Abstract
Variable strengths of signaling via the T cell antigen receptor (TCR) can produce divergent outcomes, but the mechanism of this remains obscure. The abundance of the transcription factor IRF4 increases with TCR signal strength, but how this would induce distinct types of responses is unclear. We compared the expression of genes in the TH2 subset of helper T cells to enhancer occupancy by the BATF–IRF4 transcription factor complex at varying strengths of TCR stimulation. Genes dependent on BATF–IRF4 clustered into groups with distinct TCR sensitivities. Enhancers exhibited a spectrum of occupancy by the BATF–IRF4 ternary complex that correlated with the sensitivity of gene expression to TCR signal strength. DNA sequences immediately flanking the previously defined AICE motif controlled the affinity of BATF–IRF4 for direct binding to DNA. Analysis by the chromatin immunoprecipitation–exonuclease (ChIP-exo) method allowed the identification of a previously unknown high-affinity AICE2 motif at a human single-nucleotide polymorphism (SNP) of the gene encoding the immunomodulatory receptor CTLA-4 that was associated with resistance to autoimmunity. Thus, the affinity of different enhancers for the BATF–IRF4 complex might underlie divergent signaling outcomes in response to various strengths of TCR signaling.
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Acknowledgements
We thank the Alvin J. Siteman Cancer Center at Washington University School of Medicine for use of the Center for Biomedical Informatics and Multiplex Gene Analysis Genechip Core Facility. Supported by the Howard Hughes Medical Institute (K.M.M.) and the US National Institutes of Health (RO1 AI097244-01A1 to T.E., F30DK108498 to V.D., 1F31CA189491-01 to G.E.G.-R.).
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A.I., T.L.M. and K.M.M. designed the study, and wrote the manuscript with contributions from all authors; A.I. and C.G.B. performed experiments related to cell sorting, culture and flow cytometry with advice from V.D., R.T., G.E.G.-R. and T.E.; A.I. and C.G.B. performed microarray experiments with advice from X.W. and T.L.M.; and A.I. performed and analyzed ChIP-Seq experiments with advice from V.D. and T.E.
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Supplementary Figure 1 GATA3 and CTLA-4 are differentially sensitive to graded expression of BATF and IRF4 in TH2 cells.
(a) Flow cytometry analyzing IRF4 and BATF expression on day 4 of secondary activation in WT CD4+ T cells cultured under TH2 conditions (anti-IFN-γ, anti-IL-12 and IL-4) with anti-CD28 and the indicated concentration of anti-CD3ε crosslinked by plate-bound anti-hamster IgG. Numbers indicate the percentage of live CD4+ cells in each quadrant. (b) Flow cytometry analyzing GATA3 and CTLA-4 expression on day 4 in WT CD4+ T cells cultured as in (a). (c) Overlays of flow cytometry data from (a) and (b) showing expression of the indicated proteins at various doses of plate bound anti-CD3e. Data are representative of two biological replicates (a-c). (d,e) Flow cytometry analyzing GATA3, CTLA4 and BATF on day 4 of primary activation of naïve KJ126+ CD4 T cells from DO11.10 mice activated under TH2 conditions with the indicated concentration of ovalbumin (323-339) peptide and dendritic cells from Balb/c mice. Data are representative of two biological replicates. (f) Histograms and MFI for BATF, GATA3 and CTLA4 expression from (d,e).
Supplementary Figure 2 Kinetics of the expression of BATF, IRF4, GATA3 and CTLA4 in TH2 cells.
(a) Flow cytometry analyzing IRF4 and BATF expression at the indicated time after primary activation of WT cells cultured under TH2 condition (anti-IFN-γ, anti-IL-12 and IL-4) using 10 ng/ml of anti-CD3ε and 4 μg/ml of anti-CD28 crosslinked with plate-bound anti-hamster IgG. (b) Flow cytometry analyzing CTLA-4 and GATA3 expression in TH2 cells cultured as in (a). (c) Flow cytometry analyzing BATF and GATA3 expression in TH2 cells as in (a). (d) Flow cytometry analyzing BATF and CTLA-4 expression in TH2 cells as in (a). Data are representative of three biological replicates. (e, f) Histograms and MFI for factor expression from (a-d).
Supplementary Figure 3 Analysis of Batf mutations and phenotype of Batf1-Batf3 DKO cells.
(a) Batf1/3 DKO CD4 T cells were cultured under TH17 conditions (anti-IFN-, anti-IL-12, anti-IL-4, IL-6, TGF- and IL-1 ) on plate-bound anti-CD3 and anti-CD28 and were infected on day 1 of primary stimulation with empty retrovirus or retrovirus containing WT Batf or the indicated Batf mutant (Batf-H: Batf H55Q, Batf-HK: Batf H55Q K63D, Batf-HKE: Batf H55Q K63D E77K, Batf-HKLE: Batf H55Q K63D L56A E77K.) On day 5 after secondary, cells were stimulated with PMA/ionomycin and analyzed for IL-17 expression. Shown is the activity of each mutant for reconstitution of IL-17 positive cells relative to WT Batf in the same experiment. Bars showed the mean of two experiments. (b) Batf1/3 DKO B cells were cultured with LPS and IL-4, and infected on day 1 with empty retrovirus or retrovirus containing the indicated Batf mutant described in (a). Class switch recombination to IgG1 was analyzed on day 4. Shown is the activity of each mutant for reconstitution of IgG1 positive cells relative to WT Batf in the same experiment. Bars showed the mean of two experiments. (c) Batf1/3 DKO bone marrow cells were cultured with Flt3 ligand and infected on day 1 with empty retrovirus or retrovirus containing WT Batf or the indicated Batf mutant described in (a). CD24+ CD172a− CD11c+ MHCII+ B220− SiglecH− cells were analyzed on day 9. Shown is the activity of each mutant for reconstitution of CD24+ DCs relative to WT Batf in the same experiment. Bars show the mean of three experiments. (d) Microarray expression of cytokines and transcription factors in WT and TH2 cells on day 0 or 2 days after primary activation with 10ng/ml anti-CD3 (day2) or 4 days after primary activation with 2ng/ml or 10ng/ml anti-CD3 as indicated. Shown is the mean and SEM for three biological replicates.
