BCG Vaccination in Humans Elicits Trained Immunity via the Hematopoietic Progenitor Compartment

Branko Cirovic, L Charlotte J de Bree, Laszlo Groh, Bas A Blok, Joyce Chan, Walter J F M van der Velden, M E J Bremmers, Reinout van Crevel, Kristian Händler, Simone Picelli, Jonas Schulte-Schrepping, Kathrin Klee, Marije Oosting, Valerie A C M Koeken, Jakko van Ingen, Yang Li, Christine S Benn, Joachim L Schultze, Leo A B Joosten, Nigel Curtis, Mihai G Netea, Andreas Schlitzer, Branko Cirovic, L Charlotte J de Bree, Laszlo Groh, Bas A Blok, Joyce Chan, Walter J F M van der Velden, M E J Bremmers, Reinout van Crevel, Kristian Händler, Simone Picelli, Jonas Schulte-Schrepping, Kathrin Klee, Marije Oosting, Valerie A C M Koeken, Jakko van Ingen, Yang Li, Christine S Benn, Joachim L Schultze, Leo A B Joosten, Nigel Curtis, Mihai G Netea, Andreas Schlitzer

Abstract

Induction of trained immunity by Bacille-Calmette-Guérin (BCG) vaccination mediates beneficial heterologous effects, but the mechanisms underlying its persistence and magnitude remain elusive. In this study, we show that BCG vaccination in healthy human volunteers induces a persistent transcriptional program connected to myeloid cell development and function within the hematopoietic stem and progenitor cell (HSPC) compartment in the bone marrow. We identify hepatic nuclear factor (HNF) family members 1a and b as crucial regulators of this transcriptional shift. These findings are corroborated by higher granulocyte numbers in BCG-vaccinated infants, HNF1 SNP variants that correlate with trained immunity, and elevated serum concentrations of the HNF1 target alpha-1 antitrypsin. Additionally, transcriptomic HSPC remodeling was epigenetically conveyed to peripheral CD14+ monocytes, displaying an activated transcriptional signature three months after BCG vaccination. Taken together, transcriptomic, epigenomic, and functional reprogramming of HSPCs and peripheral monocytes is a hallmark of BCG-induced trained immunity in humans.

Trial registration: ClinicalTrials.gov NCT01906853.

Keywords: BCG; epigenetic imprinting; hematopoietic stem cells; monocyte; myeloid cell; myelopoiesis; trained immunity; vaccination.

Conflict of interest statement

Declaration of Interests M.G.N. and L.A.B.J. are scientific founders of Trained Therapeutics Discovery. All other authors declare no competing interests.

