Proper immune responses are needed to control pathogen infection at mucosal surfaces. IL-22–producing CD4+ T cells play an important role in controlling bacterial infection in the gut; however, transcriptional regulation of these cells remains elusive. In this study, we show that mice with targeted deletion of the fourth DNA-binding zinc finger of the transcription factor Ikaros had increased IL-22–producing, but not IL-17–producing, CD4+ T cells in the gut. Adoptive transfer of CD4+ T cells from these Ikaros-mutant mice conferred enhanced mucosal immunity against Citrobacter rodentium infection. Despite an intact in vivo thymic-derived regulatory T cell (Treg) compartment in these Ikaros-mutant mice, TGF-β, a cytokine well known for induction of Tregs, failed to induce Foxp3 expression in Ikaros-mutant CD4+ T cells in vitro and, instead, promoted IL-22. Aberrant upregulation of IL-21 in CD4+ T cells expressing mutant Ikaros was responsible, at least in part, for the enhanced IL-22 expression in a Stat3-dependent manner. Genetic analysis using compound mutations further demonstrated that the aryl hydrocarbon receptor, but not RORγt, was required for aberrant IL-22 expression by Ikaros-mutant CD4+ T cells, whereas forced expression of Foxp3 was sufficient to inhibit this aberrant cytokine production. Together, our data identified new functions for Ikaros in maintaining mucosal immune homeostasis by restricting IL-22 production by CD4+ T cells.

Mucosal immunity requires the concerted action of innate and adaptive immune systems, among which IL-22–mediated CD4+ Th cell responses (e.g., Th17 and/or Th22 cells) are particularly important for the host to control bacterial infections in the gut, whereas regulatory T cells (Tregs) are important to limit inflammation and maintain homeostasis. Citrobacter rodentium is a murine pathogen that models human enterohemorrhagic and enteropathogenic Escherichia coli infections, which are responsible for the deaths of several hundred thousand children each year (1). Clearance of C. rodentium requires both innate and adaptive immune responses (2, 3). Although RAR-related orphan receptor gamma t (RORγt)+ group 3 innate lymphoid cells are essential for protection against infection (47), CD4+ T cell production of IL-22 is important for the host to control C. rodentium infection (8, 9). Indeed, transferring either IL-22–producing innate lymphoid cells (e.g., group 3 innate lymphoid cells) (4) or CD4+ T cells (e.g., Th22) (8) protects mice from C. rodentium infection, thereby highlighting the crucial role of IL-22 in mucosal immunity. Various proinflammatory cytokines (e.g., IL-6, IL-21, and IL-23) promote IL-22–producing CD4+ T cell responses (1015). In contrast, TGF-β was shown to inhibit IL-22 production by CD4+ T cells (1618).

The differentiation and function of CD4+ T cells are influenced by multiple transcription factors induced and/or activated by signals stemming from the local cytokine microenvironment. Activation of the nuclear receptor RORγt in response to TGF-β, in addition to Stat3-activating cytokines (e.g., IL-6, IL-21, and IL-23), is crucial for expression of the genes currently defining the Th17 cell program (e.g., IL-17 and/or IL-22) (1015). Although also induced by TGF-β, the transcription factor Foxp3, a lineage marker for Tregs, is able to suppress Th17 cell differentiation through antagonism of RORγt transcriptional activity, in part via physical interaction between the proteins (1921). Among the transcription factors implicated in Th17 cell differentiation, the ligand-dependent aryl hydrocarbon receptor (Ahr), best known for mediating the effects of environmental toxins (e.g., dioxin), is essential for IL-22 expression and is thought to enhance the expression of IL-17 by CD4+ T cells in vitro (2224). The activation of transcription factor Ahr, together with RORγt, induces IL-22 transcription (6), whereas c-Maf was shown to repress IL-22 expression by CD4+ T cells (16).

