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Dynamic regulation of T follicular regulatory cell responses by interleukin 2 during influenza infection

Abstract

Interleukin 2 (IL-2) promotes Foxp3+ regulatory T (Treg) cell responses, but inhibits T follicular helper (TFH) cell development. However, it is not clear how IL-2 affects T follicular regulatory (TFR) cells, a cell type with properties of both Treg and TFH cells. Using an influenza infection model, we found that high IL-2 concentrations at the peak of the infection prevented TFR cell development by a Blimp-1-dependent mechanism. However, once the immune response resolved, some Treg cells downregulated CD25, upregulated Bcl-6 and differentiated into TFR cells, which then migrated into the B cell follicles to prevent the expansion of self-reactive B cell clones. Thus, unlike its effects on conventional Treg cells, IL-2 inhibits TFR cell responses.

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Figure 1: Kinetics of the TFR cell response to influenza.
Figure 2: TFR cells are CD25lo.
Figure 3: CD25+FoxP3+ Treg cells downregulate CD25 and differentiate into TFR cells.
Figure 4: IL-2 inhibits TFR cell differentiation.
Figure 5: IL-2 inhibits TFR cell differentiation by a Blimp-1 mechanism.
Figure 6: Abnormal expansion of CD138+ ASCs in the absence of TFR cells.
Figure 7: CD138+ ASCs accumulate in TFR-cell-depleted mice.
Figure 8: TFR cells prevent the development of self-reactive ASC responses after infection.

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Acknowledgements

The authors would like to thank W.J. Leonard (US National Institutes of Health) for providing the Il21-mCherry-Il2-emGFP dual reporter transgenic mice, E. Meffre (Yale University) for providing the Blimp-1 reporter mice, A. Rudensky (Memorial Sloan-Kettering Cancer Center) for providing the Foxp3-DTR-GFP mice, and T.S. Simpler and U. Mudunuru for animal husbandry. This work was supported by University of Alabama at Birmingham (UAB) and National Institutes of Health grants 1R01 AI110480 to A.B.-T., R01 AI116584 to B.L., AI097357 and AI109962 to T.D.R., AIAI109962 to F.E.L., AI061061 to A.S.W., and AI049360 to A.J.Z. The X-RAD 320 unit was purchased using a Research Facility Improvement Grant, 1 G20RR022807-01, from the National Center for Research Resources, National Institutes of Health. Support for the UAB flow cytometry core was provided by grants P30 AR048311 and P30 AI027767.

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Authors and Affiliations

Authors

Contributions

A.B.-T. designed and performed the experiments with help from B.L., H.B., M.J.F., J.E.B. and D.B. A.B.-T., D.B. and B.L. analyzed the data. T.T.M.-L. and A.S.W. analyzed the RNAseq data. A.B.-T. wrote the manuscript. D.B. and B.L. contributed to data interpretation and manuscript editing. A.J.Z. performed LCMV infections. T.D.R. and F.E.L. contributed to manuscript editing and discussion, and provided reagents that were critical to this work. All of the authors reviewed the manuscript before submission.

Corresponding author

Correspondence to André Ballesteros-Tato.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 TFR cell responses.

(A) B6 mice were infected with PR8 and serial cryosections from the mLN on day 30 were stained with anti-B220 (blue), anti-FoxP3 (green) and anti-CD35 (red) or with anti-IgD (green), anti-FoxP3 (purple) and anti-CD35 (red). Stained cryosections were analyzed by fluorescence microscopy. White lines define the border of the B-cell follicle. Images are representative of three independent experiments. (B) B6 mice were infected with PR8 and serial cryosections from the mLN on day 30 were stained with anti-B220 (blue), anti-FoxP3 (red) and anti-CD4 (green). Stained cryosections were analyzed by fluorescence microscopy. Images are representative of two independent experiments. (C) B6 mice were infected with PR8 and cryosections of the mLN were stained with anti-B220 (blue), anti-FoxP3 (green) and anti-CD35 (red) on days 10 and 30. White lines define the border of the B cell follicle. Images are representative of three independent experiments. (D-E) B6 mice were immunized with 50 μg of hemaglutinin (HA) adsorbed to alum, and the frequency (D) and number (E) of Bcl-6hiCXCR5hi TFR cells were calculated in the mLN at the indicated time points. Representative plots were gated on FoxP3+CD69hiCD19- CD4+ T cells. Data are representative of two independent experiments (mean ± SD of 4-5 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined using a two-tailed Student´s t-test. (F-G) B6 mice were infected with LCMV-Armstrong and the frequency (F) and number (G) of Bcl-6hiCXCR5hi TFR cells were calculated in the mLN at the indicated time-points. Representative plots were gated on FoxP3+CD69hiCD19- CD4+ T cells. Data are representative of two independent experiments (mean ± SD of 5 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined using a two-tailed Student´s t-test.

