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Inhibition of IL-2 responsiveness by IL-6 is required for the generation of GC-TFH cells

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Science Immunology  13 Sep 2019:
Vol. 4, Issue 39, eaaw7636
DOI: 10.1126/sciimmunol.aaw7636
  • Fig. 1 Late IL-6 signaling is required for the accumulation of influenza-specific GC-TFH cells.

    (A and B) C57BL/6 (WT) and C57BL/6.Il6−/− (Il6−/−) mice were infected with PR8 and the frequency (A) and number (B) of NP-specific CD4+ T cells with a Bcl-6hiCXCR5hi TFH cell phenotype were evaluated in the mLN at the indicated time points. Representative plots gated on NP-specific CD4+ T cells are shown. Data were pooled from three independent experiments from a total of five independent experiments (data are shown as means ± SD; n = 4 to 5 mice per experiment). *P < 0.05 and ***P < 0.001. P values were determined using a two-tailed Student’s t test. (C to E) Equivalent numbers of CD45.1+CD45.2 and CD45.1+CD45.2+ OTII cells were respectively transferred into CD45.1CD45.2+ WT and CD45.1CD45.2+ Il6−/− primary recipient mice. One day later, the recipient mice were infected with PR8-OTII influenza virus. Three days after infection, CD4+ T cells from the mLN of WT and Il6−/− recipient mice were purified and mixed so that the mixture contained equivalent numbers of CD45.1+CD45.2 (WT-primed) and CD45.1+CD45.2+ (Il6−/−-primed) OTII cells. Cell numbers were then normalized to the concentration of OTII cells, and 2 × 103 cells of the 1:1 mixture of WT-primed and Il6−/−-primed OTII cells were adoptively transferred into WT and Il6−/− mice that were previously infected with PR8-OTII influenza 5 days earlier. p.i., post-infection. The frequency (D) and number (E) of WT-primed and Il6−/−-primed OTII cells with a PD-1hiCXCR5hi TFH phenotype were determined on day 11 after infection in the mLN. Data are representative of three independent experiments. Data are shown as means ± SD (n = 5 mice). **P < 0.01 and ***P < 0.001. P values were determined using a two-tailed Student’s t test. (F to L) Tcrb−/–Tcrd−/− mice were irradiated and reconstituted with a 50:50 mix of BM from CD45.1+ C57BL/6 (WT) and CD45.2+ Il6raflox/flox-lck-cre−/+ (Il6r–/–) donors. Eight weeks later, reconstituted mice were infected with PR8, and cells from the mLN were analyzed at the indicated time points. (F) Expression of CD126 (IL-6Ra) in the CD45.1+ and CD45.2+ NP-specific CD4+ T cell compartments. Representative plots on day 7 are shown. (G) Frequency of CD45.1+ and CD45.2+ NP-specific CD4+ T cells with a Bcl-6-hiCXCR5hi phenotype. Representative plots are shown. Data in the graph are shown as means ± SD (n = 5 mice per time point). (H) The ratio of WT to Il6r−/− CD4+ T cells, NP-TEFF (Bcl-6-loCXCR5lo), and NP-TFH (Bcl-6-hiCXCR5hi) cells on day 10 were calculated (means ± SD; n = 5 mice). (I) PD-1 expression within the CD45.1+ and CD45.2+ NP-specific TFH cell compartments. Representative plots on day 10 are shown. Frequency of GC-TFH (Bcl-6hiCXCR5hiPD-1hi) (J) and non–GC-TFH (Bcl-6hiCXCR5hiPD-1lo) (K) cells within the CD45.1+ and CD45.2+ NP-specific CD4+ T cell compartments. (L) The ratio of WT to Il6r−/− NP-specific GC-TFH, NP-specific non–GC-TFH, and total CD4+ T cells were calculated (means ± SD; n = 5 mice). Data are representative of three independent experiments. Data are shown as means ± SD (n = 4 to 5 mice per time point). *P < 0.05, **P < 0.01, and ***P < 0.001. P values were determined using a two-tailed Student’s t test.

  • Fig. 2 GC-TFH cells produce large amounts of IL-2 at the peak of the infection.

