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Abstract

Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (TFH cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting TFH cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust TFH cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of TFH cell differentiation and a promising target for TFH cell–skewing vaccine adjuvants.

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Acknowledgements

We thank D. Kunkel, S. Warth and A. Linke for technical advice; the flow cytometry facility of the Berlin Brandenburg Center for Regenerative Therapies (BCRT) of the Charité Berlin; S. Springer and IDT Biologika for assistance in planning the animal studies and for the Salmoporc STM vaccine; and R. Nifosí for critical review of the modelling analysis. Supported by the German Research Council (DFG grant SA1940-2/1 and SFB-TR84 TP C8 to L.E.S.; SFB-TR84 TP B1 to N.S.; SFB-TR-84 TP A1/A5 to B.O.; SFB-TR84 TP Z1b to A.D.G.; DFG-GRK 1673 project to R.R.S.; and DFG-GRK 2046, TP4 to S.H.), the European Research Council and the Federal Ministry of Education and Research (FP-7 ERA-NET / Infect-ERA consortium “HaploINFECT” / BMBF 031A402A to L.E.S., VIP + VALNEMCYS project and InfectControl 2020, consortium Art4Fun to S.H.), the European Society of Clinical Microbiology and Infectious Diseases (ESCMID research grant to L.E.S.), the Jürgen Manchot Foundation (Doctoral Research Fellowship to P.G., E.T.H. and S.M.V.), the Netherlands Nutrigenomics Center, Wageningen University, The Netherlands (to M.M. and M.B.), the Fritz Thyssen Foundation (research grant to A.H.), The Danish National Research Foundation (grant no. DNRF108 to) to Research Centre for Vitamins and Vaccines (CVIVA) supporting K.J.J., and The Novo Nordisk Foundation supporting the pig vaccination experiments at Technical University of Denmark, Federal Ministry of Education and Research.

Author information

Author notes

  1. These authors contributed equally: Matteo Ugolini, Jenny Gerhard and Sanne Burkert.

Affiliations

  1. Department of Infectious Diseases and Pulmonary Medicine, Charité–Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany

    • Matteo Ugolini
    • , Jenny Gerhard
    • , Philipp Georg
    • , Sarah M. Volkers
    • , Elisa T. Helbig
    • , Bastian Opitz
    • , Florian Kurth
    • , Norbert Suttorp
    •  & Leif E. Sander
  2. Institute of Microbiology and Hygiene, Charité–Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany

    • Sanne Burkert
    • , Shruthi Thada
    • , Saubashya Sur
    • , Nickel Dittrich
    •  & Ralf R. Schumann
  3. Research Center for Vitamins and Vaccines, Bandim Health Project, Statens Serum Institut, Copenhagen, Denmark

    • Kristoffer Jarlov Jensen
    •  & Christine S. Benn
  4. Department of Biotechnology and Biomedicine, Technical University of Denmark, Kgs Lyngby, Denmark

    • Kristoffer Jarlov Jensen
    •  & Gregers Jungersen
  5. Department of Veterinary Medicine, Institute of Immunology, Freie Universität Berlin, Berlin, Germany

    • Friederike Ebner
    •  & Susanne Hartmann
  6. Bhagwan Mahavir Medical Research Centre, Hyderabad, India

    • Shruthi Thada
    •  & Sumanlatha Gaddam
  7. Department of Veterinary Medicine, Institute of Veterinary Pathology, Freie Universität Berlin, Berlin, Germany

    • Kristina Dietert
    •  & Achim D. Gruber
  8. Chronic Immune Reactions, German Rheumatism Research Centre, a Leibniz Institute, Berlin, Germany

    • Laura Bauer
    •  & Andreas Hutloff
  9. Institute of Immunology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald—Island of Riems, Germany

    • Alexander Schäfer
    •  & Ulrike Blohm
  10. German Center for Lung Research (DZL), Berlin, Germany

    • Bastian Opitz
    • , Norbert Suttorp
    •  & Leif E. Sander
  11. Department of Genetics, Osmania University, Hyderabad, India

    • Sumanlatha Gaddam
  12. Department of Internal Medicine and Dermatology, Division of Psychosomatic Medicine, Charite–Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany

