IL-10−/− mice vaccinated with BCG alone had significantly increased production of Ag-specific IFN-γ, TNF-α, and IL-17 (Fig. 4D–F), compared with IL-10−/− mice immunized with saline. These results suggest that the absence of IL-10, similar to our data on blocking the action of IL-10R1, promotes a population of T cells capable of secreting Th1 and Th17 cytokines in the lungs.

WT mice were vaccinated s.c. with BCG/anti–IL-10R1, BCG/IgG1, or saline only. In parallel, IL-10−/− mice were vaccinated with BCG or saline. At 7 wk postimmunization, mice were challenged with a low-dose aerosol M. tuberculosis infection. As previously reported, IL-10−/− mice were capable of reducing M. tuberculosis bacterial burden in the lung to a greater extent than did WT mice (22) (Fig. 5A, saline versus (Fig. 5B, saline). BCG vaccination further reduced the bacterial burden in IL-10−/− mice by 1 log10 at 4 wk postinfection, and, importantly, this reduction was sustained for at least 52 wk postinfection (Fig. 5A).

FIGURE 5.

BCG/anti–IL-10R1 administration provides long-term protection against M. tuberculosis infection. (A and C) IL-10−/− CBA/J mice were vaccinated with saline or BCG. (B and D) WT mice were immunized with saline or BCG/anti–IL-10R1 or BCG/IgG1. Mice were infected with M. tuberculosis at 7 wk postvaccination. Lung and spleen CFU counts in IL-10−/− (A and C) or WT (B and D) mice. Survival curve for IL-10−/− (E) and WT (F) mice. Data in (B) and (C) are a combined one to three independent experiments each having five mice in each group in all data points. A Student t test was performed to determine statistical significance between BCG/anti–IL-10R1 and BCG/IgG1 (WT) or between saline- and BCG (IL-10−/−)-immunized experimental groups. *p < 0.05, **p < 0.01, ****p < 0.0001. Data in (C) and (D) represent a single experiment with 10–14 mice in each group. A log-rank test was used to determine statistical significance of survival between BCG/anti–IL-10R1 and BCG/IgG1.

FIGURE 5.

BCG/anti–IL-10R1 administration provides long-term protection against M. tuberculosis infection. (A and C) IL-10−/− CBA/J mice were vaccinated with saline or BCG. (B and D) WT mice were immunized with saline or BCG/anti–IL-10R1 or BCG/IgG1. Mice were infected with M. tuberculosis at 7 wk postvaccination. Lung and spleen CFU counts in IL-10−/− (A and C) or WT (B and D) mice. Survival curve for IL-10−/− (E) and WT (F) mice. Data in (B) and (C) are a combined one to three independent experiments each having five mice in each group in all data points. A Student t test was performed to determine statistical significance between BCG/anti–IL-10R1 and BCG/IgG1 (WT) or between saline- and BCG (IL-10−/−)-immunized experimental groups. *p < 0.05, **p < 0.01, ****p < 0.0001. Data in (C) and (D) represent a single experiment with 10–14 mice in each group. A log-rank test was used to determine statistical significance of survival between BCG/anti–IL-10R1 and BCG/IgG1.

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In WT mice, the BCG/anti–IL-10R1- and BCG/IgG1-vaccinated groups both significantly reduced the M. tuberculosis bacterial burden by ∼1 log10 at week 4 after M. tuberculosis infection compared with the unvaccinated (saline) group (Fig. 5B). As expected, WT mice that received BCG/IgG1 vaccination quickly lost protection afforded by BCG vaccination alone (37, 38), with lung bacterial burden resembling unvaccinated mice as early as 8 wk and reaching almost 8 log10 CFU in the lung by 47 wk postinfection (Fig. 5B). In contrast, WT mice vaccinated with BCG/anti–IL-10R1 further reduced the M. tuberculosis burden at week 8 postinfection, which was sustained at a significantly lower CFU for up to 47 wk postinfection, albeit at slowing increasing levels (study end point, Fig. 5B). BCG/anti–IL-10R1 also conferred protection against dissemination, as indicated by a significant decrease in bacterial burden in the spleen as compared with BCG/IgG1 at 8, 17, and 47 wk postinfection (Fig. 5D).