    (A to C) Il21-mCherry-Il2-emGFP reporter mice were infected with PR8, and cells from the mLN were analyzed at the indicated time points. (A) Frequency and (B) number of IL-2/GFP+CD4+ T cells. (C) Number of IL-2/GFP+ NP-specific CD4+ T cells. Representative plots are shown. Data in the graphs are shown as means ± SD (n = 4 mice per time point). Data are representative of three independent experiments. (D to G) Il21-mCherry-Il2-emGFP reporter mice were infected with PR8, and the frequency of IL-2/GFP+ cells within the PD-1hiCXCR5hi (TFH) and PD-1loCXCR5lo (non-TFH) CD4+ T cell populations was calculated on day 12 after infection. Representative plots gated on CD19-CD4+ T cells (data are shown as means ± SD; n = 4 mice per time point). (E) Expression of IL-21/mCherry in IL-2/GFP+ GC-TFH and IL-2/GFP+ non-TFH cells. Data are representative of three independent experiments. (F) Frequency of IL-2/GFP+ cells within the PD-1hiCXCR5hi and PD-1loCXCR5lo NP-specific CD4+ T cell populations on day 12 after infection (data are shown as means ± SD; n = 4) mice. (G and H) Frequency (G) and number (H) of IL-2/GFP+ GC-TFH cells at the indicated time points. Data are representative of three independent experiments. Data are shown as means ± SD (n = 4 mice per time point).

  • Fig. 3 IL-6 signaling is required for sustaining TFH cells developing in a high–IL-2 environment.

    (A) B6 mice were infected with PR8 and treated daily with the indicated doses of rIL-2 starting 1 day after infection. The frequency of NP-specific cells with a Bcl-6hiCXCR5hi TFH cell phenotype was determined in the mLN on day 7 after infection. Data in the graph are shown as means ± SD (n = 4 mice per group). Data are representative of three independent experiments. ***P < 0.001. P values were determined using a two-tailed Student’s t test. (B and C) B6 mice were infected with PR8 and treated daily with either 7500 U of rIL-2 or PBS alone or in combination with 250 μg of a mix of anti–IL-6 and anti–IL-6/R Abs. The frequency (B) and number (C) of Bcl-6hiCXCR5hi NP-specific TFH cells were determined in the mLN on day 7 after infection. Representative plots are shown. Data in the graph are shown as means ± SD (n = 4 to 5 mice per group). Data are representative of two independent experiments. *P < 0.05 and **P < 0.01. P values were determined using a two-tailed Student’s t test. (D and E) Foxp3-DTR–GFP mice were infected with PR8 and treated with either PBS [control (Ctrl)] or diphtheria toxin (DTx) on day 3 after infection. A third group was treated with diphtheria toxin and administered 250 μg of a mix of anti–IL-6 and anti–IL-6/R Abs on days 0, 2, and 4 after infection. The frequency (D) and number (E) of Bcl-6hiCXCR5hi NP-specific TFH cells in the mLN were calculated on day 7 after infection. Representative plots gated on NP-specific CD4+ T cells are shown. Data in the graph are shown as means ± SD (n = 4 to 5 mice per group). Data are representative of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. P values were determined using a two-tailed Student’s t test. (F) Irradiated Tcrb−/–Tcrd−/− mice were reconstituted with a 50:50 mix of BM from CD45.1+ C57BL/6 (WT) and CD45.2+ Il6raflox/flox-lck-cre−/+ (Il6r−/−) donors. Two months later, chimeric mice were infected with PR8 and treated daily with 7500 U of rIL-2 or PBS starting on day 1 after infection, and the frequency of Bcl-6hiCXCR5hi cells within the WT and Il6r−/− NP-specific CD4+ T cell compartments was calculated on day 7 after infection in the mLN. Representative plots are shown. Data in the graph are shown as means ± SD (n = 4 to 5 mice per group). Data are representative of two independent experiments. *P < 0.05 and ***P < 0.001. P values were determined using a two-tailed Student’s t test. (G) CFSE-labeled CD4+ T cells from the spleen of naïve B6 mice were activated in vitro with plate-bound anti-CD3/CD28 Abs in the presence of the indicated concentration of anti–IL-2 Abs (JES6-1A12 and S4B6), and either rIL-6 or PBS (10 ng/ml ) was added to the cultures. The expression of Bcl-6 in CFSElowCD4+ T cells was assessed at 48 hours by flow cytometry. Data are representative of four independent experiments. All values were obtained in triplicate, and the data are shown as means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001P. P values were determined using a two-tailed Student’s t test.

  • Fig. 4 IL-6 signaling limits IL-2 responsiveness by preventing CD122 expression.

