Early introduction of IL-10 weakens BCG revaccination's protection by suppressing CD4+Th1 cell responses

doi:10.21203/rs.3.rs-4715344/v1

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Early introduction of IL-10 weakens BCG revaccination's protection by suppressing CD4+Th1 cell responses

https://doi.org/10.21203/rs.3.rs-4715344/v1

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Introduction: The only authorized tuberculosis (TB) vaccine, Bacillus Calmette-Guérin (BCG), has shown limited effectiveness in protecting adolescents and adults against active TB, even boosted with either BCG itself or heterologous vaccine candidates.

Objectives: The mechanisms underlying the suboptimal efficacy of BCG revaccination against primary M.tb infection remain unknown.

Methods: C57BL/6J mice were immunized with the BCG vaccine once or twice, with lung tissue analyzed for changes in cytokine levels. Different intervals between vaccinations and detection times were also studied for IL-10 expression in various organs. IL-10 expressing cells in lung, spleen, and lymph nodes were analyzed using FACS and intracellular cytokine staining (ICS). BCG revaccinated IL-10^-/-mutant mice were compared to wild type mice for antigen-specific IgG antibody and T cell response. Protection against M.tb aerosol challenge was assessed in BCG revaccinated mice that untreated or treated with anti-IL-10R monoclonal antibody.

Results: IL-10 was found to be upregulated in the lungs of BCG revaccinated mice shortly after the booster immunization. The expression of IL-10 reached its peak in the lungs 3-6 weeks post-revaccination, and was also observed in lymph nodes and spleens as early as 2 weeks after the booster dose, regardless of the time intervals between the prime and booster vaccinations. The main sources of IL-10 in these tissues were macrophages and dendritic cells. Blocking IL-10 signaling in BCG revaccinated mice, achieved through the use of IL-10^-/-mutant mice or anti-IL-10R monoclonal antibody, increased levels of antigen-specific IFN-γ⁺ or IL-2⁺CD4⁺ T cells, central and effector memory CD4⁺ T cell responses, leading to better protection against 300 CFU of aerosol M.tb infection.

Conclusion: Our findings are crucial for formulating effective immunization strategies pertaining to the BCG vaccine, as well as developing efficacious adult TB vaccine.

BCG revaccination

BCG prime-boost strategy

adult tuberculosis

primary Mycobacterium tuberculosis infection

IL-10 inhibition

memory CD4+ T cell

Bacillus Calmette-Guérin (BCG) vaccine, attenuated from the parent Mycobacterium bovis virulent strain, has been the sole vaccine approved for tuberculosis (TB) prevention for over a century¹. BCG vaccine has been recommended for the inclusion in expanded immunization programs by the World Health Organization (WHO) since 1974². Currently, 156 countries implement the BCG vaccine, each adhering to specific national guidelines^3,4. Neonatal BCG vaccination has been extensively adopted in over 80% of these countries, with an average coverage rate exceeding 90%. Despite ongoing efforts to prevent its spread, TB continues to pose a significant threat to global public health⁵. Globally, a total of 10.6 million new TB cases were reported in 2022, and 1.3 million people died of the disease, with adolescents and adults accounting for approximately 90% of the newly reported TB cases⁶. Moreover, around 25% of the global population carries latent tuberculosis infection (LTBI), and 5–10% of them progressing to active TB over their lifetimes^5,7. Therefore, the effectiveness of neonatal BCG vaccination in preventing active TB or LTBI in adolescence and adulthood is insufficient⁸. Consequently, there is a pressing need for the development of more efficacious vaccines or immunization strategies for better controlling TB in adults.