Supplementary Figure 2 CD25loFoxp3+ cells fail to up-regulate TFR cell markers at the peak of the infection.

(A-C) B6 mice were infected with PR8 and CD69hiFoxP3+CD4+ T cells from the mLN were analyzed for expression of CD25, Bcl-6, CXCR5 and PD-1 on day 10 (A) and day 30 (B) after infection by flow cytometry. Data are representative of five independent experiments. Data are shown as the mean ± SD (n=4-5 mice). (C) B6 mice were infected with PR8 and the frequency of Bcl-6hiCXCR5hi TFR cells within the CD25loCD69hiFoxP3+CD4+ T cell population were calculated by flow cytometry at the indicated time-points. Data are shown as the mean ± SD (n=4-5 mice/time point). Data are representative of three independent experiments. (D-E). B6 mice were infected with LCMV (D) or immunized with HA adsorbed to alum (E) and the frequency of CD25hi and CD25lo FoxP3+CD69hi CD4+ T cells from the mLNs with a Bcl-6hiCXCR5hi phenotype is shown. Data are representative of two independent experiments (mean ± SD of 5 mice per group). (F) GSEA showing enrichment in the IL-2–STAT5 Hallmark-signaling pathway in Treg cells vs TFR cells (adjusted p-value <0.05, Log2fold change greater than or equal to 1). Three replicates for each cell type were obtained from three independent experiments.

Supplementary Figure 3 IL-2 production in PR8-infected and HA-alum immunized mice.

I l21-mCherry-Il2-emGFP mice were infected with PR8 (left panel) or immunized with HA adsorbed to alum (right panel) and cells from the mLN were analyzed on day 10. The frequency of GFP/IL-2+ cells within the CD4+ T-cell population is shown. Data are shown as the mean ± SD (n=3-5 mice per group). Data are representative of two independent experiments.

Supplementary Figure 4 NP-specific TFH cell response in TFR cell-deficient mice

(A-B) B6 and Bcl-6fl/flFoxp3YFP/Cre mice were infected with PR8 and cells from the mLN were analyzed by flow cytometry on day 30. The frequency (A) and number (B) of NP-specific CD4+ T cells with a CXCR5hiPD-1hi TFH cell phenotype were determined. Data are shown as the mean ± SD (n=3-5 mice). Data are representative of three independent experiments.

Supplementary Figure 5 TFH cell and B cell responses in TFR cell-depleted mice

(A-E) Tcr−/− mice were irradiated and reconstituted with a 50:50 mix of BM from CD45.1+ FoxP3-DTR and CD45.2+ B6 donors (FoxP3-B6) or from CD45.1+ FoxP3-DTR and CD45.2+ Cxcr5−/− donors (FoxP3- Cxcr5−/−). Reconstituted FoxP3-B6 and FoxP3-Cxcr5−/− chimeras were infected with PR8, treated with PBS or DT every four days starting 20 days after infection, and cells from the mLN were analyzed on day 50. Frequency (A) and number (B) of Bcl6hiCXCR5hi TFH cells. Representative plots were gated on FoxP3-CD19- CD4+ T cells. Frequency (c) and number (D) of CD19+CD138-GL-7+CD38loCD95hi GC B cells. Number (E) of CD138+ ASCs. Data are shown as the mean ± SD (n=3-5 mice). Data are representative of three independent experiments. P values were determined using a two-tailed Student´s t-test. (F-L) B6 mice were infected with PR8 and treated daily with 15,000 U of rIL-2 or PBS starting 20 days after infection. Cells from the mLN were analyzed by flow cytometry on day 30. Number (F) of TFR cells in PBS and rIL-2 treated mice. Frequency (G) and number (H) of Bcl-6hiCXCR5hi TFH cells. Representative plots were gated on FoxP3-CD19- CD4+ T cells. Frequency (I) and number (J) of NP-specific CD4+ T cells with a Bcl-6hiCXCR5hi TFH cell phenotype. Representative plots were gated on FoxP3-CD19- NP+CD4+ T cells. The frequency (K) and number (L) of CD19+CD138-GL-7+CD95hi GC B cells. Data are shown as the mean ± SD (n=4-5 mice). Data are representative of four independent experiments. P values were determined using a two-tailed Student´s t-test.

Supplementary Figure 6 Influenza-infected Bcl-6fl/flFoxp3YFP/Cre mice develop self-reactive ASC responses.

The presence of ANAs in the serum from naïve and age-matched day 30 influenza-infected B6 (top panels) and Bcl6fl/flFoxp3YFP/Cre mice (bottom panels) was determined by fluorescence microscopy using HEp-2 slides. Images are representative of two independent experiments (n=4-5 mice/group).

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Botta, D., Fuller, M., Marquez-Lago, T. et al. Dynamic regulation of T follicular regulatory cell responses by interleukin 2 during influenza infection. Nat Immunol 18, 1249–1260 (2017). https://doi.org/10.1038/ni.3837

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