    (A and B) WT and Il6−/− mice were infected with PR8, and the expression of CD25 in PD-1loCXCR5lo NP-specific TEFF cells (A) and PD-1hiCXCR5hi NP-specific GC-TFH cells (B) was examined in the mLN at the indicated time points. Data are representative of three independent experiments. Data in the graph are shown as means ± SD (n = 5 mice). *P < 0.05. P values were determined using a two-tailed Student’s t test. MFI, mean fluorescence intensity. (C) Irradiated Tcrb−/–Tcrd−/− mice were reconstituted with a 50:50 mix of BM from CD45.1+ C57BL/6 (WT) and CD45.2+ C57BL/6.Cd25−/− (Cd25−/−) donors. Two months later, chimeric mice were infected with PR8 and treated or not with 250 μg of a mix of anti–IL-6 + anti–IL-6/R Abs on days 0, 2, 4, 6, and 8 after infection, and the frequency of PD-1hiCXCR5hi GC-TFH cells within the WT and Cd25−/− NP-specific CD4+ T cells were examined on day 10 after infection. Representative plots are shown. Data in the graph are shown as means ± SD (n = 4 to 5 mice per group). Data are representative of three independent experiments. **P < 0.01 and ***P < 0.001. P values were determined using a two-tailed Student’s t test. (D and E) Kinetics demonstrating the expression of CD122 in PD-1loCXCR5lo NP-specific TEFF cells (D) and PD-1hiCXCR5hi NP-specific GC-TFH cells (E) from PR8-infected WT and Il6−/− mice. Data are representative of three independent experiments. Data in the graph are shown as means ± SD (n = 5 mice). *P < 0.05, **P < 0.01, and ***P < 0.001. P values were determined using a two-tailed Student’s t test. (F) WT/ Il6r−/− mixed BM chimeras were infected with PR8, and expression of CD122 in WT and Il6r−/− PD-1hiCXCR5hi NP-specific GC-TFH cells was determined on day 10 after infection. Data are representative of two independent experiments. Data in the graph are shown as means ± SD (n = 5). **P < 0.01. P values were determined using a two-tailed Student’s t test. (G and H) CFSE-labeled CD4+ T cells from the spleen of naïve B6 mice were activated in vitro with plate-bound anti-CD3/CD28 Abs in the presence of anti–IL-2 Abs (0.5 μg/ml; JES6-1A12 + S4B6), and either rIL-6 or PBS (10 ng/ml) was added to the cultures. (G) Expression of CD25 in CFSEloCD4+ T cells at 48 hours. (H) Expression of CD122 in CFSEloCD25lo CD4+ T cells. (I) Expression of Bcl-6 in CFSEloCD25lo CD4+ T cells. Data are representative of three independent experiments. All values were obtained in triplicate, and the data are shown as means ± SD. **P < 0.01. P values were determined using a two-tailed Student’s t test. (J to L) CFSE-labeled CD4+ T were activated in vitro in the presence of anti–IL-2 Abs (JES6-1A12 + S4B6), and rIL-6 (10 ng/ml), rIL-21 (50 ng/ml), or PBS was added to the cultures. The expression of Bcl-6 (J), CD122 (K), and Cd25 (L) in CFSElowCD4+ T cells was assessed at 48 hours. Data are representative of two independent experiments. All values were obtained in triplicate, and the data are shown as means ± SD. (M to O) WT and Il21r−/− mice were infected with PR8, and cells from the mLN were analyzed at the indicated time points. Frequency (M) and number (N) of Bcl-6hiCXCR5hi NP-specific TFH cells. (O) Frequency of PD-1hi GC-TFH cells within the NP-specific TFH cell population. (P) Expression of CD122 in NP-specific GC-TFH cells. Data are representative of two independent experiments. Data in the graph are shown as means ± SD (n = 5).

  • Fig. 5 Lack of GC-TFH cells in the absence of IL-6 signaling is STAT5 dependent.

