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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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.
References
Crotty, S. Follicular helper CD4 T cells (TFH). Annu. Rev. Immunol. 29, 621–663 (2011).
Victora, G. D. & Nussenzweig, M. C. Germinal centers. Annu. Rev. Immunol. 30, 429–457 (2012).
Crotty, S. T follicular helper cell differentiation, function, and roles in disease. Immunity 41, 529–542 (2014).
Vinuesa, C. G., Linterman, M. A., Yu, D. & MacLennan, I. C. Follicular helper T cells. Annu. Rev. Immunol. 34, 335–368 (2016).
Cannons, J. L., Lu, K. T. & Schwartzberg, P. L. T follicular helper cell diversity and plasticity. Trends Immunol. 34, 200–207 (2013).
Weinstein, J. S. et al. TFH cells progressively differentiate to regulate the germinal center response. Nat. Immunol. 17, 1197–1205 (2016).
Luthje, K. et al. The development and fate of follicular helper T cells defined by an IL-21 reporter mouse. Nat. Immunol. 13, 491–498 (2012).
Liang, H. E. et al. Divergent expression patterns of IL-4 and IL-13 define unique functions in allergic immunity. Nat. Immunol. 13, 58–66 (2011).
Ballesteros-Tato, A. et al. T follicular helper cell plasticity shapes pathogenic T helper 2 cell-mediated immunity to inhaled house dust mite. Immunity 44, 259–273 (2016).
Coquet, J. M. et al. Interleukin-21-producing CD4+ T cells promote type 2 immunity to house dust mites. Immunity 43, 318–330 (2015).
Noval Rivas, M. & Chatila, T. A. Regulatory T cells in allergic diseases. J. Allergy Clin. Immunol. 138, 639–652 (2016).
Curotto de Lafaille, M. A. et al. Adaptive Foxp3+ regulatory T cell-dependent and -independent control of allergic inflammation. Immunity 29, 114–126 (2008).
Sage, P. T. & Sharpe, A. H. T follicular regulatory cells. Immunol. Rev. 271, 246–259 (2016).
Maceiras, A. R., Fonseca, V. R., Agua-Doce, A. & Graca, L. T follicular regulatory cells in mice and men. Immunology 152, 25–35 (2017).
Sage, P. T. et al. Suppression by TFR cells leads to durable and selective inhibition of B cell effector function. Nat. Immunol. 17, 1436–1446 (2016).
Sage, P. T., Paterson, A. M., Lovitch, S. B. & Sharpe, A. H. The coinhibitory receptor ctla-4 controls B cell responses by modulating T follicular helper, T follicular regulatory, and T regulatory cells. Immunity 41, 1026–1039 (2014).
Sage, P. T., Alvarez, D., Godec, J., von Andrian, U. H. & Sharpe, A. H. Circulating T follicular regulatory and helper cells have memory-like properties. J. Clin. Invest. 124, 5191–5204 (2014).
Sage, P. T., Francisco, L. M., Carman, C. V. & Sharpe, A. H. The receptor PD-1 controls follicular regulatory T cells in the lymph nodes and blood. Nat. Immunol. 14, 152–161 (2013).
Wollenberg, I. et al. Regulation of the germinal center reaction by Foxp3+ follicular regulatory T cells. J. Immunol. 187, 4553–4560 (2011).
Wu, H. et al. Follicular regulatory T cells repress cytokine production by follicular helper T cells and optimize IgG responses in mice. Eur. J. Immunol. 46, 1152–1161 (2016).
Linterman, M. A. et al. Foxp3+ follicular regulatory T cells control the germinal center response. Nat. Med. 17, 975–982 (2011).
Fu, W. et al. Deficiency in T follicular regulatory cells promotes autoimmunity. J. Exp. Med. 215, 815–825 (2018).
Laidlaw, B. J. et al. Interleukin-10 from CD4+ follicular regulatory T cells promotes the germinal center response. Sci. Immunol. 2, eaan4767 (2017).
