Research ArticleIMMUNE REGULATION

Interleukin-10 from CD4+ follicular regulatory T cells promotes the germinal center response

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Science Immunology  20 Oct 2017:
Vol. 2, Issue 16, eaan4767
DOI: 10.1126/sciimmunol.aan4767
  • Fig. 1 Tfr cells robustly secrete IL-10 after acute viral infection.

    Analysis of the Treg cell response after LCMV infection in IL-10 reporter (10BiT Thy1.1) mice. (A) Quantification of the number of Tfr cells, non-Tfr Treg cells, pre-Tfh cells, Tfh cells, and GC B cells at days 0, 5, 8, and 12 after infection. Populations are defined as follows: Tfr cells, CD4+Ly6CPSGL1loCXCR5hiPD1hiFoxp3+; non-Tfr Treg cells, CD4+CXCR5int-loPD1int-loFoxp3+; pre-Tfh cells, CD4+CD44hiLy6CPSGL1loCXCR5intPD1intFoxp3; Tfh cells, CD4+CD44hiLy6CPSGL1loCXCR5hiPD1hiFoxp3; and GC B cells, B220+IgDloGL7+CD95+. (B) Quantification of the ratio of Tfr cells to Tfh cells or GC B cells at days 0, 5, 8, and 12 after infection. (C) Representative plot of IL-10 expression (assessed as Thy1.1) by Tfr cells (left) and non-Tfr Treg cells (middle) from mice as described in (A). Right: Frequency of Thy1.1+ cells in Tfr cells and non-Tfr Treg cells at days 0, 5, 8, and 12 after LCMV Armstrong infection. Statistical analyses were performed using unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001). Data are from two experiments representative of four experiments with three to six mice per time point after LCMV Armstrong infection.

  • Fig. 2 Regulatory CD4+ T cell–derived IL-10 is important for B cell differentiation and the GC response.

    Analysis of the B cell response in Il10f/f and Il10f/fFoxp3-Cre mice 15 days after LCMV infection. (A) Top: Representative plots of the B cell responses in Il10f/f or Il10f/fFoxp3-Cre mice. Bottom: Frequency and number of cells as indicated by the gates shown above. (B) Representative confocal images of the GC from Il10f/f or Il10f/fFoxp3-Cre mice. Right: GC sizes as measured by ImageJ. The sections were taken from four mice of each genotype. Scale bar, 100 μm. (C) Left: Frequency of plasmablasts and memory B cells defined by the expression of surface markers. Right: Absolute numbers of plasma cells and memory B cells. (D) Enzyme-linked immunosorbent assay quantification of LCMV-specific IgG2a and IgG1 antibody levels, indicated by arbitrary units (a.u.). All the analyses of the GC response were performed 15 days after acute LCMV Armstrong infection. n.s., not significant. (E) Enzyme-linked immunospot quantification of the number of LCMV-specific IgG2a and IgG1 memory B cells. All the analyses of the GC response were performed 60 days after acute LCMV Armstrong infection. Statistical analyses were performed using unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001). Data for (A) to (C) are from one experiment representative of three experiments with three to five mice per group carried out 15 days after LCMV Armstrong infection and from four experiments with three to five mice per group carried out 12 days after LCMV Armstrong infection. Data for (D) are pooled from two experiments carried out 12 or 15 days after infection. Data for (E) are pooled from two experiments with five to seven mice per group carried out 60 days after infection.

  • Fig. 3 Tfr cell–derived IL-10 is important for B cell differentiation and the GC response.

    (A) Analysis of the B cell response at day 15 after acute LCMV Armstrong infection in 50:50 Il10f/f/Bcl6f/fFoxp3-Cre or Il10f/fFoxp3-Cre/Bcl6f/fFoxp3-Cre mBMCs. Left: Schematic for the experiment. Right: Data are pooled from two experiments with four to five mice per group carried out 15 days after LCMV Armstrong infection. (B) Analysis of the B cell response at day 15 after acute LCMV Armstrong infection in 50:50 Il10f/f/SAP−/− or Il10f/fFoxp3-Cre/SAP−/− mBMCs. Left: Schematic for the experiment. Right: Data are from one experiment representative of two experiments with four to nine mice per group carried out 15 days after LCMV Armstrong infection. Statistical analyses were performed using unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001).