Ikaros is a highly conserved zinc finger protein with four amino (N)-terminal DNA-binding zinc fingers and two carboxyl (C)-terminal zinc fingers that mediate dimerization (25, 26). Ikaros is required for lymphocyte development, because its deletion completely abrogates fetal T and B lymphocytes, as well as adult B cells (27). Although Ikaros-null mice display postnatal T cells, their development is perturbed and results in clonal expansion of abnormal T cells (27). Depending on the context, Ikaros was shown to function either as a transcriptional activator or repressor (i.e., Ikaros promotes expression of Il10 or represses Il2, Tbx21, and Ifng) (2832). Although N-terminal zinc fingers 2 and 3 were shown to be required for DNA binding and function (33), recent studies suggested that Ikaros zinc fingers 1 and 4 play important roles in regulating target gene specificity (34). Specifically, it was shown that the two flanking DNA-binding zinc fingers regulate distinct sets of genes by modulating binding to different genomic sites (34). Of note, despite thousands of Ikaros-binding sites identified by chromatin immunoprecipitation (ChIP) sequencing, only a small number of genes was found to be either upregulated or downregulated in the absence of full-length wild-type Ikaros (i.e., Ikaros lacking zinc finger 1 or 4) by RNA sequencing, suggesting that Ikaros may directly regulate only a limited number of target genes involved in different hematopoietic lineages, developmental stages, and biological functions (34). Recent studies implicated Ikaros in the regulation of different stages of T cell development and in several T cell subsets (28, 30, 35). Taken together, these studies indicate the complex roles of Ikaros in multiple stages of T cell differentiation and function that are still incompletely understood. Specifically, the function of Ikaros in regulating IL-22–mediated T cell responses in gut immunity is unknown. In this study, we uncover a critical function of Ikaros in mucosal homeostasis and immunity by restricting IL-22–producing CD4+ T cells and regulating different Treg compartments. We identify a unique role for Ikaros in regulating IL-22 single-producing cells versus IL-17 and IL-22 double-producing Th17 cells.

To determine the role of Ikaros in CD4+ T cell responses both in vitro and in vivo in the steady-state and during infection, we used two Ikaros mutant mouse strains with germline-targeted deletion of Ikzf1 exons encoding zinc finger 1 (Ikzf1ΔF1/ΔF1) or 4 (Ikzf1ΔF4/ΔF4) (34). Of note, unlike Ikaros-null mice (Ikzf1−/−), which display developmental perturbation of various immune compartments, Ikzf1ΔF1/ΔF1 and Ikzf1ΔF4/ΔF4 mice have fewer and distinct immune defects (34), thus making them an appropriate model system to dissect the function of Ikaros in CD4+ T cells. By using a series of genetic and pharmacological experiments, our data reveal new functions of Ikaros in the regulation of cytokine production and transcription factor expression and/or activity in CD4+ T cells, and, thus, suggest a new role for Ikaros in limiting CD4+ T cell immune responses in vivo during mucosal intestinal infection that is controlled by IL-22.

All mice used in this study were maintained in specific pathogen–free facilities at Northwestern University. The mice were littermate controlled and were 6–10 wk old, unless otherwise indicated. Ikzf1ΔF4/ΔF4, Ikzf1ΔF1/ΔF1, Ikzf1+/−, Rorcgfp/gfp, Ahr−/−, and Stat3f/f mice were described previously (27, 34, 3638) and were all fully backcrossed to the C57BL/6 background. Cd4-cre and Rag1−/− mice were purchased from Taconic Farms or the Jackson Laboratory. All studies with mice were approved by the Animal Care and Use Committee of Northwestern University.

The isolation of intestinal lamina propria cells and flow cytometry were done as previously described (6). Splenocytes were made into a single-cell suspension, and CD4+ T cells were purified with a CD4+ T Cell Isolation Kit (Miltenyi Biotec) to 90–95% purity. Twenty-four–well plates were coated with anti-hamster Ab (MP Biomedical). CD4+ T cells were cultured in T cell media containing IMDM (Sigma-Aldrich) supplemented with soluble hamster anti-mouse CD3 (0.25 μg/ml; unless otherwise indicated), hamster anti-mouse CD28 (1 μg/ml), anti-mouse IL-4 (11B11, 2 μg/ml; Bio X Cell), and anti-mouse IFN-γ (XMG1.2, 2 μg/ml; Bio X Cell). For inducible Treg (iTreg) differentiation, 5 ng/ml TGF-β was added to the culture. For Th17 cell differentiation, TGF-β was added at 5 ng/ml, and IL-6 was added at 20 ng/ml. In some experiments, 6-formylindolo[3,2-b]carbazole (FICZ) was added at a concentration of 200 nM. For blocking RORγt or Stat3 activity, cells were cultured with 10 μM digoxin (Sigma-Aldrich) or 15 μM STA21 (Santa Cruz Biotechnology), respectively, with controls of DMSO. For IL-21 neutralization, the cells with cultured with 12.5 μg/ml anti–IL-21 (R&D Systems) or control IgG.