An independent survival study was established for BCG- or saline-vaccinated IL-10−/− mice (Fig. 5E) and for BCG/anti–IL-10R1-, BCG/IgG1-, or saline-vaccinated WT mice (Fig. 5F). BCG or saline-vaccinated IL-10−/− mice (Fig. 5E) challenged with M. tuberculosis showed that the complete absence of IL-10 resulted in 100% survival at least to 50 wk in both BCG- or saline-vaccinated mice, demonstrating the detrimental impact of IL-10 throughout M. tuberculosis infection as we described (16, 21). Although we observed no survival advantage for BCG vaccination in IL-10−/− mice, extrapolation of bacterial burden (Fig. 5A) and pathology scores (Fig. 6D) suggests that BCG vaccination would likely have extended survival further relative to IL-10−/− mice receiving saline. BCG/IgG1 in WT mice failed to extend survival, indicating that despite an early reduction in M. tuberculosis bacterial burden at week 4, BCG vaccination alone provided no survival advantage. In contrast, administration of BCG/anti–IL-10R1 vaccination in WT mice significantly increased survival, with >65% of mice surviving to the study endpoint of 50 wk.

FIGURE 6.

BCG/anti–IL-10R1 administration reduces immunopathology in lungs after M. tuberculosis infection. (AC) WT mice were immunized with saline, BCG/anti–IL-10R1, or BCG/IgG1. (DF) IL-10−/− CBA/J mice were vaccinated with saline or BCG. Mice were infected with M. tuberculosis at 7 wk postvaccination. WT and IL-10−/− mice were euthanized at predetermined time points postinfection and the caudal lung lobe was quantified for pulmonary inflammation as the percent of tissue involved. (A and D) Pulmonary inflammation in (A) WT mice at 4, 8, 12, and 17 wk postinfection and (D) IL-10−/− mice at 4, 12, and 47 wk postinfection. The percentage affected area was quantified by calculating the total area of the involved tissue over the total area of the lobe for each individual mouse and graded as 1, 2, 3, 4, and 5, which corresponded to <10%, <25%, 50%, <75%, >75% of affected tissue, respectively. (G) Representative images of H&E-stained lung sections of WT mice at 4, 8, 12, and 17 wk postinfection are shown to visualize tissue morphology (original magnification × 2). (B, C, E, and F) ELISA was performed on lung homogenates to measure the level of TNF-α, IL-17, IFN-γ and IL-10 in (B) WT mice at 4 wk postinfection, (C) WT mice at 12 wk postinfection, (E) IL-10 −/− mice at 4 wk postinfection, and (F) IL-10 −/− mice at 12 wk postinfection. Data in (A)–(C) represent the mean ± SEM of one of two independent experiments with three to five mice in each group at each time point. A Student t test was performed to determine statistical significance between BCG/anti–IL-10R1 and BCG/IgG1 (WT mice) or between saline and BCG (IL-10−/− mice) immunized experimental groups. *p < 0.05, **p < 0.01.

FIGURE 6.

BCG/anti–IL-10R1 administration reduces immunopathology in lungs after M. tuberculosis infection. (AC) WT mice were immunized with saline, BCG/anti–IL-10R1, or BCG/IgG1. (DF) IL-10−/− CBA/J mice were vaccinated with saline or BCG. Mice were infected with M. tuberculosis at 7 wk postvaccination. WT and IL-10−/− mice were euthanized at predetermined time points postinfection and the caudal lung lobe was quantified for pulmonary inflammation as the percent of tissue involved. (A and D) Pulmonary inflammation in (A) WT mice at 4, 8, 12, and 17 wk postinfection and (D) IL-10−/− mice at 4, 12, and 47 wk postinfection. The percentage affected area was quantified by calculating the total area of the involved tissue over the total area of the lobe for each individual mouse and graded as 1, 2, 3, 4, and 5, which corresponded to <10%, <25%, 50%, <75%, >75% of affected tissue, respectively. (G) Representative images of H&E-stained lung sections of WT mice at 4, 8, 12, and 17 wk postinfection are shown to visualize tissue morphology (original magnification × 2). (B, C, E, and F) ELISA was performed on lung homogenates to measure the level of TNF-α, IL-17, IFN-γ and IL-10 in (B) WT mice at 4 wk postinfection, (C) WT mice at 12 wk postinfection, (E) IL-10 −/− mice at 4 wk postinfection, and (F) IL-10 −/− mice at 12 wk postinfection. Data in (A)–(C) represent the mean ± SEM of one of two independent experiments with three to five mice in each group at each time point. A Student t test was performed to determine statistical significance between BCG/anti–IL-10R1 and BCG/IgG1 (WT mice) or between saline and BCG (IL-10−/− mice) immunized experimental groups. *p < 0.05, **p < 0.01.