The development of more efficacious TB vaccine candidates is a significant challenge, particularly almost no candidates has been demonstrated stronger protection than the current BCG immunization in preclinical and clinical trials. As a result, the replacement or elimination of the existing BCG immunization regimen for infants is impracticable^1,9. To enhance the efficacy the BCG vaccine in preventing adult TB, prioritizing BCG revaccination is considered a promising strategy due to its convenience and simplicity. Prior to 2019, a total of 78 countries, including China, had recommended the inoculations of the BCG vaccine on two and more separate occasions at different intervals based on historical data^3,4. As of 2019, seven countries have continued to include the booster doses of the BCG vaccine in their national guidelines^3,4. Even so, the WHO has not endorsed the BCG revaccination strategy^10,11. In order to overcome obstacles, an alternative approach of BCG prime-heterologous boosting regimens has seen rapid development^12,13. Unfortunately, nearly half of the heterologous boosting vaccine candidates^12,13, particularly those undergoing clinical trials¹³, have not shown improved efficacy in BCG primed-animal models against primary Mycobacterium tuberculosis (M.tb) infection in preclinical studies. Recent findings from a phase 2b clinical trial further support the conclusion that the efficacy of BCG revaccination in adults against primary M.tb infection is less than 50%¹⁴. It is noteworthy that our previous study has demonstrated that BCG primed mice and then either revaccinated with BCG ¹⁵ or heterologous boosted with subunit protein CMFO vaccine are effective approaches for preventing and controlling LTBI rather than primary M.tb infection¹⁶, attributing to upregulated IL-2 positive central memory T (T_CM) and IFN-γ⁺ effector memory T (T_EM) cell responses following the booster inoculation¹⁵. However, the underlying mechanisms contributing to the suboptimal efficacy of BCG revaccination or heterologous boosters in primary M. tb infection remain unknown.

Cellular immune response mediated by Th1 cells is generally recognized as essential in the defense against TB infection^17–19. The central question in this field of study is whether BCG revaccination or other heterologous booster vaccines may affect T cell function. To address the question, animal models in this study were established by immunizing both wild-type (WT) and mutant-type (MT) mice with the BCG vaccine once or twice, respectively. Subsequently, the lung tissue of the immunized mice was analyzed using qRT-PCR and immunohistochemical methods to identify cytokines exhibiting altered expression levels. The only anti-inflammatory cytokine, IL-10, was significantly upregulated shortly after the booster immunization of the BCG vaccine. A further analysis of the temporal and spatial expression profiles of IL-10 was performed in this study to investigate the role that IL-10 plays in modulating T cell responses and in preventing primary M. tb infection in BCG revaccinated mice. Our findings are important for better controlling adult TB by enhancing BCG immunization strategy or developing novel vaccine candidates.

Mice

This study used female C57BL/6J mice (Charles River, Beijing, China) that were specific pathogen-free and aged 6–8 weeks. Mutant type (MT) C57BL/6J mice with the IL-10 gene (IL-10⁻/⁻) knocked out were generously provided by Prof. Xiaolian Zhang from Wuhan University. Both mice were housed at the Animal Experimental Center, Tongji Medical College, Huazhong University of Science and Technology.

Ethics statement

All experiments involving animals were conducted according to the ethical policies and procedures approved by the Committee on the Ethics of Animal Experiments at Tongji Medical College, (IACUC Number: 3538).

Immunization and antibody treatment

BCG vaccine (China strain) was cultured on Middlebrook 7H11 agar plates at 37 ℃. In mice, 10⁶ CFU of the BCG vaccine were immunized subcutaneously (s.c.) with the first dose, followed by a booster dose administered at varying intervals during the experiments. Mice treated with PBS or the BCG vaccine once were used as controls. The monoclonal antibody (mAb) targeting mouse IL-10R (BioXCell, West Lebanon, USA) was injected intraperitoneally (i.p.) to inhibit IL-10 signaling as needed. The mice in each group were euthanized in the euthanasia room according to the method of gradually inflating the carbon dioxide (CO2) compressed gas cylinder, and then the follow-up analysis was carried out. All experiments were conducted in triplicate.

Different cytokines mRNAs detected by qRT-PCR

qRT-PCR was used to assess the expression levels of various cytokines in lung tissues, including IFN-γ, TNF-α, IL-17A, TGF-β, IL-10, IL-1β, IL-6, IL-2, and IL-4. In brief, total RNA was first extracted from the tissues by using TRIzol reagent (Invitrogen, Carlsbad, USA) and then reverse-transcribed into cDNA by using the ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan) following the manufacturer's instructions. Supplementary table 1 provided primer sequences for the experiments. qRT-PCR experiments were conducted using the Bio-Rad CFX96 machine and the SYBR Green Realtime PCR Master Mix reagent (Thermo Scientific, Waltham, USA). 2^−ΔΔCt method was used to quantify the relative mRNA expression levels by using GAPDH mRNA as the control. The value of -ΔΔCt was calculated by subtracting the experimental (Ct cytokine – Ct _GAPDH) value from the control (Ct cytokine – Ct _GAPDH) value. Results are expressed as the mean ± standard deviation (SD).