    • Melanie L. Conrad
  13. Odense Patient Data Explorative Network (OPEN), Odense University Hospital/Department of Clinical Research, University of Southern Denmark, Odense, Denmark

    • Christine S. Benn
  14. Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands

    • Mark V. Boekschoten
    •  & Michael Müller
  15. Norwich Medical School, University of East Anglia, Norwich, UK

    • Michael Müller

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Contributions

M.U. and J.G. designed and performed experiments; S.B. analyzed clinical data and performed experiments; K.J.J., C.S.B., G.J. and L.E.S. planned and performed animal studies; P.G., F.E., S.M.V., S.T., K.D., L.B. and E.T.H. performed experiments; M.U., J.G., P.G., F.E., S.M.V., K.D., B.O., F.K., A.D.G., A.H., S.H., M.M., N.S. and L.E.S. analyzed data and wrote the manuscript; S.S. performed structure prediction analysis; N.D., S.G., M.L.C. and R.R.S. designed and performed clinical studies; A.S. and U.B. provided samples and conceptual input; M.V.B. performed transcriptome analysis; and L.E.S. conceived of the study and designed experiments.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Leif E. Sander.

Integrated supplementary information

  1. Supplementary Figure 1 Recognition of bacterial viability induces TFH cells.

    (a) Human monocytes were stimulated with medium (ctrl), live E. coli (EC) or heat killed E. coli (HKEC) and co-cultured with autologous naïve CD4+ T cells in the presence of SEB. Expression of CXCR5, ICOS and PD-1 was measured by flow cytometry. Representative FACS plots of data shown in Fig. 1d. Numbers indicate gate frequencies as ‘% of parent population’ (in black) or as ‘% of total T cells’ (in red) (b) Same samples as in Figure 1h. Flourescence intensity values (FI) corrected for background without subtraction of control FI. (c-f) CD4+ T cells were co-cultured with APC as described in Fig. 1. T cells were sorted based on CXCR5 expression on day 5 and subsequently co-cultured with autologous naïve B cells for 7 days. Plasmablast differentiation (c, d) and Ig class-switching (e, f) measured in cultures containing only B cells, B cells + CXCR5+ T cells, and B cells + CXCR5 T cells (n=4). One-way ANOVA with post hoc correction for multiple comparison. Error bars are mean ± SEM (*; p<0.05, **; p<0.01).

  2. Supplementary Figure 2 IL-12 and in parts IL-1β, but not IFN-β are required for EC-induced TFH cell differentiation.

    (a) Proliferation of CD4+ T cells cultured as in Figure 4 in the presence of conditioned APC supernatants was measured by flow cytometry (CFSE dilution). (b, c) Same samples as in Figure 4a,b respectively. IL-17A production was measured by flow cytometry (b, n=28) and ELISA (c, n=10). (d-f) CD4+ T cells were cultured and stimulated as in Figure 3d (aIL-12; anti-IL-12 antibodies etc., rIL-12; recombinant IL-12 etc.). Bcl6 and IL-21 co-expression were measured by flow cytometry (d, n=4) and quantified (e, n=4), IL-21 production was quantified by ELISA (f, n=7). One-way ANOVA with post hoc correction for multiple comparison. Error bars are mean ± SEM. (*; p<0.05, **; p<0.01).

  3. Supplementary Figure 3 Endosomal receptors are required for the production of IL-12 in response to EC.

    (a) Human monocytes were left untreated (-) or treated with cytochalsin-D (CytD) to block actin polymerization and phagocytosis, or with bafilomycin A (Baf) to inhibit phagolysosomal acidification. Cells were subsequently stimulated with EC or HKEC. IL-6 and IL-12p40 release was measured by ELISA (n=3). (b) Expression of genes encoding for TLR1-9 was measured by qPCR in purified human monocytes, and expressed semi-quantitatively as Ct-ratio of house keeping gene beta-actin/Tlr (n=2). (c) Human monocytes were left untreated (ctrl), treated with pLA or pLA plus bacterial RNA (pLA+RNA) and upregulation of activation markers was measured by flow cytometry (n=5). Error bars are mean ± SEM.

