ARTICLES Quality of TCR signaling determined by differential affinities of enhancers for the composite BATF–IRF4 transcription factor complex Arifumi Iwata1, Vivek Durai1, Roxane Tussiwand2, Carlos G Briseño1, Xiaodi Wu1, Gary E Grajales-Reyes1, Takeshi Egawa1, Theresa L Murphy1 & Kenneth M Murphy1,3 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. The strength of TCR signaling can influence thymocyte fate ‘choice’1 motif: the AICE (‘activator protein 1 (AP-1)–interferon-regulatory and the effector outcome of T cells2, but how signal strength con- factor (IRF) composite element’)13–16. IRF4 is also recruited to EICE trols different gene programs has remained unclear3. Antigen dose motifs (‘E twenty-six (ETS)–IRF composite element’) through interac- 4,5 can alter the TH1 cell–TH2 cell balance , TH1 cell–follicular helper tions with the ETS family members PU.1 and SpiB in B cells and den- 6 T cell (TFH cell) balance and the production of interleukin 10 (IL-10) dritic cells (DCs), but not in T cells, due to the low abundance of ETS 7 11,17 by TH1 cells . The transcriptional repressor BCL-6 and transcription transcription factors . In plasma cells, which have low expression factor BLIMP-1, which support the development of TFH cells or TH1 of BATF, IRF4 has high expression and binds to interferon-sensitive cells, respectively, do show graded abundance at different TCR signal response elements18. strengths, but this cannot explain all graded T cell responses2, and The BATF subfamily of AP-1 factors includes BATF, BATF2 how differences in the strength of TCR signaling regulate the differ- and BATF3, which all bind DNA as heterodimers with Jun fac- ential abundance of these factors is unknown. tors17. BATF expression is restricted to the immune system and is 17,19 The transcription factor IRF4 might mediate some aspects of vari- required for the differentiation of TH9, TH17 and TFH cells and able TCR signaling3, including BLIMP-1 abundance8. The abundance for the differentiation and population expansion of effector CD8+ T of IRF4 increases in proportion to TCR signal strength and corre- cells20. Batf−/− CD8+ T cells produce less interferon-γ (IFN-γ) than lates with T cell population expansion and gene expression for meta- do wild-type cells, which indicates that BATF also regulates activa- bolic and biosynthetic pathways8–10. IRF4 is required for the effector tion20. BATF is also required for the germinal-center reaction and function of T cells11 and for the development, class-switch recom- for class-switch recombination in B cells21. BATF3 is expressed in bination and plasma-cell differentiation of B cells12. IRF4 binds the DCs and is required for the development of CD24+ DCs22. BATF DNA sequence GAAA but requires heterodimerization with other and BATF3 are expressed in different cell types but can compensate factors for high-affinity binding. In B cells and T cells, IRF4 forms for each other when expressed in the same cells13,17. BATFs enable a complex with a heterodimer composed of the transcription fac- IRF4- and IRF8-dependent transcription by binding cooperatively tor BATF (‘basic leucine zipper transcription factor, ATF-like’) and to two variants of AICEs: AICE1 (TTTCNNNNTGASTCA, where transcription factor Jun, which binds DNA at a specific sequence ‘N’ indicates any nucleotide, and ‘S’ indicates cytosine or guanine) 1Department of Pathology and Immunology, Washington University in St. Louis, School of Medicine, St. Louis, Missouri, USA. 2Department of Biomedicine, University of Basel, Basel, Switzerland. 3Howard Hughes Medical Institute, Washington University in St. Louis, School of Medicine, St. Louis, Missouri, USA. Correspondence should be addressed to K.M.M. ([email protected]). Received 4 August 2016; accepted 23 February 2017; published online 27 March 2017; doi:10.1038/ni.3714 NATURE IMMUNOLOGY VOLUME 18 NUMBER 5 MAY 2017 563 ARTICLES and AICE2 (GAAATGASTCA)14,15,17. BATF and IRF4 are both a induced within 4 h of TCR stimulation and thus might initiate the Anti-CD3ε: 0.5 ng/ml 1.0 ng/ml 2.2 ng/ml 4.6 ng/ml 10 ng/ml 105 0 0 4 2 10 46 0 85 0 100 expression of many genes encoding products associated with activa- 4 23 10 tion and differentiation . 