  • Fig. 4 IL-10 acts on B cells to regulate the GC response.

    (A) GC B cells and DCs, but not Tfh cells, are responsive to IL-10. Analysis of IL-10 responsiveness in GC B cells, DCs (CD11chiMHCII+), and Tfh cells. Splenocytes isolated from mice at day 12 after LCMV infection were stimulated for 30 min with IL-10, and pSTAT3 levels were determined. Data are from one experiment representative of three experiments with four mice per group. (B) Analysis of the B cell response in Il10rαf/f, Il10rαf/fCd19-Cre, Il10rαf/fCd11c-Cre, and Il10rαf/fCd4-Cre mice 15 days after LCMV infection. Top: Representative plots of B cell response. Bottom: Quantification of B cell response shown above. Statistical analyses were performed using unpaired two-tailed Student’s t test (*P < 0.05; **P < 0.01). Data are from one experiment representative of two to three experiments with three to seven mice per group carried out 15 days after LCMV Armstrong infection. Mice for the control Il10rαf/f group were pooled from littermate controls produced from the independent crosses used to generate the experimental groups.

  • Fig. 5 GC B cells in mice lacking regulatory CD4+ T cell–derived IL-10 display an enhanced light zone GC B cell gene signature.

    (A) RNA-seq analysis of select DEGs among mRNA isolated from GC B cells pooled from Il10f/f and Il10f/fFoxp3-Cre mice 12 days after LCMV Armstrong infection presented as expression (log2) in Il10f/fFoxp3-Cre cells relative to that in Il10f/f cells (key below; columns indicate paired replicates). (B) GSEA of light zone and dark zone signatures in GC B cells, based on published gene sets (6, 41). A positive enrichment score (ES) signifies enrichment in the Il10f/fFoxp3-Cre sample relative to the Il10f/f condition of a given gene set, that is, more highly expressed. (C) Normalized ES (NES) for select pathways identified from the Reactome Pathway Database (R), Kegg Pathway Database (K), and published gene sets (G), where dot size represents P values adjusted by the family-wise error rate (FWER). All gene sets attain significant enrichment [false discovery rate (FDR) < 0.001], with the exception of the dark zone gene set (FDR = 0.5). Data are from three independent experiments with three mice per group pooled for each sample. (D) Analysis of the light zone and dark zone GC B cell response in Il10f/f and Il10f/fFoxp3-Cre mice 15 days after LCMV infection. Representative plots (left) of GC B cells as gated in fig. S1B. The numbers of the outlined area indicate dark zone (top left) and light zone (bottom right) based on the expression of the surface markers CXCR4 and CD86. Middle: Frequency of GC B cells from light zone gate defined by CXCR4+CD86 and dark zone gate defined by CXCR4CD86+ from Il10f/f or Il10f/fFoxp3-Cre mice. Right: Expression of CXCR4 in GC B cells. Statistical analyses were performed using unpaired two-tailed Student’s t test (*P < 0.05). Data are from one experiment representative of two experiments with at least four mice per group carried out 15 days after LCMV Armstrong infection. MFI, mean fluorescence intensity.

  • Fig. 6 Reduced FOXO1 expression in GC B cells from mice lacking regulatory CD4+ T cell–derived IL-10.