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We also evaluated the effect of vaccination on lung pathology by assessing the degree of tissue involvement through quantification of cellular aggregation relative to the total size of the lung.

All WT mice, independent of experimental groups, had similar and minimal tissue involvement (>10%) in the lung at 4 wk postinfection (Fig. 6A), although some proinflammatory cytokines in homogenates were significantly reduced in the BCG/anti–IL-10R1 group compared with BCG/IgG1-immunized mice at that time (Fig. 6B). These results indicate that even though there was no difference in M. tuberculosis burden or pathology, an early increased proinflammatory cytokine response in the BCG/IgG1-vaccinated group may augment lung pathology at later time points. This was confirmed at week 12 postinfection where BCG/IgG1-vaccinated mice had abundant cellular infiltration and inflammation, with ∼50% of the lung involved (Fig. 6A), although Th1 cytokine levels remained similar to the 4 wk postinfection time point (Fig. 6C). Increased pathology scores were associated with high M. tuberculosis burden in the lungs of BCG/IgG1-treated mice at later time points (Fig. 5B). These findings contrast with BCG/anti–IL-10R1-vaccinated mice that maintained a reduced lung involvement (Fig. 6A), correlating with less M. tuberculosis burden (Fig. 5B), which was maintained even at later time points. BCG/anti–IL-10R1-vaccinated mice also had significantly reduced proinflammatory cytokines levels (TNF-α, IL-17), as well as immunomodulatory cytokines (IL-10), in their lungs compared with the BCG/IgG1-vaccinated group at both 4 and 12 wk postinfection (Fig. 6B, 6C). BCG/anti–IL-10R1-vaccinated mice also had significantly less IFN-γ in their lungs compared with the BCG/IgG1-vaccinated group at 12 wk postinfection. (Fig. 6C).

Similar to WT mice, both saline- and BCG-immunized IL-10−/− mice had minimal tissue involvement in the lungs at week 4 postinfection; however, BCG immunization showed reduced inflammation scores (<25%) at week 4 postinfection, which was sustained through week 47 postinfection (Fig. 6D), consistent with lower M. tuberculosis burden in the lung (Fig. 5A). IL-10−/− mice receiving BCG showed a reduction in some Th1/proinflammatory cytokines in lung homogenates with a significant reduction in IFN-γ at week 4 and in IL-17 at week 12 postinfection as compared with saline group (Fig. 6E, 6F).

Following vaccination with BCG/anti–IL-10R1 and prior to M. tuberculosis challenge, we observed a modest increase in CD4+ and in CD8+ central and effector memory T cells in the lung (Fig. 3). We therefore evaluated the same T cell subsets in the lung at weeks 4 and 8 after M. tuberculosis infection (Fig. 7). Results showed modest differences in total CD4+ and CD8+ T cell subsets in the lung between BCG/anti–IL-10R1- and BCG/IgG1-vaccinated WT mice at week 4 after M. tuberculosis infection (Fig. 7A, 7E). At 4 wk postinfection, we detected a significant increase in the total number of both CD4+ and CD8+ central memory T cells in mice that received BCG/anti–IL-10R1 vaccination (Fig. 7C, 7G), which was sustained through week 8 postinfection (Fig. 7K, 7O). These data suggest that a single and simultaneous vaccination with BCG/anti–IL-10R1 pre–M. tuberculosis challenge can induce central memory cells that are associated with long-term protection against M. tuberculosis infection. CD4+ and CD8+ effector memory T cells were detected at similar or modestly increased levels by both BCG/anti–IL-10R1- and BCG/IgG1-vaccinated WT mice at 4 and 8 wk (Fig. 7D, 7H). Corresponding cell frequencies are presented for week 4 (Fig. 7Q–X) and week 8 (Fig. 7Y–AF) postinfection. Cell frequencies of central memory CD4+ cells (Fig. 7S, 7AA) showed a significant increase similar to the absolute counts at weeks 4 and 8 postinfection. The frequency of central memory CD8+ T cells showed an increasing trend (Fig. 7W, 7AE) but did not reach significance as observed in absolute numbers (Fig. 7G, 7O). In contrast to the absolute numbers of CD4+ (Fig. 7D, 7L) and CD8+ T effector memory cells (Fig. 7H, 7P), the frequency of CD4 and CD8+ T effector memory subsets (Fig. 7T, 7AB and (Fig. 7X, 7AF) showed a significant reduction in the BCG/anti–IL-10R1 group compared with the BCG/IgG1 group both at 4 and 8 wk postinfection.