Detection of IL-10 expression in lung tissues through immunohistochemistry

Lung tissue from each euthanised mouse was fixed in 4% formalin, sectioned, and immunohistochemically analyzed for IL-10 protein expression with anti-IL-10 antibodies (Servicebio, Wuhan, China) and an HRP-conjugated secondary antibody. Results were observed using a diaminobenzidine substrate and hematoxylin counterstaining. Eight fields of view were examined under a light microscope at a magnification of 400×, and the number of cells exhibiting a brownish-yellow color was determined. Results are expressed as the mean ± SD.

FACS and intracellular cytokine staining (ICS) for the detection of IL-10 positive cells

2×10⁶cells, prepared from spleen, lung, or inguinal lymph nodes of each mouse were seeded into each well of the 96-well plates and incubated with a cell activation cocktail containing Brefeldin (Cat#423304, Biolegend, Santiago, USA) for 6 hours. After washing with ice-cold PBS, the cells were incubated with anti-rat CD16/CD32 antibody (Cat#553141, BD, USA) at 4 ℃ for 15 minutes. Dead cell staining was done with fixable viability stain 620 (Cat#564996, BD, USA) and stopped with PBS containing 5% FBS. Cells were then stained with various surface biomarkers, including anti-CD25-PE-CY7 (Cat#561780), anti-CD11c-BV650 (Cat#561241), anti-CD8-BV510 (Cat#563068), anti-F4/80-BV421 (Cat#565411), anti-CD4-APC-CY7 (Cat#552051), anti-B220-BV605 (Cat#3554467), and anti-CD3-FITC (Cat#553061), as well as intracellular biomarkers including anti-IFN-γ-PE-CY7 (Cat#557649), anti-IL17A-APC (Cat#560184), anti-Foxp3-APC (Cat#560401) antibodies and anti-IL-10-PE antibodies (Cat#563708) obtaining from BD, USA. FlowJo software was used to determine the proportion of IL-10 positive DC (CD11c⁺), Macrophage (F4/80⁺), B (B220⁺), T (CD3⁺), Th (CD3⁺ CD4⁺), Th1 (CD3⁺ CD4⁺ IFN-γ⁺), Th17 (CD3⁺ CD4⁺ IL17A⁺), Tc (CD3⁺ CD8⁺) and Treg (CD25⁺ Foxp3⁺) cells in each organ, using an LSRII multicolor flow cytometer from BD Biosciences. Results are shown as mean ± SD.

Detection of CMFO-specific T cells by ICS and FACS

BCG revaccinated WT and IL-10^−/− C57BL/J mice were used as experimental models to identify CMFO-specific T cells by using ICS and FACS, as previously described¹⁵. In brief, 5×10⁶ splenocytes or lung cells from each mouse were incubated with 20 µg CMFO and 1 µg anti-mouse CD28 antibody (Cat#102116, Biolegend, USA) for 4 hours, followed by the addition of 2 µM monensin solution (Cat#420701, Biolegend, USA) for 12 hours. Fixable Viability Stain 450 (Cat#562247, BD, USA) was utilized to exclude dead cells. Cells were stained with surface and intracellular biomarkers obtained from BD, including anti-CD44-FITC (Cat#561859), anti-CD62L-PerCP-Cy5.5 (Cat#560513), anti-CD4-APC-Cy7 (Cat#552051), anti-CD8a-BV510 (Cat#563068), anti-IL-2-APC (Cat#554429), and anti-IFN-γ-PE (Cat#557649). FlowJo software was used to determine the absolute number of CMFO-specific IFN-γ or IL-2 positive T cells, effector memory T cells (T_EM, CD62L^lo CD44^hi), and central memory T cells (T_CM, CD62L^hi CD44^hi) in each organ. Results are shown as mean ± SD of each group.