    (A) IPA canonical pathway analysis. The number on the x axis indicates the negative log P values adjusted by the Benjamini-Hochberg (B-H) procedure, which is the significance of the probability that the genes in the data set described here are associated with the canonical pathway listed. The orange line represents the threshold of significance for a B-H multiple testing correction P value of 0.05. (B) IPA upstream regulator analysis. The top upstream regulators that are predicted to be responsible for the gene expression changes observed in the Il10f/fFoxp3-Cre sample relative to the Il10f/f sample are shown. A negative activation z score indicates that the upstream regulator is predicted to be inhibited in the Il10f/fFoxp3-Cre sample relative to the Il10f/f sample, and a positive score indicates that the upstream regulator is predicted to be activated. (C) Expression of FOXO1 in GC B cells from Il10f/f or Il10f/fFoxp3-Cre mice 15 days after LCMV Armstrong infection. Data are from one experiment representative of two experiments with at least four mice per group carried out 15 days after LCMV Armstrong infection. (D) Assessment of the percentage of nuclear translocated FOXO1 after IL-10 stimulation in IgDlo B cells 5 days after LCMV infection as determined by Amnis ImageStream. Statistical analyses were performed using paired two-tailed Student’s t test (*P < 0.05; **P < 0.01). Data are pooled from three experiments with four mice per group carried out 5 days after LCMV Armstrong infection. (E) Assessment of the number of mutations in the CDR region (left) and selection strength (right) in both the CDR region and FWR of a large number of the VH genes of GC B cells from Il10f/f or Il10f/fFoxp3-Cre mice 15 days after LCMV infection. Data are pooled from two experiments with three to four mice per group carried out 15 days after LCMV Armstrong infection.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/16/eaan4767/DC1

    Supplementary Methods

    Fig. S1. Flow cytometric gating strategy for Tfh, pre-Tfh, GC B, Treg, and Tfr cells, with IL-10 expression by these populations.

    Fig. S2. Mice lacking regulatory CD4+ T cell–derived IL-10 do not have defects in steady-state lymphoid cell populations.

    Fig. S3. Temporal development of the GC B cell response in mice lacking regulatory CD4+ T cell–derived IL-10.

    Fig. S4. Systemic IL-10 is not required for the GC response.

    Fig. S5. Regulatory CD4+ T cell–derived IL-10 is not required for effector CD4+ T cell differentiation.

    Fig. S6. Heat map of DEGs based on RNA-seq.

    Fig. S7. GC B cells in mice lacking regulatory CD4+ T cell–derived IL-10 display similar levels of proliferation and death.

    Fig. S8. IL-10 does not induce FOXO1 nuclear translocation in IgDhi B cells.

    Fig. S9. The VH CDR3 region of GC B cells in mice lacking regulatory CD4+ T cell–derived IL-10 displays altered amino acid physiochemical properties.

    Table S1. Tabulated data for Figs. 1 to 6 and figs. S1 to S9.

    References (5768)

  • Supplementary Materials

    Supplementary Material for:

    Interleukin-10 from CD4+ follicular regulatory T cells promotes the germinal center response

    Brian J. Laidlaw, Yisi Lu, Robert A. Amezquita, Jason S. Weinstein, Jason A. Vander Heiden, Namita T. Gupta, Steven H. Kleinstein, Susan M. Kaech, Joe Craft*

    *Corresponding author. Email: joseph.craft{at}yale.edu

    Published 20 October 2017, Sci. Immunol. 2, eaan4767 (2017)
    DOI: 10.1126/sciimmunol.aan4767

    This PDF file includes:

    • Supplementary Methods
    • Fig. S1. Flow cytometric gating strategy for Tfh, pre-Tfh, GC B, Treg, and Tfr cells, with IL-10 expression by these populations.
    • Fig. S2. Mice lacking regulatory CD4+ T cell–derived IL-10 do not have defects in steady-state lymphoid cell populations.
    • Fig. S3. Temporal development of the GC B cell response in mice lacking regulatory CD4+ T cell–derived IL-10.
    • Fig. S4. Systemic IL-10 is not required for the GC response.
    • Fig. S5. Regulatory CD4+ T cell–derived IL-10 is not required for effector CD4+ T cell differentiation.
    • Fig. S6. Heat map of DEGs based on RNA-seq.
    • Fig. S7. GC B cells in mice lacking regulatory CD4+ T cell–derived IL-10 display similar levels of proliferation and death.
    • Fig. S8. IL-10 does not induce FOXO1 nuclear translocation in IgDhi B cells.
    • Fig. S9. The VH CDR3 region of GC B cells in mice lacking regulatory CD4+ T cell–derived IL-10 displays altered amino acid physiochemical properties.
    • References (57–68)

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Tabulated data for Figs. 1 to 6 and figs. S1 to S9.

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