CD16/32 Ab was used to block the nonspecific binding to FcR before surface staining in all gut lamina propria lymphocytes and some splenocyte experiments. Abs were purchased from eBioscience, BD Pharmingen, or R&D Systems. For nuclear staining, cells were fixed and permeabilized using a Mouse Regulatory T Cell Staining Kit (eBioscience). For intracellular staining, cells were fixed and permeabilized using a Mouse Regulatory T Cell Staining Kit (eBioscience) or the BD Cytofix/Cytoperm Kit. For cytokine production, cells were stimulated ex vivo by 50 ng/ml PMA and 500 ng/ml ionomycin for 4 h. Brefeldin A (2 μg/ml) was added 2 h before cells were harvested for analysis. Dead cells in the gut were excluded from the analysis using a LIVE/DEAD Violet Viability Kit (Invitrogen). Live lymphocytes, isolated from the spleen or thymus, were gated on forward scatter (FSC)/side scatter. Flow cytometry data were collected using a FACSCanto II or FACSCalibur (BD Biosciences) and analyzed with FlowJo software (TreeStar). The amount of IL-21 in the culture supernatant was measured by an ELISA kit (R&D Systems). The assays were performed in duplicate, according to the manufacturer’s instructions.

RNA from sorted cell populations was isolated with TRIzol reagent (Invitrogen). cDNA was synthesized using a GoScript Reverse Transcription Kit (Promega). Real-time RT-PCR was performed using SYBR Green (Bio-Rad) and different primer sets (Supplemental Table I).

Reactions were run using the MyiQ2 Two-Color Real-Time PCR Detection System (Bio-Rad). The results were analyzed by the comparative CT method and displayed as relative gene expression values normalized to β-actin.

Approximately 5 × 106 CD4+ T cells under the indicated polarizing conditions (i.e., none or TGF-β) were fixed in 1% formaldehyde in IMDM (Sigma Aldrich) for 10 min at room temperature. The reaction was stopped by adding glycine solution (final concentration, 0.125 M). Fixed cells were washed three times with cold PBS and resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1]) with protease inhibitor mixture (Sigma). Chromatin was sheared by sonication in a water Bioruptor using four cycles of 15 min each at 4°C, and the insoluble fraction was removed by centrifugation. Then, ChIP was performed with a Millipore ChIP kit (cat. no. 17-295) using Millipore H3K4me3 or control IgG Ab (cat. no. 17-614), according to protocol. After proteinase K digestion, DNA was extracted using phenol-chloroform (Invitrogen) and precipitated for quantitative real-time PCR analyses using specific primers (Supplemental Table I).

MIG (MSCV-IRES-GFP) plasmid expressing Foxp3 was described previously (19). Plasmids were transfected into Phoenix cells for viral preparation, and supernatant containing retroviruses was collected at 48 and 72 h and used for primary CD4+ T cell transduction. Briefly, CD4+ T cells were stimulated with anti-CD3 and anti-CD28 in the presence of gentamicin, anti–IFN-γ, and anti–IL-4 for 24 h. Cells were transduced on two consecutive days by spin infection at 2500 rpm for 90 min at 30°C in the presence of supernatant containing retroviruses together with 8 μg/ml polybrene. After the second transduction, the retroviral supernatant was replaced with T cell culture medium with cytokines required for CD4+ T cell differentiation before analysis.