FIGURE 7.

BCG/anti–IL-10R1 administration enhances central memory T cell responses in lung of M. tuberculosis–infected mice. WT mice were s.c. immunized with saline, BCG/anti–IL-10R1, or BCG/IgG1. (AP) Seven weeks later, mice were challenged with M. tuberculosis and euthanized at the 4 (A–H) and 8 wk (I–P) postinfection. Figure represents (A–P) absolute number and (QAF) cellular frequency of (A, I, Q, and Y) CD4+, (B, J, R, and Z) CD4+CD44hi, (C, K, S, and AA) CD4+CD44hiCD62L+CCR7+ central memory, (D, L, T, and AB) CD4+CD44hiCD62LCCR7 effector memory, (E, M, U, and AC) CD8+, (F, N, V, and AD) CD8+CD44hi, (G, O, W, and AE) CD8+CD44hiCD62L+CCR7+ central memory, and (H, P, X, and AF) CD8+CD44hiCD62LCCR7 effector memory cells. Data are the mean ± SEM of one of the two independent experiment with three to five mice in each group. A Student t test was performed to determine the statistical significance between BCG/anti–IL-10R1 or BCG/IgG1 experimental groups. *p < 0.05, **p < 0.01.

FIGURE 7.

BCG/anti–IL-10R1 administration enhances central memory T cell responses in lung of M. tuberculosis–infected mice. WT mice were s.c. immunized with saline, BCG/anti–IL-10R1, or BCG/IgG1. (AP) Seven weeks later, mice were challenged with M. tuberculosis and euthanized at the 4 (A–H) and 8 wk (I–P) postinfection. Figure represents (A–P) absolute number and (QAF) cellular frequency of (A, I, Q, and Y) CD4+, (B, J, R, and Z) CD4+CD44hi, (C, K, S, and AA) CD4+CD44hiCD62L+CCR7+ central memory, (D, L, T, and AB) CD4+CD44hiCD62LCCR7 effector memory, (E, M, U, and AC) CD8+, (F, N, V, and AD) CD8+CD44hi, (G, O, W, and AE) CD8+CD44hiCD62L+CCR7+ central memory, and (H, P, X, and AF) CD8+CD44hiCD62LCCR7 effector memory cells. Data are the mean ± SEM of one of the two independent experiment with three to five mice in each group. A Student t test was performed to determine the statistical significance between BCG/anti–IL-10R1 or BCG/IgG1 experimental groups. *p < 0.05, **p < 0.01.

Close modal

IL-17 and IFN-γ are critical for mediating immunity against M. tuberculosis infection as well as vaccine-induced protection against the development of TB (39, 40). Lung cells from vaccinated and M. tuberculosis-challenged mice were cultured ex vivo with CFPs, and IFN-γ and IL-17 production was measured. Relative to unstimulated cells, Ag-specific IFN-γ and IL-17 secretion was increased for both BCG/anti–IL-10R1- and BCG/IgG1-vaccinated groups at weeks 4 and 8 after M. tuberculosis infection (Figs. 8). There was no significant differences between the BCG/anti–IL-10R1 and BCG/IgG1 groups (Fig. 8), with the exception of IL-17 at week 4 (Fig. 8B), although significance was lost by week 8 after M. tuberculosis infection (Fig. 8D). IL-17 secretion in unstimulated cultures remained high, perhaps representing a non–T cell source of IL-17 (Fig. 8B, 8D).

