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Antigen-specific B cells direct T follicular-like helper cells into lymphoid follicles to mediate Mycobacterium tuberculosis control

Nature Immunology volume 24, pages 855–868 (2023)Cite this article

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Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a global cause of death. Granuloma-associated lymphoid tissue (GrALT) correlates with protection during TB, but the mechanisms of protection are not understood. During TB, the transcription factor IRF4 in T cells but not B cells is required for the generation of the TH1 and TH17 subsets of helper T cells and follicular helper T (TFH)-like cellular responses. A population of IRF4+ T cells coexpress the transcription factor BCL6 during Mtb infection, and deletion of Bcl6 (Bcl6fl/fl) in CD4+ T cells (CD4cre) resulted in reduction of TFH-like cells, impaired localization within GrALT and increased Mtb burden. In contrast, the absence of germinal center B cells, MHC class II expression on B cells, antibody-producing plasma cells or interleukin-10-expressing B cells, did not increase Mtb susceptibility. Indeed, antigen-specific B cells enhance cytokine production and strategically localize TFH-like cells within GrALT via interactions between programmed cell death 1 (PD-1) and its ligand PD-L1 and mediate Mtb control in both mice and macaques.

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Fig. 1: Analysis of IRF4 expression in animal models and in human tuberculosis.
Fig. 2: Irf4 expression in T cells is indispensable for Mtb control.
Fig. 3: IRF4+ BCL6+ T cells increase during Mtb infection.
Fig. 4: TFH-like cells but not germinal center B cells mediate control of Mtb.
Fig. 5: Dispensable B cell effector functions in Mtb control.
Fig. 6: Mtb-specific B cells control chronic Mtb infection.
Fig. 7: PD-1+ TFH-like cells localize within GrALT for Mtb control.
Fig. 8: B cells mediate control in MtbΔsigH-vaccinated macaques.

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Data availability

Bulk RNA-seq data are available in the NCBI GEO with accession numbers GSE66578 and SRX960261–SRX960267. All macaque bulk RNA-seq reads are available in the NCBI Sequence Read Archive, Bioproject PRJNA523820. scRNA-seq data for B6 mice are available on the GEO under accession number GSE200639. Source data are provided with this paper.

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Acknowledgements

This work was supported by Washington University in St. Louis and University of Chicago; NIH grants HL105427 and AI111914 to S.A.K.; AI123780 to S.A.K., D.K., T.S. and M.M.; AI134236 to S.A.K. and D.K.; R01AI134240, R01AI138587, P51OD011133, P51OD011104, U42OD010442, S10OD028732 and C06OD030079 to D.K.; and NIH grant AR069655 (Center for Musculoskeletal Research, University of Rochester). We thank M. Veschak, M. D. Dunlap and S. Thirunavukkarasu of Washington University of St. Louis, Department of Molecular Microbiology, and A. Seeger and L. Reese of University of Rochester, Department of Surgical Pathology for technical support and assistance.

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Author notes

  1. Ananya Gupta, Sadia Akter, Mushtaq Ahmed & Shabaana A. Khader

    Present address: Department of Microbiology, University of Chicago, Chicago, IL, USA

  2. Taylor W. Foreman

    Present address: AstraZeneca, Washington DC–Baltimore, MD, USA

  3. These authors contributed equally: Rosemary V. Swanson, Ananya Gupta.

Authors and Affiliations

  1. Department of Molecular Microbiology, Washington University in St. Louis, St. Louis, MO, USA

    Rosemary V. Swanson, Ananya Gupta, Lan Lu, Sadia Akter, Shibali Das, Mushtaq Ahmed & Shabaana A. Khader

  2. Divisions of Bacteriology and Parasitology, Tulane National Primate Research Center, Covington, LA, USA

    Taylor W. Foreman & Smriti Mehra

  3. Laboratory of Immunobiology and Genetics, Instituto Nacional de Enfermedades Respiratorias Ismael Cosio Villegas, Mexico City, Mexico

    Jose Alberto Choreno-Parra & Joaquin Zuniga

  4. South African Tuberculosis Vaccine Initiative (SATVI), Institute of Infectious Disease and Molecular Medicine and Division of Immunology, Department of Pathology, University of Cape Town, Cape Town, South Africa

