Abstract
After a century of using the Bacillus Calmette–Guérin (BCG) vaccine, our understanding of its ability to provide protection against homologous (Mycobacterium tuberculosis) or heterologous (for example, influenza virus) infections remains limited. Here we show that systemic (intravenous) BCG vaccination provides significant protection against subsequent influenza A virus infection in mice. We further demonstrate that the BCG-mediated cross-protection against influenza A virus is largely due to the enrichment of conventional CD4+ effector CX3CR1hi memory αβ T cells in the circulation and lung parenchyma. Importantly, pulmonary CX3CR1hi T cells limit early viral infection in an antigen-independent manner via potent interferon-γ production, which subsequently enhances long-term antimicrobial activity of alveolar macrophages. These results offer insight into the unknown mechanism by which BCG has persistently displayed broad protection against non-tuberculosis infections via cross-talk between adaptive and innate memory responses.
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Data availability
Bulk RNA-seq data have been deposited in the Gene Expression Omnibus and are publicly available under accession number GSE246201. Data can be interactively viewed on a webserver available at http://dinglab.rimuhc.ca:8080/mcgill/Maz/kimtran/webserver/dataExpr/. Source data are provided with this paper.
Change history
01 February 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41590-024-01773-5
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Acknowledgements
We acknowledge technical help from staff at the RI-MUHC immunophenotyping platform. We thank M. J. Richer for his invaluable insight throughout this study. This work was supported by Canadian Institute of Health Research (CIHR) Project grants (168884 and 168885, to M.D.). M.D. holds a Fonds de Recherche du Québec–Santé Award and the Strauss Chair in Respiratory Diseases and is a fellow member of the Royal Society of Canada. K.A.T. is supported by a Fonds de Recherche du Québec–Santé studentship. The funders had no role in study design, data collection and analysis, the decision to publish or the preparation of the manuscript. The graphical abstract was created using BioRender.com.
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M.D. and K.A.T. conceived the project and designed the experiments. E.P., M.S., J. Downey, J.C., E.L., O.T. and E. K. performed the experiments. K.A.T., J. Ding and M.D. analyzed the data. K.A.T. and J. Ding performed RNA-seq bioinformatics analysis. K.A.T. and M.D. wrote the paper. M.D. supervised the project.
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Extended data
Extended Data Fig. 1 Reduction of viral replication and enrichment of CX3CR1hi T cells in BCG-IV mice.
(a) Confocal microscopy of vaccinated mice infected with Ruby-NS1 3 days post-infection, lung parenchyma sections (500 PFU). (b) Frequency of CD3+ and (c) CD19+ cells in blood of mice after 1 month BCG vaccination (n = 3-4) (d, e) Frequency and absolute number of CD11b+CX3CR1+ cells in the blood (n = 4). (f, g) Frequency and absolute number of CD11b+CX3CR1+ cells in the lung parenchyma (n = 4). (h, i) Frequency and absolute number of CX3CR1hi cells in the lung vasculature following 1 month BCG vaccination (n = 4-5). (j, k) Frequency and absolute number of CX3CR1hi cells in the bone marrow of mice following 1 month BCG vaccination. (n = 4-5). (l, m) Frequency and absolute number of CX3CR1hi cells in spleen of mice after 1 month BCG vaccination (n = 4-5). (n, o) Frequency and absolute number of CX3CR1hi cells in mediastinal lymph node of mice following month BCG vaccination (n = 4-5). (p, q) Kinetic of CD3+ cells and CD3+CX3CR1hi cells in blood of mice following BCG vaccination (n = 5). (r, s) Kinetic of CD3+ cells and CD3+CX3CR1hi cells in the lung parenchyma of mice following BCG vaccination (n = 5) (t, u) Frequency of CD4+ and CD8+ CX3CR1hi cells in blood and lung parenchyma following BCG vaccination (n = 5). Data are presented as mean ± s.e.m with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data were analyzed using One-way ANOVA followed by Tukey’s multiple comparisons test.
Extended Data Fig. 2 Transcriptional reprogramming of CX3CR1hi T cells.