FIGURE 8.

BCG/anti–IL-10R1 administration enhances Ag=specific IFN-γ and IL-17 levels in lungs after M. tuberculosis infection. (AD) WT mice were s.c. immunized with saline, BCG/anti–IL-10R1, or BCG/IgG1. At 7 wk later, mice were challenged with M. tuberculosis and euthanized at 4 and 8 wk postinfection. Isolated lung mononuclear cells were ex vivo stimulated with medium or M. tuberculosis CFPs for 48 h. Culture supernatants of WT mice were analyzed for the production of (A and C) IFN-γ and (B and D) IL-17 by ELISA. Data represent the mean ± SEM of three to five mice in each group. A Student t test was performed to determine the statistical significance between the medium and CFP stimulation within an experimental group and between the groups. *p < 0.05, **p < 0.01.

FIGURE 8.

BCG/anti–IL-10R1 administration enhances Ag=specific IFN-γ and IL-17 levels in lungs after M. tuberculosis infection. (AD) WT mice were s.c. immunized with saline, BCG/anti–IL-10R1, or BCG/IgG1. At 7 wk later, mice were challenged with M. tuberculosis and euthanized at 4 and 8 wk postinfection. Isolated lung mononuclear cells were ex vivo stimulated with medium or M. tuberculosis CFPs for 48 h. Culture supernatants of WT mice were analyzed for the production of (A and C) IFN-γ and (B and D) IL-17 by ELISA. Data represent the mean ± SEM of three to five mice in each group. A Student t test was performed to determine the statistical significance between the medium and CFP stimulation within an experimental group and between the groups. *p < 0.05, **p < 0.01.

Close modal

In this study, we demonstrate that a single coadministration of anti–IL-10R1/BCG substantially improved the long-term protective efficacy of BCG against M. tuberculosis infection for up to 47 wk in the CBA/J mouse model, a mouse strain that is defined as relatively susceptible to M. tuberculosis (41, 42). We therefore demonstrate that a single-dose host-directed therapy combined with BCG can improve vaccine efficacy. We also identify the specific time of BCG vaccination for a single-dose IL-10 blockade intervention to positively influence the development of long-term protective immunity against M. tuberculosis infection.

Our vaccination strategy was based on prior observations from our group that early anti–IL-10R1 treatment of M. tuberculosis–infected mice could substantially improve control of M. tuberculosis infection in CBA/J mice (22). We hypothesized that anti–IL-10R1 treatment blocks the negative influence of IL-10 on priming and development of long-term memory immunity. Because the presence of IL-10 can also influence maintenance of M. tuberculosis infection as we have described (21), we separated immune cell priming from M. tuberculosis infection by blocking the action of IL-10 during anti–IL-10R1/BCG vaccination instead of at M. tuberculosis infection. The superior control of M. tuberculosis infection that we observed in anti–IL-10R1/BCG-vaccinated mice demonstrates that IL-10 inhibits the early development of long-term memory immunity. Further supporting our findings, we have previously demonstrated that selective delipidation of BCG, which induces less IL-10, generates enhanced protection against M. tuberculosis challenge, minimizing tissue damage when compared with conventional BCG (43).