Kim, J. M., Rasmussen, J. P. & Rudensky, A. Y. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat. Immunol. 8, 191–197 (2007).
Hou, S. et al. FoxP3 and Ezh2 regulate TFR cell suppressive function and transcriptional program. J. Exp. Med. 216, 605–620 (2019).
Sayin, I. et al. Spatial distribution and function of T follicular regulatory cells in human lymph nodes. J. Exp. Med. 215, 1531–1542 (2018).
Dema, B. et al. Immunoglobulin E plays an immunoregulatory role in lupus. J. Exp. Med. 211, 2159–2168 (2014).
Dema, B. et al. Autoreactive IgE is prevalent in systemic lupus erythematosus and is associated with increased disease activity and nephritis. PLoS ONE 9, e90424 (2014).
Sage, P. T. et al. Dendritic cell PD-L1 limits autoimmunity and follicular T cell differentiation and function. J. Immunol. 200, 2592–2602 (2018).
Bettelli, E., Baeten, D., Jager, A., Sobel, R. A. & Kuchroo, V. K. Myelin oligodendrocyte glycoprotein-specific T and B cells cooperate to induce a Devic-like disease in mice. J. Clin. Invest. 116, 2393–2402 (2006).
Krishnamoorthy, G., Lassmann, H., Wekerle, H. & Holz, A. Spontaneous opticospinal encephalomyelitis in a double-transgenic mouse model of autoimmune T cell/B cell cooperation. J. Clin. Invest. 116, 2385–2392 (2006).
Mulero, P., Midaglia, L. & Montalban, X. Ocrelizumab: a new milestone in multiple sclerosis therapy. Ther. Adv. Neurol. Disord. 11, 1756286418773025 (2018).
McHeyzer-Williams, L. J., Milpied, P. J., Okitsu, S. L. & McHeyzer-Williams, M. G. Class-switched memory B cells remodel BCRs within secondary germinal centers. Nat. Immunol. 16, 296–305 (2015).
Zhu, J., Jankovic, D., Grinberg, A., Guo, L. & Paul, W. E. Gfi-1 plays an important role in IL-2-mediated Th2 cell expansion. Proc. Natl Acad. Sci. USA 103, 18214–18219 (2006).
Tomlinson, K. L., Davies, G. C., Sutton, D. J. & Palframan, R. T. Neutralisation of interleukin-13 in mice prevents airway pathology caused by chronic exposure to house dust mite. PLoS ONE 5, e13136 (2010).
Botta, D. et al. Dynamic regulation of T follicular regulatory cell responses by interleukin 2 during influenza infection. Nat. Immunol. 18, 1249–1260 (2017).
Crawford, G. et al. Epithelial damage and tissue gammadelta T cells promote a unique tumor-protective IgE response. Nat. Immunol. 19, 859–870 (2018).
Yao, Y. et al. Allergen immunotherapy improves defective follicular regulatory T cells in patients with allergic rhinitis. J. Allergy Clin. Immunol 144, 118–128 (2019).
He, J. S. et al. IgG1 memory B cells keep the memory of IgE responses. Nat. Commun. 8, 641 (2017).
Xiong, H., Dolpady, J., Wabl, M., Curotto de Lafaille, M. A. & Lafaille, J. J. Sequential class switching is required for the generation of high affinity IgE antibodies. J. Exp. Med. 209, 353–364 (2012).
Bettelli, E. et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, 235–238 (2006).
Litzenburger, T. et al. B lymphocytes producing demyelinating autoantibodies: development and function in gene-targeted transgenic mice. J. Exp. Med. 188, 169–180 (1998).
Sage, P. T. & Sharpe, A. H. In vitro assay to sensitively measure TFR suppressive capacity and TFH stimulation of B cell responses. Methods Mol. Biol. 1291, 151–160 (2015).
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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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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DOI: https://doi.org/10.1038/s41590-019-0472-4
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