(a) Splenocytes from BCG-IV mice were sorted in CX3CR1lo and CX3CR1hi T cells. 5 ×104 cells were stimulated for 5 hours with CD3/CD28 beads before Bioenergetic Flux Assay (Mito Stress Test) (n = 3). (b) Top enriched pathways between CX3CR1hi and CX3CR1lo T cells isolated from BCG-IV mice (n = 4). (c) Z score heatmap of T cell function and migration genes of CX3CR1hi T cells isolated from BCG-IV mice compared to CX3CR1hi and CX3CR1lo isolated from PBS-IV mice (n = 4). Data are presented as mean ± s.e.m with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data were analyzed using unpaired two-tailed t test (a), One-sided Fisher’s exact test (b) or Mann-Whitney two-tailed test (b).
Extended Data Fig. 3 Kinetics of CX3CR1hi T cells in the circulation and lungs.
(a–c) Absolute number of CD8+ CX3CR1hi T cells post IAV-PR8 infection in mice following 1 month BCG vaccination in the blood, lung parenchyma and BAL (left to right) (50 PFU; n = 4-5) (d) Pulmonary levels of CX3CL1 at day 3 post IAV-PR8 infection in mice after 1 month BCG vaccination (n = 5). (e) Frequency of CD4+ CX3CR1hi T cells in the blood mice following 2 months, 3 months and 6 months BCG-vaccination, shown as frequency of viable cells (n = 5). (f) Absolute number of CD11b+ CX3CR1+ cells in the blood and lung parenchyma of CX3CR1gfp/gfp mice after 1 month BCG vaccination (n = 3-5). (g) BCG CFUs in antibiotic-treated and BCG-vaccinated mice (n = 5). Data are presented as mean ± s.e.m with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data were analyzed using One- or Two-way ANOVA followed by Tukey’s multiple comparisons test.
Extended Data Fig. 4 BCG-induced cross-protection against IAV is mediated via bystander memory T cell activation.
(a) Absolute number of CD8+ NP+ memory T cells and (b) CD4+ NP + memory T cells at day 9 post IAV-PR8 infection in the lung parenchyma of mice following 1 month BCG vaccination (50 PFU; n = 4-5). (c, d) Frequency of IFNγ-producing NK1.1+ and CD11b+ cells at day 1 post IAV-PR8 infection in IfngeYFP mice following 1 month BCG vaccination (50 PFU; n = 4-5). (e) TNFα -producing CD4+ T cells isolated from the lung tissue of BCG vaccinated mice and stimulated in vitro with PMA/ionomycin for 5 h (n = 5). (f) Frequency of TNFα-producing splenic T cells isolated from BCG-vaccinated mice following 5 h in vitro stimulation. (n = 5) (g) Total concentration of TNFα in splenic T cells isolated from BCG-vaccinated mice after 5-hour in vitro stimulation (n = 5). (h) MFI of MHCII of AMs in the BAL following BCG vaccination and IFNγ neutralization treatment (n = 3). (i) IFNγ serum concentrations in IFNγ ARE del mice (n = 5). Data are presented as mean ± s.e.m with * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data were analyzed using One-way ANOVA followed by Tukey’s multiple comparisons test (a-h) or unpaired two-tailed t-test (i).
Extended Data Fig. 5 Gating strategy for CX3CR1hi T cells.
Single viable cells were separated for vascular leukocytes and parenchymal leukocytes, then gated for T cells (CD11b− CD3+) and CX3CR1hi. Sample obtained from 4 week-vaccinated BCG-IV mouse, mouse was intravascularly stained to discriminate between lung vasculature and parenchyma. Data was analysed by FlowJo software.
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Tran, K.A., Pernet, E., Sadeghi, M. et al. BCG immunization induces CX3CR1hi effector memory T cells to provide cross-protection via IFN-γ-mediated trained immunity. Nat Immunol 25, 418–431 (2024). https://doi.org/10.1038/s41590-023-01739-z
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DOI: https://doi.org/10.1038/s41590-023-01739-z
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