IL-10R1 blockade during vaccination has been tested in various short-term experimental or disease models (4446), including a mouse model of human papillomavirus virus 16 E7-transformed TC-1 tumor growth, where inclusion of anti–IL-10R with vaccination enhanced specific cytolytic (CTL) responses (47). Similarly, blockade of IL-10R1 permitted an otherwise ineffective DNA vaccine to become highly efficient at stimulating CD4+ and CD8+ T cell responses, leading to accelerated clearance of lymphocytic choriomeningitis virus in mice (48). Moreover, IL-10 blockade was shown to enhance the magnitude and quality of Th1 responses sufficient to reduce vaccination/boost frequency from three vaccine doses to only one dose (49). Indeed, anti–IL-10R1 has also been used in combination with BCG to induce anti-cancer immunity, which protected mice from bladder cancer (50). Thus, these combined studies highlight the potential for IL-10 modulation as a host-directed therapy during vaccination to improve protective immunity against a variety of different disease models, including TB, and we postulate that studies of IL-10 modulation during initial vaccination should be revisited as a mechanism to boost host protective immunity with vaccines that have known diminished long-term protection efficacy, such as BCG. The absence of IL-10 has been shown to cause colitis and intestinal inflammation in human and animal models (5153), whereas a temporary IL-10 blockade via administration of anti–IL-10R Ab in mice, non-human primates, and in humans was found to be safe, with no abnormal findings having been observed (28, 48, 50, 5457). This suggests that blockade of IL-10R could be a safe and well-tolerated in vivo therapeutic in humans, and it is clearly supported by studies in different animal models.

Protective immunity generated by BCG vaccination wanes over time in humans and in most experimental animal models (37, 38, 5860). Interestingly, BCG vaccination induces strong Th1 responses (6164), and both M. tuberculosis infection and BCG immunization induce effector T cells (6469). However, long-lived central memory T cells are associated with superior efficacy of experimental TB vaccines (7075). In contrast to many short-term microbial infections where a rapid effector T cell response can resolve infection, BCG must stimulate long-term memory immunity to maintain effective control of a recent or a latent M. tuberculosis infection, often in the absence of sterilizing immunity. Adding additional complexity, BCG also induces IL-10 production, which diminishes long-term Ag-specific Th1 immune responses (62, 7679). In the current study, we determined immune responses to anti–IL-10R1/BCG vaccination at week 1 to observe the immediate impact of IL-101R blockade, and at week 7 postvaccination to characterize the immune status in the lung at the time of M. tuberculosis challenge. At week 1, BCG vaccination alone increased IL-10 in the lung, which remained high 7 wk later. Interestingly, anti–IL-10R1/BCG vaccination reduced IL-10 and led to a concomitant reduction in proinflammatory cytokines in the lung. The reduction in proinflammatory cytokines was independent of BCG burden, as we did not detect BCG in the lungs, and it was accompanied by a modest increase in central and effector memory CD4+ and CD8+ T cell subsets in the lungs, with enhanced Ag-specific Th1 and Th17 responses. Thus, a single anti–IL-10R1/BCG mixture dose was capable of modifying the local lung environment immediately following vaccination.

IL-10 is a pleiotropic cytokine secreted by various immune cells, with innate cells being a source of IL-10 production during the early phase of BCG vaccination (80). Innate cells can also respond to IL-10 and alter their function (81). Thus, the immediate impact of IL-101R blockade during BCG vaccination may reflect changes in innate cell function including enhanced and prolonged Ag presentation, resulting in altered T cell priming and generation of long-lived T cell responses (27, 82). Indeed, IL-10 from non–B cell or non–T cell sources has been shown to regulate dendritic cell–driven Th1/Th2 responses in vivo (82), where, similar to our findings, early IL-10 blockade enhanced Th1/Th17 responses associated with accelerated fungal clearance in mice (83).

Our studies extend observations by Pitt et al. (28) where anti–IL-10R1 treatment given at the time of BCG vaccination, and for an additional 6 wk thereafter, promoted Ag-specific Th1/Th17 immunity in the lungs, and reduced M. tuberculosis burden, defining an association between enhanced protection and Th1/Th17 responses. However, the extended period of anti–IL-10R1 treatment (for 6 wk postvaccination) could not fully separate the timing of the negative influence of IL-10 on the early generation of long-term protective immunity from effector functions. Our single-dose anti–IL-10R1/BCG vaccine strategy confirms that IL-10 can negatively impact the initial generation of long-lasting protective immunity against M. tuberculosis infection. Given the short half-life of anti–IL-10R1/IgG1, a direct pleiotropic influence of anti–IL-10R1 at week 7 postvaccination (our selected time for M. tuberculosis challenge) was unlikely (84).