Supplementary Figure 4 TH2 cell–related genes have low sensitivity to the binding of BATF and IRF4.
(a) ChIP-seq for BATF in WT TH2 cells and IRF4 in WT and Batf1/3 DKO TH2 cells (DKO) and in Batf1/3 DKO TH2 cells reconstituted with retroviral Batf-HKE (Batf-HKE). Cells were prepared for ChIP after PMA/ionomycin stimulation on day 4 of secondary stimulation. (b) IRF4 ChIP-seq in WT TH2 cells cultured with 10 ng/ml of anti-CD3ε for 4 days, 2.2 ng/ml of anti-CD3ε for 4 days, or 10 ng/ml of anti-CD3ε for 2 days. (c) ChIP-seq for BATF3 in Batf−/− TH2 cells and IRF4 in WT and Batf−/− TH2 cells within genes from Cluster I/II and Cluster III. ChIP-seq was performed after PMA/ionomycin activation on day 4 of secondary stimulation and is compared with data from Figure 4a. (d) ChIP-seq for BATF3 in Batf−/− TH2 cells and IRF4 in WT and Batf−/− TH2 cells within TH2 related genes. ChIP-seq was performed after PMA/ionomycin activation on day 4 of secondary stimulation and is compared with data from Supplementary Figure 4a. *Peaks that do not bind BATF3 in Batf−/− TH2 cells.
Supplementary Figure 5 Affinity of IRF4 ChIP-seq peaks correlates with BATF-dependent gene expression, but peak affinity cannot be distinguished by de novo motif analysis.
(a) Merged IRF4 ChIP-seq peaks from three conditions of primary stimulation were categorized as high-affinity (present in three conditions), intermediate-affinity (two conditions) and low-affinity (one condition). Color map shows the intensity of IRF4 binding centered on the peak ±1 kb. (b) The number of high-affinity peaks, intermediate-affinity peaks and low-affinity peaks within ±50 kb from TSS of Cluster I and II genes or Cluster III genes. Chi-square test, p = 3.417e-08, standardized residuals, high affinity peaks: 5.74, low sensitivity peaks: -5.61. (c) De novo motif analysis for the top 3000 IRF4 peaks in each category ranked by sum of tag counts of three conditions. P, final enrichment p-value; T, number of target sequences with motif as percent of total targets; B, number of background sequences with motif as percent of total background.
Supplementary Figure 6 The flanking region regulates the affinity of a motif for the BATF-IRF4 complex.
(a-d) ChIP-seq for IRF4 using the indicated primary activation condition of mouse TH2 cells. Shown are pairs of AICE1-containing peaks, Enpp6 -45 kb and Ptchd3 -26 kb, or Bcor +65 kb and Mzt1 +230 kb with identical AICE1 sequences but different flanking regions (a) or pairs of AICE2-containing peaks, Prdm1 +14 kb and Ctla4 -33 kb, or Ccr4 +8 kb and Snrpe +38 kb with identical AICE2 sequences but different flanking regions (b). (c) EMSA using nuclear extracts of HEK293FT cells expressing BATF, JUNB and IRF4 with probes based on sequences in (a). Open triangle: BATF/JUNB. Solid triangle: BATF/JUNB/IRF4. (d) EMSA using nuclear extract of HEK293FT cells expressing BATF, JUNB and IRF4 with probes based on sequences in (b). Data were representative of two experiments (c,d). Full sequence of probes are shown in Supplementary Table 2 (c,d).
Supplementary Figure 7 Multiple AICE motifs within single IRF4 ChIP-seq peaks.
(a) Proportion of IRF4 peaks with multiple AICE motifs identified by de novo motif analysis. (b) ChIP-seq analysis and ChIP-exo analysis of BATF and IRF4 in WT TH2 cells. Black bars: predicted AICE sites. (c,d) Example of site from (a) with ChIP-exo binding.
Supplementary Figure 8 AICE motif sites identified by de novo motif analysis show ChIP-exo binding, but ETS motif sites do not.
(a) Mean ChIP-exo tag counts of BATF and IRF4 binding on AICE1, AICE2 and ETS motifs within IRF4 ChIP-seq peaks that were identified by de novo motif analysis. (b) Mean ChIP-exo tag counts of BATF and IRF4 on consensus ETS motifs within IRF4 ChIP-seq peaks showing ±50 bp flanking regions; red box: exo binding region of IRF4, light red box: control region for IRF4, blue box: exo binding region of BATF, light blue box: control region for BATF. (c) AICE1 motifs predicted by de novo motif analysis of IRF4 ChIP-seq. (d) AICE1 motifs identified from sites with higher ChIP-exo tag count on exo binding region of both BATF and IRF4 than control region.
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Iwata, A., Durai, V., Tussiwand, R. et al. Quality of TCR signaling determined by differential affinities of enhancers for the composite BATF–IRF4 transcription factor complex. Nat Immunol 18, 563–572 (2017). https://doi.org/10.1038/ni.3714
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DOI: https://doi.org/10.1038/ni.3714
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