Detection of CMFO specific cytokines secreted by splenocytes using CBA method

5 × 10⁶ splenocytes from each mouse were stimulated with 20 µg CMFO and 1 µg anti-mouse CD28 (Cat#102116, Biolegend) for 72 hours. Various cytokines secreted by splenocytes in culture supernatants, such as IFN-γ, TNF-α, IL-6, IL-17A, IL-2, IL-4, and IL-10, were quantified using mouse Th1/Th2/Th17/Treg cytokine kits (Cat#LX-560485, BD, USA). Results are expressed as mean ± SD for each group.

ELISA detection of CMFO-specific IgG and subclass

IgG (Cat#151276, Abcam, USA), IgG2a (Cat#157720), and IgG1 (Cat#133045) were used to detect CMFO-specific endpoint titers in serum samples from individual mice via ELISA, as previously described¹⁶. The antibody titers for each mouse were determined as log₁₀ (endpoint titer) of each mouse, and results are shown as mean ± standard error of the mean (SEM) for each group.

Evaluation of the protective effect against virulent M.tb H37Rv infection

Mice were infected with virulent M.tb H37Rv via aerosol exposure (Jingnuo Biotech., Shanghai, China) and then housed in an ABSL-3 laboratory. The next day after being infected, five mice that were not immunized were euthanized, and their lungs were aseptically extracted to quantify the colony forming units (CFU) and determine the actual infection dose. Four weeks post-infection, protection levels were evaluated as previously described¹⁶. In brief, the lung tissue homogenates from each infected mouse were prepared and serially diluted before being plated on Middlebrook 7H11 agar. After incubated for 3–4 weeks at 37 ℃, CFU was enumerated and results are shown as the mean log₁₀ (CFU) ± SEM. Additionally, either hematoxylin and eosin (HE)or acid-fast (AF) stains was used to stain lung tissue samples.

Statistical analyses

GraphPad Prism 9 was used to collect and analyze the data. A P value below 0.05 was used to determine statistical significance by using two-tail Student's t-test or one-way ANOVAs, as required.

Revaccination with BCG quickly causes higher IL-10 levels in the lungs

Immunomodulatory effects of cytokines have a significant impact on immunity and infection²⁰. In order to identify anti-inflammatory cytokines that affect the protective efficacy of BCG revaccination, C57BL/6J mice were initially immunized s.c. with 10⁶ CFU BCG, followed by a booster dose nine weeks later (BCG revaccination group). Controls mice treated with either PBS or immunized with the BCG vaccine once. Three weeks later, the mRNA expression levels of different cytokines in the lung tissue of mice were detected using qRT-PCR (Fig. 1A). Although mRNA levels of TNF-α, IFN-γ, IL-2, IL-6, TGF-β, IL-4, and IL-1β were consistent in all groups, the BCG revaccination group significantly induced higher levels of IL-10 and IL-17A than PBS and BCG groups (Fig. 1B). Immunohistochemical analysis also showed increased IL-10 protein expression in the lung tissue after BCG revaccination (Fig. 1C-D). According to these results, IL-10, a cytokine with anti-inflammatory properties, is rapidly increased within the lung tissue following the re-inoculation with the BCG vaccine.

IL-10's lung expression peaks 3–6 weeks post BCG revaccination

To further investigate the impact of the interval duration between the priming and subsequent boost immunization on IL-10 expression in the lung, mice previously primed with BCG were given a second dose of the BCG vaccine at weeks 5, 9, or 14, respectively. Three weeks following the boost immunization, IL-10 expression in lung tissue was analyzed using qRT-PCR (Fig. 2A). Notably, BCG that were revaccinated mice displayed higher IL-10 levels in lung tissue, regardless of the specific interval duration, when compared with the control groups receiving either PBS or BCG alone (Fig. 2B).Furthermore, the expression pattern of IL-10 in the lung was examined after a 9-week interval between the prime and boost vaccinations to explore the effect of time of detection (1, 2, 3, 6, 9, 12, and 28 weeks) following the boost inoculation (Fig. 2C). Interestingly, IL-10 mRNA levels in the lungs increased significantly at weeks 3 and 6 after BCG revaccination, but there was no difference among three groups at other time points of detection (Fig. 2D-E). Therefore, enhanced IL-10 expression in the lungs due to repeated BCG immunization peaks three to six weeks after the second dose and is not influenced by the interval time between both doses.