Copyright © 2020 Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
BCG Vaccination Elicits Trained Innate Immunity in Healthy Individuals (A) Study design including the experimental arm with BCG-vaccinated healthy individuals (n = 15) and placebo-treated control arm (diluent-treated, n = 5). Blood and BM aspirations were analyzed before (D0), two weeks (D14, blood only), and three months (D90) after vaccination. (B) Cytokine measurement in supernatants of PBMC challenged ex vivo with C. albicans for 24 h. Data are presented as mean and SD. Fold changes relative to baseline (D0) are shown. Mann-Whitney test was used to compare fold induction at D90 in BCG versus controls (∗p < 0.05). Rectangular symbol indicates a single data point exceeding axis limits and the actual value next to it. (C) Hallmark GSEA of transcriptomic data derived from the adherent PBMC fraction prepared before (D0) and after (D90) vaccination from the same individuals and treated ex vivo with C. albicans for 24 h (n = 5 per group). NES, normalized enrichment score; pval, p value; padj, adjusted p value. Top ten hallmark pathways enriched after vaccination are listed. (D) Enrichment plots from GSEA for the two significantly enriched terms after BCG vaccination (see C). See also Tables 1 and S1.
Figure 2
Figure 2
BCG Does Not Alter the Composition of Immune Cells and Progenitors in Blood and BM (A) Whole blood counts of neutrophils, lymphocytes, and monocytes as fold change relative to D0. (B and D) Uniform manifold approximation and projection (UMAP) representation of analyzed cell lineages of the alive Lin (CD3, CD7, CD10, CD15, CD19, and CD20)− CD45+ compartment in PBMC (B) and Lin (CD3, CD7, CD15, CD19, and CD20)− CD45+ BM MNC (D) (representative donor, 5 × 105 sampled cells). (C and E) Quantification of cell types depicted in (B) and (D) as proportion of Lin−CD45+ relative to D0 for all time points (mean and SD; D0/D90, BCG, n = 15, Ctrl, n = 5; D14, BCG, n = 7, Ctrl, n = 2). Unpaired t test was used in (A), (C), and (E) to compare BCG versus Ctrl D90 (all p values > 0.05). See also Figure S1.
Figure 3
Figure 3
BCG Vaccination Induces Persistent Changes in BM HSPCs (A) Heatmap visualizing Euclidean distance measurement of transcript abundance between HSPC transcriptomes from D90 (Ctrl, n = 5; BCG, n = 15). (B) Expression heatmap of DEGs in HSPCs (BCG, D0, n = 14 versus D90, n = 15). Samples are separated in columns based on time point and experimental arm. Pearson correlation is used as distance measure to cluster genes in rows. Names of a subset of genes previously associated with trained immunity or myeloid biology are shown. For the statistical details, see STAR Methods. See also Figure S2 and Tables S2 and S3.
Figure 4
Figure 4
BCG Vaccination Elicits Myeloid Cell Fate Priming (A) GOEA of upregulated genes in HSPCs. Top ten enriched terms (designated to letters “a” to “j”) are listed (BCG, D0, n = 14 versus D90, n = 15). (B) Network representation of GO terms significantly enriched in upregulated DEGs in HSPCs (BCG, D0 versus D90, p 9/L) of infants in the MIS BAIR study (BCG-vaccinated, n=385; BCG-naive, n = 409; ∗p < 0.05). See also Tables S4–S7.
Figure 5
Figure 5
HNF1 TFs Represent Regulatory Factors in Trained Immunity (A) GSEA of enriched signatures associated with distinct TF-binding motifs in genes ranked according to expression fold change in HSPCs at D0 versus D90. TFs associated with the binding motifs are listed. NES, normalized enrichment score; pval, p value; padj, adjusted p value. (B) Expression heatmap of all leading-edge genes derived from TF-binding site gene sets as found in (A). Black lines in the first six columns indicate association of genes to the respective TF signature gene sets listed in the column header. (C) Ex vivo training of PBMCs with β-glucan or BCG and IL6/TNF cytokine measurements after LPS challenge. SNPs ± 250 kb within the genomic locus of HNF1A (green) or HNF1B (purple) and variants thereof significantly leading to a change in cytokine production for any of the conditions are listed. Threshold for inclusion, p < 0.01. Top two expression quantitative trait loci (eQTLs) are annotated as pink and bright blue. (D) Detailed TNF cytokine level after BCG training and LPS challenge for the top two eQTLs (annotated in C as pink and bright blue). Number of individuals displaying distinct SNP variants are shown in brackets. Analysis in (C) and (D) is based on 141 individuals in total. (E) Fold changes of IL1B after ex vivo C. albicans restimulation and serum SERPINA1 (D0 versus D90) correlated in the same individuals. Data points from BCG-vaccinated or Ctrl-treated individuals are shown in black or gray, respectively. See also Figure S3 and Table S8.
Figure 6
Figure 6
Epigenetic Changes in Blood Monocytes Are a Feature of BCG-Induced Training (A) PCA based on normalized peak counts of open regions derived from ATAC-seq in peripheral blood CD14+ monocytes. Open or filled symbols indicate D0 or D90 time points of BCG-vaccinated individuals, respectively. Oval shapes denote confidence intervals at 0.85 for the two groups. (B) Heatmap of normalized ATAC-seq reads focusing on the core set of 47 differentially accessible regions annotated according to the closest gene. Peaks are included reaching an adjusted p value (padj) CXCL6 region. Called peaks are visualized in violet, differentially accessible (DA) peaks in red and identified TF-binding sites (TFBS) within called peaks in yellow. Summarized reads from CD14+ monocytes before and after vaccination are shown in blue and green, respectively. (D) ATAC-seq heatmap of 909 peaks within promoters closest to genes transcriptionally upregulated in HSPCs after 3 months from the vaccinated group (n = 13 per group). See also Figure S4 and Tables S9, S10, and S11.