CD4+ T cells from pooled spleens of littermate wild-type or Ikzf1ΔF4/ΔF4 mice were purified with a CD4+ T Cell Isolation Kit (Miltenyi Biotec) and transferred by retro-orbital i.v. injection (2 × 106 cells/mouse) into littermate Rag1−/− recipients. Reconstitution efficiency was evaluated 2 wk postinjection, and mice were subsequently inoculated by gavage with 1010 CFU C. rodentium in 200 μl PBS. C. rodentium strain DBS100 (ATCC 51459; American Type Culture Collection) was prepared by culturing in Luria–Bertani broth overnight, and bacterial concentration was determined by measuring the OD at 600 nm. Body weight was measured daily. Fecal pellets were collected, weighed, and homogenized in sterile PBS. Serially diluted homogenates were plated on MacConkey agar plates. C. rodentium colonies were identified based on morphology after 18–24 h of incubation at 37°C. Tissues from middle colon were dissected, fixed with 10% formalin, embedded in paraffin, sectioned at 4 μm, and stained with H&E. Sections were analyzed blindly by a trained gastrointestinal pathologist for lamina propria inflammation, goblet cell loss, abnormal crypts, crypt abscesses, mucosal erosion or ulceration, submucosal spread to transmural inflammation, and neutrophil counts. Disease score was assessed as previously described (3941).

Unless otherwise noted, statistical analysis was performed with the unpaired two-tailed Student t test on individual biological samples with GraphPad Prism.

Members of the Ikaros family of transcription factors have been implicated in Treg and Th17 cell differentiation in vitro (35, 4244). We hypothesized that Ikaros may play an important role in vivo, especially in mucosal tissues in which Th17 cells and Tregs are abundant. To understand the role of Ikaros in vivo, we used recently developed Ikaros-mutant mice with genetic deletion of exons encoding either DNA-binding zinc finger 1 (Ikzf1ΔF1/ΔF1) or DNA-binding zinc finger 4 (Ikzf1ΔF4/ΔF4) of Ikaros (34). Two benefits of using these mice rather than Ikaros-null mice are the ability to study the phenotypes of homozygous mutant Ikzf1ΔF1/ΔF1 and Ikzf1ΔF4/ΔF4 mice on a C57BL/6 background and the knowledge that these finger mutants disrupt expression of only a subset of Ikaros target genes (34), thus potentially avoiding some confounding global defects of Ikaros-null mice.

We first examined the T cell composition in the spleen of Ikaros zinc finger 1 or 4 mutant mice. Although the percentages of T cells (i.e., CD3+ cells) and CD4+ T cells were increased as a result of downregulation of B cells (i.e., CD19+ cells) (Supplemental Fig. 1A, 1B) (34), we found that the total number of T lymphocytes (e.g., CD3+ cells), as well as the number of CD4+ T cells, in the Ikaros-mutant mice was reduced (Supplemental Fig. 1C, 1D), consistent with a decrease in total splenocytes in the Ikaros isoform mutants (Supplemental Fig. 1E). To further investigate the T cell function in vivo, we examined CD4+ T cell cytokine expression under steady-state conditions. In agreement with the abundant presence of naturally occurring splice isoform of Ikaros lacking DNA-binding zinc finger 1 (e.g., Ik-2) in T cells (26, 45, 46), CD4+ T cells isolated from the spleen and intestines of Ikzf1ΔF1/ΔF1 mice had similar production of IL-17 and IL-22 compared with those from wild-type littermate mice (Supplemental Fig. 1F). However, CD4+ T cells from the spleen and intestines of Ikzf1ΔF4/ΔF4 mice aberrantly expressed higher levels of IL-22, but not IL-17, compared with those from wild-type littermate mice (Fig. 1A, 1B). Given the relatively normal function of CD4+ T cells in Ikzf1ΔF1/ΔF1 mice, we focused our remaining studies on using Ikzf1ΔF4/ΔF4 mice to elucidate the role of Ikaros in CD4+ T cells. Considering the elevated cytokine production by CD4+ T cells under steady-state conditions in Ikzf1ΔF4/ΔF4 mice, we next examined the Treg compartment. Total Foxp3+ Tregs isolated from the spleen, thymus, and intestines of Ikzf1ΔF4/ΔF4 mice were increased in the absence of wild-type full-length Ikaros in Ikzf1ΔF4/ΔF4 mice (Fig. 1C, Supplemental Fig. 1G), suggesting that the cytokine upregulation (i.e., IL-22) observed in Ikzf1ΔF4/ΔF4 mice was not due to a general loss of Tregs in vivo.