  4. Supplementary Figure 4 Bacterial RNA induces BCL6/IL-21 expression, ST and HKST equally induce TBET and FOXP3 expression.

    (a) Purified CD14+ porcine monocytes were stimlated with bacterial RNA complexed with pLa, Pam3CSK4 (200ng/ml) or LPS (2ug/ml). Supernatatnts were collected after 24h and analyzed by ELISA (n=3, 4). Error bars, maximum and minimum (*; p<0.05, **; p<0.01, ***; p<0.001). (b, c) Tbet/FoxP3 expression was detected by flow cytometry in CD3+CD4+ T cells or CD3+CD4+CD8+ T cells from pigs immunized subcutaneously with saline (ctrl), live attenuated S. enterica serovar Typhimurium vaccine (ST) or heat killed ST (HKST). (d, e) Quantification of (b) and (c) respectively (n=6,5,5). One-way ANOVA with post hoc correction for multiple comparison. Error bars are mean ± SEM. Error bars are mean ± SEM. (*; p<0.05).

  5. Supplementary Figure 5 Vaccination with ST induces B cell follicles with active germinal centers.

    (a) Sections of paraffin embedded spleen tissues were stained for KI67. Scale bars: upper panels; 5mm; lower panels; 500µm (b) Sections of paraffin embedded lymph nodes were stained for PAX5, KI67 and BCL-2. Scale bar= 200µm. (c) Co-immunofluorescence staining of PAX5 (red) and KI67 (green) on spleen section of pigs vaccinated with ST, HKST, or saline injected control animals (ctrl). Nuclei were counterstained with DAPI (blue). Control sections stained only with DAPI to control for autofluorescence are shown in the bottom row. Note the high level of autofluorescence of the red pulp (erythrocytes), whereas no interfering autofluorescence signal was detected in the lymphoid follicles. Scale bar; 50µm.

  6. Supplementary Figure 6 Structure prediction models of TLR8-A and TLR8-G.

    (a, b) Ribbon diagram of 3D structures for TLR8 Isoform B TLR8-A variant (a) and Isoform B TLR8-G truncated variant (b). (c, d) Surface charge distribution of TLR8 Isoform B TLR8-A variant (c) and Isoform B TLR8-G truncated variant (d). Negatively and positively charged surface areas are colored red and blue.

  7. Supplementary Figure 7 Altered clefts and cavities in the TLR8-G variant.

    (a, b) CASTp analysis showing 148 functional pockets of TLR8 Isoform B TLR8-A variant (a) and 135 for Isoform B TLR8-G truncated variant (b). Pockets are coloured differently. (c, d) Rear view of TLR8 structures. Isoform B TLR8-A variant (c) and Isoform B TLR8-G truncated variant (d) showing alteration of residues in pockets associated with dimerization. Surface diagram showing major functional clefts and cavities in TLR8. (e,f) Isoform B TLR8-A variant (e) and Isoform B TLR8-G truncated variant (f). Colours represent different clefts arranged according to volume from largest to smallest. The TLR8-G variant is 42.9% rigid (i.e., more flexible) compared to 58% for TLR8-A. (g) Comparison of the volumes of clefts between the two gene variants.

  8. Supplementary Figure 8 TLR8-G displays a slight gain-of-function phenotype.

    PBMC from healthy donors, screened for either TLR8-A or TLR8-G, variant stimulated with (a) the TLR8 agonist CL075 (at the indicated concentrations), (b) Mycobacterium tuberculosis RNA or LPS as a negative control for 18h. Cytokine release was analyzed by ELISA (n=16, TLR8-A n=7, TLR8-G n=9). (c) HEK-Blue Null-I reporter cells were stably transfected with either TLR8-A or TLR8-G variants and stimulated with TLR8 agonist (CL075, R848) or LPS as a negative control for 18h. NF-kB-activation was determined in the SEAP Reporter Gene Assay. Induction values were normalized to stimulation with saline. (n=6). Two-way ANOVA with post hoc correction for multiple comparisons. Error bars are mean ± SEM. (**; p<0.01, ****; p<0.0001).

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https://doi.org/10.1038/s41590-018-0068-4