The CBA/J mouse strain, the strain background for our studies, has limited protection afforded by BCG (41), making it an ideal WT strain to determine whether the anti–IL-10R1/BCG mixture can improve protective efficacy and identify potential mechanisms of action. M. tuberculosis infection of anti–IL-10R1/BCG-vaccinated CBA/J WT mice had a sustained enhanced control of M. tuberculosis infection and extended lifespan beyond 50 wk. In contrast, BCG vaccination alone failed to extend survival relative to non-vaccinated CBA/J WT mice. A single dose of anti–IL-10R1/BCG vaccine was also associated with reduced lung inflammation as determined by histopathology, and reduced proinflammatory cytokines in the lung at weeks 4 and 8 after M. tuberculosis infection. Proinflammatory cytokine production can depend on the bacterial load, yet both BCG- and anti–IL-10R1/BCG-vaccinated mice had similar M. tuberculosis burden at week 4 postinfection, suggesting that anti–IL-10R1/BCG vaccination generates an immune response that is less inflammatory, identifying a potential causal relationship between reduced inflammation at the time of vaccination and the generation and prolonged maintenance of IL-17/IFN-γ–producing central memory T cells, as described by others (43). An inflammatory environment can alter the development of effector and/or memory response generating short-lived effector cells (85, 86). Thus, our results support the concept that a strong inflammatory response regulates T cell sensitivity, proliferation, and migration of both effector and established memory T cells populations (87, 88).

Our findings in WT mice treated with anti–IL-10R1/BCG were corroborated by similar studies in IL-10 knockout mice on the same CBA/J mouse strain background (22). BCG-immunized IL-10−/− CBA/J mice had superior control of M. tuberculosis infection and significantly reduced lung immunopathology for up to 50 wk postinfection. Interestingly, both non-immunized and BCG-immunized IL-10−/− mice had 100% survival for at least 50 wk postinfection (study endpoint), albeit with different immunopathology scores, suggesting a possible split in survival much later. Modulation of IL-10R1 at late stages of M. tuberculosis infection is already known to improve outcomes (21). However, the use of a non-conditional knockout model, while complementary, cannot spatially separate this from the influence of IL-10R1 during T cell priming but it can substantiate IL-10 as the primary driver of our observed phenotypes. In contrast to CBA/J mice, we have not observed enhanced BCG efficacy in C57BL/6 mice vaccinated with anti–IL-10R1/BCG (data not shown). C57BL/6 mice are poor inducers of IL-10, which increases gradually as M. tuberculosis infection progresses (21), suggesting a possible need to maintain continuous IL-10 blockade during the whole vaccination phase as demonstrated by Pitt et al. (28).

Although a single vaccination of anti–IL-10R1/BCG showed an increase in the CD4+ and CD8+ memory cells subsets and elevated Ag-specific Th1 and Th17 cytokines before and after M. tuberculosis infection, a detailed and precise mechanism to correlate immune changes with enhanced protection needs to be evaluated in future studies. Additionally, because IL-10 signaling was blocked at the time of BCG immunization, a detailed characterization of the APC and T cell kinetics during the early phase of vaccination may also be helpful to determine the mechanism of long-term sustained protection. IL-10 is secreted by several cell types and regulates myeloid and T cells (33, 89) and, therefore, blocking of IL-10 signaling may involve more than one mechanism.

Overall, our studies indicate that temporal and spatial blocking of IL-10R1 is sufficient to generate long-term protective immunity against M. tuberculosis infection in the relatively susceptible CBA/J mouse strain. Our studies identify IL-10, or its downstream effector functions, as a putative target for the development of improved vaccines for TB, and they identify a single-dose vaccine strategy against M. tuberculosis that can result in long-term protective immunity and reduced TB disease.

We thank the animal resources personnel and Biosafety Level 3 Program at both The Ohio State University and Texas Biomedical Research Institute. The following reagent was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases, National Institutes of Health: Mycobacterium tuberculosis, strain H37Rv, culture filtrate protein, NR-14825.

This work was supported by a pilot grant from the College of Medicine, The Ohio State University (to J.T.), Texas Biomedical Research Institutional funds (to J.T.), and by the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation (to J.B.T.).

Abbreviations used in this article:

BCG

bacillus Calmette–Guérin

CFP

culture filtrate protein

TB

tuberculosis

WT

wild-type.

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The authors have no financial conflicts of interest.