References

    1. Aaby P., Roth A., Ravn H., Napirna B.M., Rodrigues A., Lisse I.M., Stensballe L., Diness B.R., Lausch K.R., Lund N. Randomized trial of BCG vaccination at birth to low-birth-weight children: beneficial nonspecific effects in the neonatal period? J. Infect. Dis. 2011;204:245–252.
    1. Armendariz A.D., Krauss R.M. Hepatic nuclear factor 1-alpha: inflammation, genetics, and atherosclerosis. Curr. Opin. Lipidol. 2009;20:106–111.
    1. Arts R.J.W., Moorlag S.J.C.F.M., Novakovic B., Li Y., Wang S.Y., Oosting M., Kumar V., Xavier R.J., Wijmenga C., Joosten L.A.B. BCG vaccination protects against experimental viral infection in humans through the induction of cytokines associated with trained immunity. Cell Host Microbe. 2018;23:89–100.e5.
    1. Assarsson E., Lundberg M., Holmquist G., Björkesten J., Thorsen S.B., Ekman D., Eriksson A., Rennel Dickens E., Ohlsson S., Edfeldt G. Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability. PLoS One. 2014;9:e95192.
    1. Baldwin A.S. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu. Rev. Immunol. 1996;14:649–683.
    1. Bankoti R., Ogawa C., Nguyen T., Emadi L., Couse M., Salehi S., Fan X., Dhall D., Wang Y., Brown J. Differential regulation of effector and regulatory T cell function by Blimp1. Sci. Rep. 2017;7:12078.
    1. Becht E., McInnes L., Healy J., Dutertre C.-A., Kwok I.W.H., Ng L.G., Ginhoux F., Newell E.W. Dimensionality reduction for visualizing single-cell data using UMAP. Nat Biotechnol. 2019;37:38–44.
    1. Bekkering S., Blok B.A., Joosten L.A., Riksen N.P., van Crevel R., Netea M.G. In vitro experimental model of trained innate immunity in human primary monocytes. Clin. Vaccine Immunol. 2016;23:926–933.
    1. Bekkering S., Arts R.J.W., Novakovic B., Kourtzelis I., van der Heijden C.D.C.C., Li Y., Popa C.D., Ter Horst R., van Tuijl J., Netea-Maier R.T. Metabolic induction of trained immunity through the mevalonate pathway. Cell. 2018;172:135–146.e9.
    1. Benn C.S., Netea M.G., Selin L.K., Aaby P. A small jab - a big effect: nonspecific immunomodulation by vaccines. Trends Immunol. 2013;34:431–439.
    1. Biering-Sørensen S., Aaby P., Lund N., Monteiro I., Jensen K.J., Eriksen H.B., Schaltz-Buchholzer F., Jørgensen A.S.P., Rodrigues A., Fisker A.B., Benn C.S. Early BCG-Denmark and neonatal mortality among infants weighing <2500 g: a randomized controlled trial. Clin. Infect. Dis. 2017;65:1183–1190’.
    1. Cheng S.C., Quintin J., Cramer R.A., Shepardson K.M., Saeed S., Kumar V., Giamarellos-Bourboulis E.J., Martens J.H.A., Rao N.A., Aghajanirefah A. mTOR- and HIF-1α-mediated aerobic glycolysis as metabolic basis for trained immunity. Science. 2014;345:1250684.
    1. Christ A., Günther P., Lauterbach M.A.R., Duewell P., Biswas D., Pelka K., Scholz C.J., Oosting M., Haendler K., Baßler K. Western diet triggers NLRP3-dependent innate immune reprogramming. Cell. 2018;172:162–175.e14.
    1. Clark I.A., Allison A.C., Cox F.E. Protection of mice against Babesia and Plasmodium with BCG. Nature. 1976;259:309–311.
    1. Coombes J.L., Siddiqui K.R., Arancibia-Cárcamo C.V., Hall J., Sun C.M., Belkaid Y., Powrie F. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism. J. Exp. Med. 2007;204:1757–1764.
    1. Crișan T.O., Netea M.G., Joosten L.A. Innate immune memory: implications for host responses to damage-associated molecular patterns. Eur. J. Immunol. 2016;46:817–828.
    1. Freyne B., Marchant A., Curtis N. BCG-associated heterologous immunity, a historical perspective: intervention studies in animal models of infectious diseases. Trans. R. Soc. Trop. Med. Hyg. 2015;109:52–61.
