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Follicular regulatory T cells control humoral and allergic immunity by restraining early B cell responses

Abstract

Follicular regulatory T (TFR) cells have specialized roles in modulating follicular helper T (TFH) cell activation of B cells. However, the precise role of TFR cells in controlling antibody responses to foreign antigens and autoantigens in vivo is still unclear due to a lack of specific tools. A TFR cell-deleter mouse was developed that selectively deletes TFR cells, facilitating temporal studies. TFR cells were found to regulate early, but not late, germinal center (GC) responses to control antigen-specific antibody and B cell memory. Deletion of TFR cells also resulted in increased self-reactive immunoglobulin (Ig) G and IgE. The increased IgE levels led us to interrogate the role of TFR cells in house dust mite models. TFR cells were found to control TFH13 cell-induced IgE. In vivo, loss of TFR cells increased house-dust-mite-specific IgE and lung inflammation. Thus, TFR cells control IgG and IgE responses to vaccines, allergens and autoantigens, and exert critical immunoregulatory functions before GC formation.

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Fig. 1: Development of a TFR cell-specific deleter model.
Fig. 2: TFR cells potently regulate early GC formation.
Fig. 3: TFR cells control autoreactive IgG and IgE during foreign antibody responses.
Fig. 4: TFR cells regulate antibody memory responses.
Fig. 5: HDM antigen generates distinct populations of TFH and TFR cells.
Fig. 6: TFR cells regulate TFH13 cell-mediated IgE responses in vitro.
Fig. 7: TFR cells regulate HDM-specific IgE responses in vivo.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Transcriptomic data have been deposited in the Gene Expression Omnibus with the accession code GSE134153.

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Acknowledgements

We would like to thank T. Chatila, R. Anthony, D. Wesemann and M. Carroll for helpful discussions, the MICRON imaging core for help with microscopy and H. Wekerle and S. Zamvil for reagents. This work was supported by the US National Institutes of Health through grant nos. K22AI132937 (P.T.S.), P01AI056299 (A.H.S.), R37AI34495 (B.R.B.) and R01HL11879 (B.R.B.), and the Evergrande Center for Immunologic Diseases.

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R.L.C, J.D., M.T.M. and P.T.S. performed the experiments. R.L.C, A.D., S.B.L. and P.T.S. analyzed the data. B.R.B., V.K.K. and A.H.S provided key technical help and reagents. P.T.S. conceived of the project and wrote the manuscript. All the authors edited the manuscript.

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Correspondence to Peter T. Sage.

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Peer review information: Z. Fehervari was the primary editor on this article, and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 Characterization of the TFR-DTR strain.

a) Gating strategy to identify TFH, TFR and CXCR5 Treg cells in draining lymph nodes of mice immunized with NP-OVA 7 days previously. b) DTR expression on TFH, TFR and CXCR5- Treg cells gated as in (a) in indicated mouse strains. Full stain = all antibodies used along with secondary reagent for DTR staining. No DTR = all antibodies except anti-DTR primary was not added. c) Quantification of activated Treg cells in TFR-DTR mice. Control or TFR-DTR mice were immunized and given DT to delete TFR cells as in Fig. 1f. CXCR5-Ki67+FoxP3+ activated Treg cells were quantified. Representative gating (left) and analysis (right) are shown. d) Quantification of TFR cells by flow cytometry (left) and quantification of FoxP3+ cells within individual GCs by microscopy (right) in TFR-DTR mice as in (c). Column graphs represent the mean with error bars indicating standard error. P value indicates two-tailed student’s T test. Data are from an individual experiment and are representative of two experimental repeats (a-c), or are combined data from two experiments (d).