    Stanley Kimbung Mbandi & Thomas J. Scriba

  5. Division of Infectious Diseases, Department of Internal Medicine, Washington University in St. Louis, St. Louis, MO, USA

    Bruce A. Rosa & Makedonka Mitreva

  6. McDonnell Genome Institute, Washington University in St. Louis, St. Louis, MO, USA

    Bruce A. Rosa & Makedonka Mitreva

  7. Division of Allergy, Immunology and Rheumatology, Department of Medicine, University of Rochester Medical Center, Rochester, NY, USA

    Maria de la Luz Garcia-Hernandez & Javier Rangel-Moreno

  8. Southwest National Primate Research Centre (SNPRC) at Texas Biomedical Research Institute, San Antonio, TX, USA

    Dhiraj K. Singh, Smriti Mehra & Deepak Kaushal

  9. Department of Pathology and Immunology, Washington University in St. Louis, St. Louis, MO, USA

    Ekaterina Esaulova & Maxim N. Artyomov

  10. St. George Campus, University of Toronto, Toronto, Ontario, Canada

    Jennifer Gommerman

  11. Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Campus Mexico City, Mexico

    Joaquin Zuniga

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Contributions

R.V.S., A.G., D.K. and S.A.K. designed experiments, analyzed data, constructed figures and wrote the manuscript. D.K. and S.A.K. initiated the study and supervised all aspects. R.V.S. and A.G. performed the experiments. T.W.F., L.L., J.A.C.-P., S.K.M., B.A.R., S.A., S.D., M.A., M.L.G.-H., D.K.S., E.E., M.N.A., J.G., S.M., J.Z., M.M., T.J.S. and J.R.-M. performed specific experiments, contributed resources and/or data analysis. M.M., D.K., T.J.S. and S.A.K provided funding. All authors reviewed, edited and approved the manuscript.

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Correspondence to Deepak Kaushal or Shabaana A. Khader.

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Nature Immunology thanks Andreas Kupz and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editor: L. A. Dempsey, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Irf4 expression by CD19+ B cells is required for accumulation of FO B cells in the lung.

Irf4fl/fl, CD4creIrf4fl/fl, and CD19creIrf4fl/fl mice (n = 5–10 mice/group) were infected with Mtb HN878 and euthanized at 25 and 50 dpi. (a) MHC-IIhi AMs, (b) neutrophils, (c) total B cells, (d) GC B cells, and (e) FO B cells were determined by flow cytometry. Sera collected from mice (50 dpi; dilution shown 1:30) was analyzed via ELISA for levels of (f) IgG2a and IgG2b specific for Mtb antigen CFP-10. Data represent mean + SD, analysis was performed using one-way ANOVA with Tukey’s multiple comparison test (a, c, d, f), and Kruskal-Wallis ANOVA with Dunn’s multiple comparison test (b, e). *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005.

Extended Data Fig. 2 Bcl6 expression by CD19+ B cells is required for optimal infiltration by B cell subsets in the Mtb-infected lung.

scRNA-seq of cells isolated from the lungs of C57BL/6 mice, either uninfected (n = 2 mice), or Mtb-infected at 50 dpi (n = 3 mice) or 100 dpi (n = 3 mice). (a) Swarm plot showing expression of Bcl6, a downstream target of Irf4, in CD4+, CD8+ T and B cells. Bcl6fl/fl, CD4creBcl6fl/fl, and CD19creBcl6fl/fl mice (n = 5–9 mice/group) were infected with Mtb HN878, euthanized at 50 and 100 dpi, and lungs analyzed by flow cytometry for (b) MHC-IIhi AMs, (c) neutrophils, (d) total B cells, (e) GC B cells, and (f) FO B cells. (g) IgG2a and IgG2b antibody specific against Mtb antigen CFP-10 in peripheral blood serum (diluted 1:30) collected at 50 dpi. (h) Tfh-like cells in the lungs of Bcl6fl/fl vs CD19creBcl6fl/fl mice. Data represent mean + SD, analysis was performed using Kruskal-Wallis ANOVA with Dunn’s multiple comparison test (b), one-way ANOVA with Tukey’s multiple comparison test (c to g), and two-sided unpaired t-test (h). *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005.

