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Suppression by TFR cells leads to durable and selective inhibition of B cell effector function

Nature Immunology volume 17, pages 1436–1446 (2016)Cite this article

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Abstract

Follicular regulatory T cells (TFR cells) inhibit follicular helper T cell (TFH cell)–mediated antibody production. The mechanisms by which TFR cells exert their key immunoregulatory functions are largely unknown. Here we found that TFR cells induced a distinct suppressive state in TFH cells and B cells, in which effector transcriptional signatures were maintained but key effector molecules and metabolic pathways were suppressed. The suppression of B cell antibody production and metabolism by TFR cells was durable and persisted even in the absence of TFR cells. This durable suppression was due in part to epigenetic changes. The cytokine IL-21 was able to overcome TFR cell–mediated suppression and inhibited TFR cells and stimulated B cells. By determining mechanisms of TFR cell-mediated suppression, we have identified methods for modulating the function of TFR cells and antibody production.

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Figure 1: Suppressed B cells undergo early activation.
Figure 2: Suppressed TFH cells and B cells retain transcriptional programs except for inhibition of genes encoding specific effector molecules.
Figure 3: Inhibition of the Myc and mTOR pathways suppresses B cell effector function.
Figure 4: TFR cells inhibit multiple metabolic pathways in B cells.
Figure 5: Suppression by TFR cells results in sustained inhibition and epigenetic changes in B cells.
Figure 6: IL-21 can overcome TFR cell–mediated suppression of B cell metabolism and antibody production.
Figure 7: IL-21 directly stimulates B cells and inhibits TFR cells.

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Acknowledgements

We thank the Nikon Imaging Center at Harvard Medical School for help with light microscopy and the MBIB flow cytometry core for help with flow cytometry. Supported by the US National Institute of Health (T32HL007627 to P.T.S.; and R37AI38310, P01AI56299 and P01AI065687 to A.H.S.) and the Evergrande Center for Immunologic Diseases.

Author information

Authors and Affiliations

  1. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA

    Peter T Sage, Vikram R Juneja, Seth Maleri & Arlene H Sharpe

  2. Evergrande Center for Immunologic Diseases, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts, USA

    Peter T Sage, Vikram R Juneja, Seth Maleri, Waradon Sungnak, Vijay K Kuchroo & Arlene H Sharpe

  3. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA

    Noga Ron-Harel & Marcia Haigis

  4. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Debattama R Sen & W Nicholas Haining

  5. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Waradon Sungnak & Vijay K Kuchroo

  6. FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA

    Nicolas Chevrier

  7. Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA

    Arlene H Sharpe

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Contributions

P.T.S. performed all experiments and analyzed data; N.R.-H. performed metabolic-flux analysis; N.R.-H. and M.H. provided technical help on metabolic pathways; V.R.J. provided technical help on RNA-seq experiments; D.R.S. and W.N.H. prepared ATAC-seq samples and provided technical assistance on ATAC-seq analysis; S.M. provided technical help; W.S. and V.K.K. provided Il21r−/− mice and technical help; N.C. provided the RNA-seq library-preparation protocol and provided technical help; and P.T.S. and A.H.S. conceived of the project and wrote the manuscript.

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Correspondence to Arlene H Sharpe.

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Integrated supplementary information

Supplementary Figure 1 Sorting gates for TFH cells, TFR cells and B cells.

(a) Schematic of Suppression assay. FoxP3-GFP reporter mice were immunized with NP-OVA and 7 days later dLN were harvested and CD19+ B cells and CD4+CXCR5+ICOS+FoxP3-CD19- TFH cells were cultured with or without CD4+CXCR5+ICOS+FoxP3+CD19- TFR cells in the presence of anti-CD3/IgM. (b) Sort strategy for sorting TFH and TFR cells. (c) Class switch to IgG1 in suppression assays in which B and TFH cells were cultured with or without TFR cells along with NP-OVA.

Supplementary Figure 2 Additional characterization of activated and suppressed B cells based on RNA-seq analysis.

(a) Schematic of experiment. B and TFH cells, sorted from NP-OVA immunized FoxP3GFP mice, were cultured alone (“Activated”) or with TFR cells (“Suppressed”) sorted from FoxP3GFP ActinCFP mice, in the presence of NP-OVA. After 4 days CD19+IA+CD4-CFP- B and CD4+CD19-IA-CFP- TFH cells were sorted and processed for RNA-seq analysis. (b) Sorting gates for B cells. (c) Volcano plots showing genes in B and TFH cells in the context of activated or suppressed cultures. (d) Heat map of genes differentially expressed (FDR corrected p<0.05) in B and TFH cells from activated versus suppressed cultures. (e) Genes differentially expressed in both TFH and B cells in activated versus suppressed cultures.

Supplementary Figure 3 Metabolic pathway analysis of activated B cells versus suppressed B cells.

Metabolic map of essential enzymes in glycolysis, 1-carbon metabolism and serine biosynthesis. Blue indicates transcripts that are statistically lower in suppressed versus activated B cells. Gray indicates no significant change. Red indicates transcripts that are more abundant in suppressed versus activated B cells (none were found).

Supplementary Figure 4 Expanded analysis of metabolic pathways.

(a) Heat maps of individual genes in metabolic pathways (shown in Fig. 4a ) from suppression assays in which B and TFH (Act B) or B, TFH and TFR (Supp B) were cultured.

Supplementary Figure 5 Additional analysis of altered metabolic pathways in TFR cell–suppressed B cells.

(a) Glut1 staining in B cells for experiments shown in Fig. 4b. (b) (left) Gating of division number for experiments shown in Fig. 4c. (right) IgG1+ staining in B cells gated by division number. (c) IgG1+ staining in B cells that were added to 3 day cultures and harvested 20 hours later as in Fig. 4d. (d) Glutamine uptake measured from culture supernatants from cultures as in Fig 4b. CD4+ICOS–CXCR5–FoxP3+ Treg cells were added in some conditions. (e) IgG1+ B cells in culture supernatants from cultures as in (a) with the addition of glutaminolysis inhibitor BPTES.

Supplementary Figure 6 Antibody measurements in reactivation of suppressed B cells.

(a) Schematic of restimulation of suppressed B cells. (b) Antibody measurements from restimulation of suppressed B cells. B cells were sorted from suppressed cultures and cultured with new TFH cells in the presence of anti-CD3/IgM for 6 days. IgG from culture supernatants were measured by ELISA.

Supplementary Figure 7 Expanded analysis of ‘IL-21 rescue’ experiments.

(a) Cell count (left), IgG1+ staining (middle) and Glut1 expression (right) on B cells from suppression assays in which IL21, IL6 or IL4 were added. (b) Cell count (left), IgG1+ staining (middle) and Glut1 expression (right) on B cells from suppression assays in which WT or Il21r-/- B cells were cultured as well as IL-21. (c) Heat map of genes downregulated in activated versus suppressed B cells, but not rescued with the addition of IL-21.

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Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1874 kb)

Supplementary Video 1

Time lapse movie showing TFR cell dynamics (MOV 850 kb)

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Sage, P., Ron-Harel, N., Juneja, V. et al. Suppression by TFR cells leads to durable and selective inhibition of B cell effector function. Nat Immunol 17, 1436–1446 (2016). https://doi.org/10.1038/ni.3578

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