    1. Gong Y., Ma Z., Patel V., Fischer E., Hiesberger T., Pontoglio M., Igarashi P. HNF-1beta regulates transcription of the PKD modifier gene Kif12. J. Am. Soc. Nephrol. 2009;20:41–47.
    1. Higgins J.P.T., Soares-Weiser K., López-López J.A., Kakourou A., Chaplin K., Christensen H., Martin N.K., Sterne J.A.C., Reingold A.L. Association of BCG, DTP, and measles containing vaccines with childhood mortality: systematic review. BMJ (Clin. Res. Ed.) 2016;355:i5170.
    1. Jaensson E., Uronen-Hansson H., Pabst O., Eksteen B., Tian J., Coombes J.L., Berg P.L., Davidsson T., Powrie F., Johansson-Lindbom B., Agace W.W. Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans. J. Exp. Med. 2008;205:2139–2149.
    1. Jensen K.J., Larsen N., Biering-Sørensen S., Andersen A., Eriksen H.B., Monteiro I., Hougaard D., Aaby P., Netea M.G., Flanagan K.L., Benn C.S. Heterologous immunological effects of early BCG vaccination in low-birth-weight infants in Guinea-Bissau: a randomized-controlled trial. J. Infect. Dis. 2015;211:956–967.
    1. Jovic S., Linge H.M., Shikhagaie M.M., Olin A.I., Lannefors L., Erjefält J.S., Mörgelin M., Egesten A. The neutrophil-recruiting chemokine GCP-2/CXCL6 is expressed in cystic fibrosis airways and retains its functional properties after binding to extracellular DNA. Mucosal Immunol. 2016;9:112–123.
    1. Kaufmann E., Sanz J., Dunn J.L., Khan N., Mendonça L.E., Pacis A., Tzelepis F., Pernet E., Dumaine A., Grenier J.C. BCG educates hematopoietic stem cells to generate protective innate immunity against tuberculosis. Cell. 2018;172:176–190.e19.
    1. Kleinnijenhuis J., Quintin J., Preijers F., Joosten L.A.B., Ifrim D.C., Saeed S., Jacobs C., van Loenhout J., de Jong D., Stunnenberg H.G. Bacille calmette-guerin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes. Proc. Natl. Acad. Sci. USA. 2012;109:17537–17542.
    1. Kleinnijenhuis J., Quintin J., Preijers F., Benn C.S., Joosten L.A.B., Jacobs C., van Loenhout J., Xavier R.J., Aaby P., van der Meer J.W.M. Long-lasting effects of BCG vaccination on both heterologous Th1/Th17 responses and innate trained immunity. J. Innate Immun. 2014;6:152–158.
    1. Lawrence T. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb. Perspect. Biol. 2009;1:a001651.
    1. Linge H.M., Collin M., Nordenfelt P., Mörgelin M., Malmsten M., Egesten A. The human CXC chemokine granulocyte chemotactic protein 2 (GCP-2)/CXCL6 possesses membrane-disrupting properties and is antibacterial. Antimicrob. Agents Chemother. 2008;52:2599–2607.
    1. McInnes L., Healy J. UMAP: uniform manifold approximation and projection for dimension reduction. arXiv. 1802 arXiv:1802.03426v2.
    1. Merad M., Sathe P., Helft J., Miller J., Mortha A. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu. Rev. Immunol. 2013;31:563–604.
    1. Mitroulis I., Ruppova K., Wang B., Chen L.S., Grzybek M., Grinenko T., Eugster A., Troullinaki M., Palladini A., Kourtzelis I. Modulation of myelopoiesis progenitors is an integral component of trained immunity. Cell. 2018;172:147–161.e12.
    1. Moraes-Vieira P.M., Yore M.M., Dwyer P.M., Syed I., Aryal P., Kahn B.B. RBP4 activates antigen-presenting cells, leading to adipose tissue inflammation and systemic insulin resistance. Cell Metab. 2014;19:512–526.
    1. Murphy J.R. Host defenses in murine malaria: nonspecific resistance to Plasmodium berghei generated in response to Mycobacterium bovis infection or Corynebacterium parvum stimulation. Infect. Immun. 1981;33:199–211.
    1. Netea M.G., Quintin J., van der Meer J.W.M. Trained immunity: A memory for innate host defense. Cell Host Microbe. 2011;9:355–361.
    1. Netea M.G., Joosten L.A., Latz E., Mills K.H., Natoli G., Stunnenberg H.G., O’Neill L.A., Xavier R.J. Trained immunity: A program of innate immune memory in health and disease. Science. 2016;352:aaf1098.