IL-10 is quickly induced in mouse lymph nodes and spleen via macrophages and DCs following BCG revaccination

Mice were immunized s.c. with the BCG vaccine twice at week 0 and 9. At weeks 1, 2, 3, and 28 post-booster dose, the IL-10 expression in peripheral immune organs, such as lymph nodes and spleen, was measured by qRT-PCR (Fig. 3A). There was no statistically significant difference in levels of IL-10 mRNA in lymph nodes and spleen between the BCG group and the PBS group across different time points. Remarkably, only at week 2 and week 3 after the booster dose, IL-10 mRNA levels were statistically significantly higher in the BCG revaccination group when compared with PBS and BCG groups (Fig. 3B-E). ICS and FACS analyses were performed to identify the specific cell types that produce IL-10 following repeated BCG immunization, including B cells, dendritic cells (DCs), macrophages, T cells, as well as T cell subsets such as Th1, Th17, helper (Th), cytotoxic (Tc) and regulatory T cells (Treg) (Fig. 4A, Supplementary Fig. 1). Two weeks post-second dose, mice that received the second dose of the BCG vaccine displayed a greater number of IL-10⁺ DCs, IL-10⁺ macrophages, and IL-10⁺ B cells in the lymph nodes when compared with PBS and BCG control groups (Fig. 4B). Three weeks after the booster dose, BCG revaccinated mice showed a notable increase in IL-10⁺ DCs and IL-10⁺ macrophages in the spleen and lung, as well as IL-10⁺ B cells in the spleen (Fig. 4C-D). In addition, compared to the PBS control group, BCG revaccinated mice significantly increased the number of IL-10⁺ T, Th, and Th1 cells in their spleens (Fig. 4C). Therefore, these results demonstrate that macrophages and dendritic cells in the lymph nodes and spleen are primarily responsible for IL-10 production shortly after BCG revaccination.

IL-10 knockout mice enhanced antigen-specific T cell responses following BCG revaccination compared to wild-type mice

The effect of IL-10 on the T cell response in BCG revaccinated mice was further explored. Six to 8-week-old WT mice and IL-10^−/− mice were immunized s.c. with the BCG vaccine twice at weeks 0 and 9, respectively. Three weeks following the second dose, the numbers of CMFO-specific T cells in the spleen and lung of each vaccinated mice were determined using ICS and FACS (Fig. 5A, Supplementary Fig. 2). Compared with the WT group, BCG revaccinated IL-10^−/− mice induced higher levels of IFN-γ⁺ CD4⁺ T cells, IFN-γ⁺ CD4⁺ T_EM cells, and IL-2⁺ CD4⁺ T_CM cells in the both spleen (Fig. 5B) and lung (Fig. 5C), as well as IL-2⁺ CD4⁺ T cells in the spleen. Furthermore, BCG-revaccinated IL-10^−/− mice exhibited higher levels of IL-2, IFN-γ, TNF-α, IL-17A, and IL-6 in the supernatant of splenocytes upon stimulation with CMFO than the WT mice (Fig. 5D). In addition, both BCG revaccinated IL-10^−/− and WT mice induced similar levels of CMFO-specific IgG, IgG2a, and IgG1 in the serum (Supplementary Fig. 3A-C), and IgG2a/IgG1 ratio was comparable between both groups (Supplementary Fig. 3D). Thus, BCG- revaccinated IL-10^−/− mice enhance antigen-specific CD4⁺ T cells, especially memory CD4⁺ T cell responses in comparison to the WT mice.