    (A) Cells obtained from the mLN of PR8-infected WT and Il6−/− mice were stimulated with the indicated amounts of rIL-2 for 15 min, and STAT5 phosphorylation in PD-1hiBcl-6hiCXCR5hiCD4+B220 GC-TFH, CD4+B220Bcl-6loCXCR5loCD25+Foxp3+ Treg, and in PD-1loBcl-6loCXCR5loCD4+B220CD25Foxp3 naïve CD4+ T cells was determined by flow cytometry on day 10. Data are representative of three independent experiments. Data are shown as means ± SD (n = 8 to 10 mice). (B) Kinetic of STAT5 phosphorylation in GC-TFH at different times after infection. Data were pooled from three independent experiments. Data are shown as means ± SD. (C to E) Irradiated Tcrb−/–Tcrd−/− mice were reconstituted with a 50:50 mix of BM from CD45.1+ C57BL/6 (WT) and CD45.2+ Stat5a/bfl/fl-Cd4cre/+ (Stat5−/−) donors (C). Two months later, chimeric mice were infected with PR8 and treated or not with 250 μg of a mix of anti–IL-6 + anti–IL-6/R Abs on days 0, 2, 4, 6, and 8 after infection, and cells from the mLN were analyzed on day 10 after infection. (D) Frequency of PD-1hiCXCR5hi GC-TFH cells within the WT and Stat5−/− CD4+ T cell compartments. (E) Frequency of PD-1hiCXCR5hi GC-TFH cells within the WT and Stat5−/− NP-specific CD4+ T cell compartments. Representative plots are shown. Data in the graph are shown as means ± SD. Data were pooled from three independent experiments. *P < 0.05 and ***P < 0.001. P values were determined using a two-tailed Student’s t test.

  • Fig. 6 IL-6 signaling limits CD122 expression by inhibiting STAT5 association to the Il2rb locus.

    (A) OTII (CD45.1+) cells were adoptively transferred into WT and Il6−/− recipient mice. One day later, recipient mice were infected with PR8-OTII. Seven days after infection, donor-derived CD45.1+CXCR5hiSLAMloCD4+CD19 TFH and CD45.1+CXCR5loSLAMhiCD4+CD19 TEFF cells were sorted from the mLN of WT and Il6−/− recipient mice, and ATAC-seq was performed. UCSC genome browser tracks displaying chromatin accessibility peaks at the Il2rb locus are shown. Results are representative of two biological replicates from two independent experiments. (B) WT/Stat5−/− mixed BM chimeras were infected with PR8 and treated or not with 250 μg of a mix of anti–IL-6 + anti–IL-6/R Abs on days 0, 2, 4, 6, and 8 after infection, and expression of CD122 in WT and Stat5−/− PD-1hiCXCR5hi NP-specific GC-TFH cells was determined on day 10 after infection. Data in the graph are shown as means ± SD (n = 3 to 5 mice per group). Data are representative of three independent experiments. ***P < 0.001. P values were determined using a two-tailed Student’s t test. (C to E) CD4+ T cells from the spleen of naïve B6 mice were activated in vitro with plate-bound anti-CD3/CD28 Abs in the presence of rIL-6 or control PBS. (C) The expression of Il2rb was determined by RT-PCR on day 3. Data are normalized to Rps18 and represented as fold change in expression relative to the control condition. Data are shown as means ± SD (n = 5). (D and E) CD4+ T cells activated in the presence of IL-6 or control PBS were cross-linked with paraformaldehyde at 72 hours. Chromatin samples were immunoprecipitated with anti-STAT5 (D) or anti-STAT3 (E), and the indicated regions of the Il2rb locus were monitored by quantitative PCR. Data are shown as means ± SEM (n = 3). Samples were normalized to the total input, followed by subtraction of the isotype control to account for unspecific Ab binding and represented as relative fold change enrichment. Data are shown as means ± SEM (n = 3). Data are representative of three independent experiments. *P < 0.05 and ***P < 0.001. P values were determined using a two-tailed Student’s t test. n.s., not significant.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/39/eaaw7636/DC1

    Materials and Methods

    Fig. S1. TFH and GC B cell responses in the absence of IL-6.

    Fig. S2. IL-2 production by TFH cells.

    Fig. S3. IL-6 signaling fine-tunes IL-2 responsiveness of TFH cells.

    Fig. S4. IL-6 signaling prevents CD122 expression.

    Fig. S5. STAT3 and STAT5 binding to the Il2rb locus.

    Data file S1. Raw data file (Excel).

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. TFH and GC B cell responses in the absence of IL-6.
    • Fig. S2. IL-2 production by TFH cells.
    • Fig. S3. IL-6 signaling fine-tunes IL-2 responsiveness of TFH cells.
    • Fig. S4. IL-6 signaling prevents CD122 expression.
    • Fig. S5. STAT3 and STAT5 binding to the Il2rb locus.

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    Other Supplementary Material for this manuscript includes the following:

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