    1. Netea M.G., Joosten L.A.B., van der Meer J.W.M. Hypothesis: stimulation of trained immunity as adjunctive immunotherapy in cancer. J. Leukoc. Biol. 2017;102:1323–1332.
    1. Picelli S., Faridani O.R., Björklund A.K., Winberg G., Sagasser S., Sandberg R. Full-length RNA-seq from single cells using smart-seq2. Nat Protoc. 2014;9:171–181.
    1. Rasmussen A.L., Wang I.M., Shuhart M.C., Proll S.C., He Y., Cristescu R., Roberts C., Carter V.S., Williams C.M., Diamond D.L. Chronic immune activation is a distinguishing feature of liver and PBMC gene signatures from HCV/HIV coinfected patients and may contribute to hepatic fibrogenesis. Virology. 2012;430:43–52.
    1. Rieckmann A., Villumsen M., Sørup S., Haugaard L.K., Ravn H., Roth A., Baker J.L., Benn C.S., Aaby P. Vaccinations against smallpox and tuberculosis are associated with better long-term survival: a Danish case-cohort study 1971-2010. Int. J. Epidemiol. 2017;46:695–705.
    1. Rollini P., Fournier R.E. The HNF-4/HNF-1alpha transactivation cascade regulates gene activity and chromatin structure of the human serine protease inhibitor gene cluster at 14q32.1. Proc. Natl. Acad. Sci. USA. 1999;96:10308–10313.
    1. Saeed S., Quintin J., Kerstens H.H.D., Rao N.A., Aghajanirefah A., Matarese F., Cheng S.C., Ratter J., Berentsen K., van der Ent M.A. Epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity. Science. 2014;345:1251086.
    1. Sander J., Schmidt S.V., Cirovic B., McGovern N., Papantonopoulou O., Hardt A.L., Aschenbrenner A.C., Kreer C., Quast T., Xu A.M. Cellular differentiation of human monocytes is regulated by time-dependent interleukin-4 signaling and the transcriptional regulator NCOR2. Immunity. 2017;47:1051–1066.e12.
    1. Savelkoul P.H., Catsburg A., Mulder S., Oostendorp L., Schirm J., Wilke H., van der Zanden A.G., Noordhoek G.T. Detection of Mycobacterium tuberculosis complex with real time PCR: comparison of different primer-probe sets based on the IS6110 element. J. Microbiol. Methods. 2006;66:177–180.
    1. Siddiqui K.R.R., Powrie F. CD103+ GALT DCs promote Foxp3+ regulatory T cells. Mucosal Immunol. 2008;1:S34–S38.
    1. Storgaard L., Rodrigues A., Martins C., Nielsen B.U., Ravn H., Benn C.S., Aaby P., Fisker A.B. Development of BCG scar and subsequent morbidity and mortality in rural Guinea-Bissau. Clin. Infect. Dis. 2015;61:950–959.
    1. Sun J.C., Lopez-Verges S., Kim C.C., DeRisi J.L., Lanier L.L. NK cells and immune “memory”. J. Immunol. 2011;186:1891–1897.
    1. Ter Horst R., Jaeger M., Smeekens S.P., Oosting M., Swertz M.A., Li Y., Kumar V., Diavatopoulos D.A., Jansen A.F.M., Lemmers H. Host and environmental factors influencing individual human cytokine responses. Cell. 2016;167:1111–1124.e13.
    1. Varol C., Mildner A., Jung S. Macrophages: development and tissue specialization. Annu. Rev. Immunol. 2015;33:643–675.
    1. Villumsen M., Sørup S., Jess T., Ravn H., Relander T., Baker J.L., Benn C.S., Sørensen T.I.A., Aaby P., Roth A. Risk of lymphoma and leukaemia after Bacille Calmette-Guérin and smallpox vaccination: a Danish case-cohort study. Vaccine. 2009;27:6950–6958.
    1. Walk J., de Bree L.C.J., Graumans W., Stoter R., van Gemert G.J., van de Vegte-Bolmer M., Teelen K., Hermsen C.C., Arts R.J.W., Behet M.C. Outcomes of controlled human malaria infection after BCG vaccination. Nat. Commun. 2019;10:874.
    1. Zimmermann P., Finn A., Curtis N. Does BCG vaccination protect against nontuberculous mycobacterial infection? A systematic review and meta-analysis. J. Infect. Dis. 2018;218:679–687.

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