Blocking IL-10 signaling enhances BCG-revaccinated mice's resistance to primary M.tb infection

To investigate the effect of IL-10 on the protective efficacy of BCG revaccinated mice, mice were immunized s.c. with the BCG vaccine twice at weeks 0 and 9. IL-10 signaling was inhibited through intraperitoneal administration of anti-IL-10R antibodies one day before the booster dose, with subsequent weekly injections prior to the challenge with virulent M.tb. At the twelfth week, mice were challenged with 300 CFU M.tb H37Rv via aerosol exposure. Four weeks later, the lungs of each mouse were aseptically harvested for assessment of protective efficacy (Fig. 6A). BCG -revaccinated mice treated with IL-10R mAb more significantly decreased bacterial loads in the lungs (Fig. 6B) and had less lung histopathological changes (Fig. 6C)than the untreated control group. Thus, blocking IL-10 signaling in BCG revaccinated mice results in the enhanced protection against primary M. tb infection.

Currently, adolescents and adults are the primary demographics affected by the TB epidemic. Given that infants typically receive immunization with the BCG vaccine, it is a potentially effective strategy to protect adults from TB by administering a booster immunization with either a homologous BCG vaccine or a heterologous vaccine candidate of alternative other types during adolescence and adulthood. Nevertheless, the suboptimal efficacy and the uncertain mechanism of action pose significant obstacles to the advancement of this strategy for preventing TB in adults. Thus, there is an urgent need to investigate pertinent mechanisms and essential technologies in order to overcome this barrier and facilitate the progress and implementation of an effective adult TB vaccine. In this study, qRT-PCR was used to determine the differential cytokines expression in mice following BCG revaccination, demonstrating for the first time that the expression of IL-10, the only anti-inflammatory cytokine, is significantly increased in the lungs following revaccination. Interestingly, elevated IL-10 expression in the lungs was transient, lasting for a period of 3–6 weeks after revaccination, regardless of the time interval between the prime and booster doses. Lymph nodes and spleen were also detected with IL-10 expression as early as the second week after the boosting vaccination. In these tissues, IL-10 is primarily produced by antigen-presenting cells (APCs), such as macrophages and dendritic cells. An inhibition of IL-10 signaling resulted in higher levels of antigen-specific CD4⁺ T cells and memory CD4⁺ T cell responses in the spleens and lungs of mice revaccinated with the BCG vaccine, and thus BCG revaccinated mice became more resistance to primary M.tb infection. Therefore, our findings help formulate a rational immunization approach by utilizing the BCG vaccine for TB prevention in adult populations.

As a result of the subcutaneous administration of the BCG vaccine, APCs take up the BCG in local lymph nodes, leading to the activation of CD4⁺ T cells through the phagocytosis-lysosomal pathway and MHC class II molecules^21,22. Despite this, the immunological effects of repeated BCG immunizations have not been widely recognized until now. This study is the first to demonstrate that IL-10⁺ DCs and IL-10⁺ macrophages in lymph nodes are induced as early as the second week after the second dose of the BCG vaccine, and subsequently, these cells were also found to be significantly present in the spleen and the lung during the third week. Previous studies have suggested that IL-10 may have detrimental effects on the host's ability to combat M.tb infection due to its inhibitory and anti-inflammatory properties. Furthermore, studies have shown that the BCG vaccine’s protective efficacy against M.tb infection is compromised by IL-10 after a single vaccination^23–27. Specifically, IL-10 can suppress MHC II expression and impairs the ability of macrophages and DCs to present antigens^28–31, thereby decreasing the secretion of TNF-α and IL-12 and impeding the establishment of the Th1 response. In addition, lymph nodes and spleen of mice revaccinated with the BCG vaccine were found to contain IL-10⁺ Th1 cells and IL-10⁺ B cells in our study. There is evidence that IL-10 prevents Th1 cell migration from lymph nodes to infection sites, and reduces the production of antigen-specific T_EM cells and T_CM cells. Additionally, IL-10⁺ B cells may inhibit the production of Th1 cells and promote the survival of M. tb³². It has also been noted that IL-10 contributes to compromised T cell function in individuals with TB³³. Therefore, anti-IL-10R mAb for the prevention and treatment of TB has been explored extensively, which has yielded significant findings in various animal models^{24–26,34,35}. For instance, after being vaccinated with the BCG vaccine once, IL-10⁻/⁻ BALB/c mice showed increased IFN-γ⁺ CD4⁺ T cells in the lungs²⁴. Anti-IL-10R mAb inhibiting IL-10 signaling significantly increased the number of memory CD4⁺ T cells in CBA/J mice vaccinated with the BCG vaccine once³⁴. These results also highlight the importance of M.tb-specific memory CD4⁺ T cell response in protecting against TB infection, a concept that is well-established in the literature ^33,36. It has been noted that inhibiting IL-10 signaling boosts CD4⁺ Th1 cell response, leading to a marked improvement in the enduring protection conferred by the BCG vaccinated mice once against M.tb infection²⁶. Therefore, our study offers a rationale for elucidating why BCG revaccination does not provide immunity against primary M.tb infection. BCG vaccine components, like manLAM, can induce APCs to produce IL-10^32,37. Therefore, the phenomenon could be explained by the intrinsic upregulation of IL-10 production following the second dose of the BCG vaccine, thus leading to a dampened memory CD4⁺ T cell response and diminished resistance to primary M.tb infection. In addition, in accordance with previous researches^38,39, BCG revaccinated mice exhibited a heightened level of IL-17. Numerous studies have suggested that IL-17 might be involved in the pathogenesis and progression of TB, however, individuals who have received the BCG vaccine once are able to induce IL-17 and Th17 cells^38–40, which have been linked to the BCG vaccine-induced protection against primary M.tb infection^18,41. Further investigation is necessary to clarify the significance of elevated IL-17 expression in BCG revaccinated mice and its potential impact on anti-TB immunity.