Extended Data Fig. 3 Blimp1 deficiency in B cells impairs antibody production in mice.

iABfl/fl, and CD19creiABfl/fl mice (n = 6–8 mice/group) were infected with Mtb HN878, euthanized at 50 and 100 dpi, and lungs analyzed by flow cytometry for (a) Tfh-like cells. Blimp1fl/fl, and CD19creBlimp1fl/fl mice (n = 6–8 mice/group) were infected with Mtb HN878, euthanized at 50 and 100 dpi. Lungs analyzed by flow cytometry for (b) Tfh-like cells. (c) Immunoglobulin specificity against Mtb antigen CFP-10 in peripheral blood serum (diluted 1:30) collected at 50 dpi were detected by ELISA. Il10fl/fl, and CD19creIl10fl/fl mice (n = 6–8 mice/group) were infected with Mtb HN878 and euthanized at 50 and 100 dpi. (d) Tfh-like cells were enumerated by flow cytometry. Data represent mean + SD and statistical analysis was performed with two-sided unpaired t-test. *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005.

Extended Data Fig. 4 IghelMD4 mice show reduced Mtb-specific antibody production.

B6 and IghelMD4 mice (n = 5–10 mice/group) were infected with Mtb HN878, euthanized at 50, 75 and 100 dpi and organs were collected and processed. (a) CD44hiCD4+ T cells, (b) IFNγ-producing CD4+ T cells, (c) TNFα-producing CD4+ T cells, (d) total B cells, (e) GC B cells, and (f) FO B cells were determined by flow cytometry. (g) Immunoglobulin specificity against Mtb antigen CFP-10 in peripheral blood serum (diluted 1:15) collected at 100 dpi. Data is mean + SD, analysis was performed using two-sided unpaired t-test (a to f), and two-sided Mann-Whitney U-test (g). *, p ≤ 0.05; **, p ≤ 0.005.

Extended Data Fig. 5 B cell depletion in Mtb-infected mice compromises the accumulation of Tfh-like cells in the lung.

C57BL/6 mice (n = 3–5 mice/group) were infected with Mtb, and infection established over 50 days. At 50 dpi, mice either received B cell depleting antibodies (α-B220 and α-CD19) or isotype via the intratracheal route every 3–4 days, over a 25 days period. Mice were euthanized at 75 dpi, and the lungs collected and processed. (a) Experimental scheme. Flow cytometry was used to enumerate (b) CD19+ B220hi B cells, and (c) immunoglobulin specificity against Mtb antigen CFP-10 in peripheral blood serum (diluted 1:30) was determined via ELISA. (d) Bacterial burden was determined in the lungs of Mtb-infected mice receiving either isotype or B cell-depleting antibodies. Formalin-fixed lung lobes were cut and stained to analyze (e) inflammation (average area) and (f) average area of GrALT. Flow cytometry was used to enumerate (g) activated CD4+ T cells, (h) activated CD4+ Tfh-like cells, (i) IL17+ CD4+ Tfh-like cells and (j) IFNγ+ CD4+ Tfh-like cells in the lungs. Data represents mean ± SD, and statistical analyses performed using two-sided unpaired t-test. *, p ≤ 0.05; **, p ≤ 0.005.

Extended Data Fig. 6 Strategic positioning of PD1+ Tfh-like cells within GrALT structures mediates Mtb control.

(a) Average area of GrALT collated from all mouse models (n = 3–10 mice/group) used in studies at 50 dpi and 128 dpi. (b) Representative images from FFPE lung sections stained with CD3 (red), PD-1 (green) and CD45R/B220 (white) in indicated models at 50 or 128 dpi. Data represent mean + SD and statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test (3 groups), and two-tailed unpaired t-test (2 groups). *, p ≤ 0.05; **, p ≤ 0.005.

Extended Data Fig. 7 Mtb 16sRNA is located outside GrALT areas and Pd1+ T cells are critical to control Mtb in mice.