Supplementary Figure 2 TFR cells regulate early GC responses.

a) Antibody responses in TFR-DTR mice with pre-GC TFR deletion strategies. TFR-DTR mice were immunized with NP-OVA and received DT on days 5,7 and 9. Serum was collected on day 21 and NP-specific IgG (left), total IgA (middle) and total IgE (right) were measured. b) Phenotype of TFR cells in TFR-DTR mice with early (left) or pre-GC (right) start TFR deletion strategies. TFR-DTR mice were immunized with NP-OVA and received DT on days 2,4,6 (left) or 5,7 and 9 (right). Expression of CXCR5, ICOS, PD-1 or Ki67 was measured on day 7 (left) or day 21 (right). c) Assessment of B cell responses in TFR-DTR mice with post-GC TFR deletion strategies. TFR-DTR mice were immunized with NP-OVA and received DT on days 10,12 and 14. Serum was collected on day 21 and NP-specific IgG (left), total IgA (middle) and total IgE (right) were measured. d) Expression of CXCR5, ICOS and Ki67 on TFR cells in control or TFR-DTR mice in post-GC deletion experiments. FoxP3Cre Cxcr5wt control or TFR-DTR mice which were immunized with NP-OVA, given DT on days 10,12, and 14 and draining lymph nodes harvested on day 21. e) Evaluation of B cell responses early during pre-GC TFR deletion strategies. TFR-DTR mice were immunized with NP-OVA and received DT on days 5,7 and 9. Draining lymph nodes were collected on day 14 for the indicated analysis. TFR (CD4+ICOS+CXCR5+FoxP3+CD19-) cells, GC B (CD19+GL7+FAS+) cells, Plasma cells (CD138+) and class switched B cells (CD19+GL7+IgG1+) cells were quantified. f) Evaluation of B cell responses late during post-GC TFR deletion strategies. TFR-DTR mice were immunized with NP-OVA and received DT on days 10,12 and 14. Draining lymph nodes and serum was collected on day 26 for analysis. GC B (CD19+GL7+FAS+) cells from lymph nodes, NP specific IgG from serum, and total IgG from serum were quantified. Column graphs represent the mean with error bars indicating standard error. P value indicates two-tailed student’s T test. Data are from an individual experiments and are representative of two experimental repeats.

Supplementary Figure 3 Additional analysis of memory responses in TFR-DTR mice.

a) NP2/16 ratio in TFR-DTR mice with pre-GC TFR deletion strategies. TFR-DTR mice were immunized with NP-OVA/MF59-Addavax and received DT on days 5,7 and 9. Serum was collected on day 30 (pre-boost). b) NP2/16 ratio in TFR-DTR mice with pre-GC TFR deletion strategies. TFR-DTR mice were immunized with NP-OVA and received DT on days 5,7 and 9. Serum was collected on day 21. Explain boost. c) NP2/16 ratio in TFR-DTR mice with TFR deletion post-GC formation. TFR-DTR mice were immunized with NP-OVA and received DT on days 10,12 and 14. Serum was collected on day 21. Column graphs represent the mean with error bars indicating standard error. P value indicates two-tailed student’s T test. Data are from 3 combined experiments.

Supplementary Figure 4 TFR cells suppress TFH13-mediated class switching.

a) FoxP3-GFP mice were immunized with NP-OVA subcutaneously or HDM intranasally according to materials and methods. 7 days later draining lymph nodes were harvested and B, TFH and TFR cells were isolated. Indicated populations were cultured together with the indicated antigen for 6 days. Plots are pregated on CD19+CD4-. Data are from an individual experiment and are representative of two experimental repeats.

Supplementary Figure 5 Additional analysis of allergic inflammation in TFR-DTR mice.

a) Quantification of Ki67+ CXCR5- activated Treg cells in TFR-DTR mice as in Fig. 7a, b. Representative gating (left) and representative gating (right) are shown. b) Representative micrographs of lungs from FoxP3Cre Cxcr5wt control or TFR-DTR mice stained for Actin (green), Siglec F (white), Gr1 (red) or I-A (blue) as in Fig. 7a. Scale bars indicate 500 microns. Column graphs represent the mean with error bars indicating standard error. P value indicates two-tailed student’s T test. Data are from an individual experiment and are representative of three experimental repeats (a), or are from one experiment (b).

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Clement, R.L., Daccache, J., Mohammed, M.T. et al. Follicular regulatory T cells control humoral and allergic immunity by restraining early B cell responses. Nat Immunol 20, 1360–1371 (2019). https://doi.org/10.1038/s41590-019-0472-4

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