103 2 The role of BATF in TH2 differentiation has remained unclear due 10 0 100 0 93 1 31 13 3 11 0 0 13,24,25 IRF4 to differing results . BATF was shown not to be required for 2 3 4 5 13,21,25 0 10 10 10 10 the development of TH2 cells , but other studies have reported BATF −/− 24 impaired TH2 development in distinct Batf mice . Batf3 expression b Anti-CD3 : 0.5 ng/ml 1.0 ng/ml 2.2 ng/ml 4.6 ng/ml 10 ng/ml compensates for loss of BATF in TH2 development, maintaining the ε 5 expression of IL-4 and IL-10 but not of CTLA-4, and Batf−/−Batf3−/− 10 0 0 5 0 53 15 10 79 2 94 104 (Batf1-Batf3 DKO) T cells lack expression of IL-4, IL-10 and CTLA-4 103 (ref. 13). This suggests that genes that are targets of BATFs include 102 0 100 0 94 1 31 1 3 8 0 4 some that are sensitive to compensation by endogenous BATF3, GATA-3 0 102 103 104 105 but that varying conditions of activation24 might influence the CTLA-4 amount BATF3 or the sensitivity of target genes to compensation by c BATF3. In either case, the basis for such differential sensitivity has 0.5 ng/ml 1.0 ng/ml 2.2 ng/ml 4.6 ng/ml 10 ng/ml 105 remained unclear. 104 103 Here, we first documented clearly distinct sensitivities of several 4 −/− 102 genes to compensation by BATF3 in Batf TH2 cells. We found that 0 GATA3 IRF4 CTLA- enhancers that controlled BATF-dependent TCR-inducible genes 0 102 103 104 105 GATA3 responded to different levels of BATF–IRF4. For genes that were BATF CTLA-4 BATF BATF highly sensitive to low levels of total BATF, endogenous BATF3 was Figure 1 GATA-3 and CTLA-4 are differentially sensitive to graded −/− able to compensate for BATF in Batf TH2 cells, while for genes expression of BATF and IRF4 in TH2 cells following increasing strength of whose expression required higher levels of BATF, it was unable to do TCR stimulation. (a) Flow cytometry analyzing the expression of IRF4 and so. By ChIP followed by deep sequencing (ChIP-seq) and ChIP-exo BATF on day 4 of primary activation in wild-type CD4+ T cells cultured analysis, we found that the sensitivity of enhancers in these genes under TH2 conditions (anti-IFN-γ, anti-IL-12 and IL-4) with antibody to to BATF was regulated by sequences surrounding AICE motifs that the co-receptor CD28 (anti-CD28) and various concentrations (above plots) of anti-CD3ε crosslinked by plate-bound antibody to hamster influenced affinity for the BATF–IRF4 ternary complex. ChIP-exo immunoglobulin G (IgG). Numbers in quadrants indicate percent cells in analysis helped to identify a previously unknown AICE2 motif that each throughout. (b) Flow cytometry analyzing the expression of GATA-3 conferred high affinity for BATF–IRF4 that might control a SNP in and CTLA-4 in wild-type CD4+ T cells on day 4 of culture as in a. the CTLA4 locus known to result in a lower incidence of autoimmune (c) Overlay of flow cytometry data from a and b showing expression diseases26,27. of IRF4, BATF, GATA-3 and CTLA-4 at various doses (key) of plate bound anti-CD3ε. Data are representative of two experiments with five RESULTS biological replicates. GATA-3 and CTLA-4 respond to distinct signal strengths We assessed the expression of BATF, IRF4, GATA-3 and CTLA-4 in Differential compensation of BATF targets in TH2 cells by Batf3 TH2 cells activated with a ‘graded’ level of TCR signaling. IRF4 was Expression of CTLA-4 in TH2 cells was partially dependent on BATF −/− −/− induced in a gradual and uniform manner in proportion to TCR sig- but was completely lacking in Batf Batf3 (Batf1-Batf3 DKO) TH2 nal strength (Fig. 1a), consistent with published reports8–10. BATF cells, even at a high dose of antibody to the TCR invariant chain CD3ε expression was also graded and had dose-dependent responses to (anti-CD3ε) in a secondary stimulation, as reported13 (Fig. 2a,b). In TCR signaling similar to those of IRF4 (Fig.