Although this study confirmed previous trials in Brazil and Malawi which found no improvement in BCG efficacy in preventing primary M.tb infection by revaccination ^42–45, BCG revaccination has shown benefits in preventing LTBI in mice, and has been found to be safe and immunogenic in individuals with positive interferon-gamma release assay (IGRA⁺) results³⁹. Further research is necessary to assess the efficacy and feasibility of integrating BCG revaccination in individuals with LTBI, which can be achieved through LTBI screenings using IGRA methods in both infected and uninfected populations. Most importantly, the levels of IL-10-expressing APC cells in lymph nodes, spleen, or lungs after BCG prime-boost immunization could be performed to identify promising booster vaccine candidates, which can advance the development of adult TB vaccines.

Conflict of Interest

The authors declare no conflicts of interest.

Authors’ Contributions

Fan X designed the study. Lei Q, Yao ZJ, Fu H, Zhou ZJ and Lin XS performed experiments and collected the data. Lei Q, Yao ZJ, Wang YQ, Yuan Y, Ouyang Q, Xu XY, Cao JG and Gan MZ analyzed the data. Lei Q and Fu H draw the figures and wrote the original manuscript, and Fan X revised the manuscript.

Acknowledgments

This work was supported by grants from the Natural Science Foundation of China (No. 81971909) and the National Key R & D project of China (2022YFA1303503).

Data availability statement

All data are contained in manuscripts or supplementary information files provided.

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Early introduction of IL-10 weakens BCG revaccination's protection by suppressing CD4+Th1 cell responses

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Abstract

Figures

Introduction

Materials and methods

Mice

Ethics statement

Immunization and antibody treatment

Different cytokines mRNAs detected by qRT-PCR

Detection of IL-10 expression in lung tissues through immunohistochemistry

FACS and intracellular cytokine staining (ICS) for the detection of IL-10 positive cells

Detection of CMFO-specific T cells by ICS and FACS

Detection of CMFO specific cytokines secreted by splenocytes using CBA method

ELISA detection of CMFO-specific IgG and subclass

Evaluation of the protective effect against virulent M.tb H37Rv infection

Statistical analyses

Results

Revaccination with BCG quickly causes higher IL-10 levels in the lungs

IL-10's lung expression peaks 3–6 weeks post BCG revaccination

IL-10 is quickly induced in mouse lymph nodes and spleen via macrophages and DCs following BCG revaccination

IL-10 knockout mice enhanced antigen-specific T cell responses following BCG revaccination compared to wild-type mice

Blocking IL-10 signaling enhances BCG-revaccinated mice's resistance to primary M.tb infection

Discussion

Declarations

Conflict of Interest

Authors’ Contributions

Acknowledgments

Data availability statement

References

Supplementary Files

Status:

Version 1