FFPE lungs sections from Mtb-infected B6 and gene deficient mice were stained for the presence of Mtb 16S rRNA via in situ hybridization (ISH) at (a) 30, 50, and 75 dpi. FFPE lungs sections from Rag1KO Mtb-infected mice that either received WT T cells:WT B cells or Pd1KO T cells:WT B cells, were stained for (b) the presence of Mtb 16S rRNA via in situ hybridization (ISH) at 30 dpi, (c) presence of GrALT via immunofluorescence; CD3 (red); B220 (white); PD-1 (green).

Extended Data Fig. 8 Compromised accumulation of Tfh-like cells in macaques and mice lungs dampens vaccine mediated protection against Mtb.

Macaques were aerosol exposed to MtbΔsigH prior to challenge with virulent Mtb CDC1551 and received either CD20 depleting (n = 4 NHPs) or IgG isotype control antibodies (n = 2 NHPs). Clinical samples were collected throughout the study and at necropsy. (a) FACS profiles for detection of B cells in PBMCs (left panel), BAL (middle panel), and lung (right panels) in CD20-depleted (upper panels) and control macaques (lower panels). (b) The percentage of total peripheral blood B cells (flow cytometry) and (c) Levels of C-reactive protein (CRP) were determined from samples collected throughout the study. (d) FACS profile for detection of T cell subsets in macaque lungs. (e) ESAT6+ CD4+ T cells producing IFNγ, IL2, IL17 and TNFα in lung tissue were enumerated by flow cytometry at the final end-point. (f) CFP-10 specific antibody levels in serum (diluted 1:15) collected at indicated time points. Data represent mean + SD and statistical analysis was performed with two-sided Mann-Whitney U-test. ***, p≤0.0005. Bcl6fl/fl and CD4creBcl6fl/fl mice (n = 4 to 7 mice/group) were mucosally vaccinated with BCG and rested for thirty days following which mice were infected with Mtb HN878. Mice were euthanized at 20 dpi. (g) Experimental scheme. Lungs were collected and bacterial burden was determined in (h) Bcl6fl/fl and CD4creBcl6fl/fl mice. Data represent mean + SD and statistical analysis was performed with two-sided unpaired t-test. **, p ≤ 0.005.

Extended Data Fig. 9 Gating strategy for B cells and T cells flow-cytometric analysis.

Representative FACS plots with gates defining (a) total B cells, GC B cells, and FO B cells, and (b) CD4 T cells, cytokine producing Th1 cells, Tfh-like cells and cytokine producing Tfh-like cells.

Extended Data Fig. 10 Graphical abstract: Mtb specific B cells and Tfh-like cells interact to mediate protection against Mtb.

B cell effector functions including formation of GC B cells, IL-10 production and antibody/plasma cells have non-essential roles during in vivo Mtb infection. However, Mtb antigen-specific B cells interact with Tfh-like cells likely through PDL1-PD1 to drive differentiation and effective localization of cytokine-producing Tfh-like cells within GrALT to mediate Mtb control (left). Key transcription factors Irf4 and Bcl6 (blue text) are essential for differentiation of Tfh-like cells from naive CD4+ T cells. PD1-PDL1 (blue text) receptor-ligand axis likely drive Tfh-like cell localization and homing into GrALT but this interaction is independent of TCR-MHCII interaction, IL-10 production or antibody response (black text). Thus, Tfh-like cells and B cell interactions play critical roles in mediating Mtb control within GrALT.

Supplementary information

Reporting Summary

Supplementary Table 1

Ten cohort studies and their accession numbers, used for IRF4 expression analysis.

Supplementary Table 2

Top transcription factors coexpressed with Irf4 upon Mtb infection in mouse.

Source data

Source Data

Statistical source data for main figures.

Source Data

Statistical source data for extended data figures.

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Swanson, R.V., Gupta, A., Foreman, T.W. et al. Antigen-specific B cells direct T follicular-like helper cells into lymphoid follicles to mediate Mycobacterium tuberculosis control. Nat Immunol 24, 855–868 (2023). https://doi.org/10.1038/s41590-023-01476-3

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