FIGURE 1.

Absence of full-length wild-type Ikaros results in aberrant IL-22 expression by CD4+ T cells and altered Treg compartments in vivo. CD4+ T cells were isolated from the spleen, thymus, small intestinal lamina propria (SI), and large intestinal lamina propria (LI) of steady-state littermate mice of the indicated genotypes. Cytokine (A and B) or Foxp3 (CE) expression was measured by intracellular staining and flow cytometry. Neuropilin-1 (D) or Helios (E) was measured on cells that were gated on CD4+TCRβ+Foxp3+. Data in (A) and (C)–(E) are representative of at least three independent experiments. Data in (B) are compiled from independent experiments (each symbol represents one mouse); mean ± SEM is shown for cytokine-expressing cells gated on total CD4+TCRβ+ cells from the indicated organs. *p < 0.05, **p < 0.01.

FIGURE 1.

Absence of full-length wild-type Ikaros results in aberrant IL-22 expression by CD4+ T cells and altered Treg compartments in vivo. CD4+ T cells were isolated from the spleen, thymus, small intestinal lamina propria (SI), and large intestinal lamina propria (LI) of steady-state littermate mice of the indicated genotypes. Cytokine (A and B) or Foxp3 (CE) expression was measured by intracellular staining and flow cytometry. Neuropilin-1 (D) or Helios (E) was measured on cells that were gated on CD4+TCRβ+Foxp3+. Data in (A) and (C)–(E) are representative of at least three independent experiments. Data in (B) are compiled from independent experiments (each symbol represents one mouse); mean ± SEM is shown for cytokine-expressing cells gated on total CD4+TCRβ+ cells from the indicated organs. *p < 0.05, **p < 0.01.

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To better assess the Foxp3+ Tregs in vivo, we costained Foxp3+ CD4+ T cells with the intracellular marker Helios or the surface marker neuropilin-1 (Nrp-1), both of which were identified to be expressed at high levels on thymic-derived Tregs (tTregs) but not on Tregs induced by TGF-β (iTregs) or peripherally derived Tregs (pTregs) (43, 4749). Although total Foxp3+ T cells were markedly enhanced under in vivo steady-state conditions, the Foxp3+Helios and Foxp3+Nrp-1 populations that are characteristic of pTregs were reduced in Ikzf1ΔF4/ΔF4 mice (Fig. 1D, 1E). These data indicate that Ikaros may differentially regulate tTregs and pTregs in vivo.

Although less severe than in Ikaros-null mice, Ikzf1ΔF4/ΔF4 mice also have various deficiencies in the immune system [e.g., early development of thymic lymphoma, lack of lymph nodes and Peyer’s patches, and fetal lymphoid tissue inducer cells (34)] that might complicate more detailed in vivo studies in these mice. Therefore, to further examine the role of Ikaros in T cell–mediated mucosal immunity, we chose a T cell adoptive-transfer model to determine the in vivo function of the CD4+ T cells lacking full-length wild-type Ikaros upon bacterial infection. Based on our observations of enhanced IL-22 production by Ikaros-mutant T cells, we chose C. rodentium, which is a murine bacteria that requires IL-22 for its clearance (4, 8, 50). Specifically, we examined the T cell responses directly in the gut of Rag1−/− mice that were initially reconstituted with wild-type or Ikzf1ΔF4/ΔF4 mutant total CD4+ T cells and then subjected to C. rodentium infection. Although there was no significant difference in the survival or the weight changes among the Rag1−/− mice reconstituted with the wild-type or Ikaros-mutant T cells (data not shown), the mice that received Ikaros-mutant CD4+ T cells showed an increased percentage and number of IL-22–producing CD4+ T cells in the gut compared with the mice reconstituted with wild-type CD4+ T cells (Fig. 2A–C). Furthermore, Rag1−/− mice reconstituted with Ikzf1ΔF4/ΔF4 CD4+ T cells displayed enhanced clearance of the bacteria and alleviated intestinal histopathology postinfection compared with those reconstituted with wild-type CD4+ T cells (Fig. 2D–F). These data indicate that the enhanced generation (e.g., expansion and/or differentiation) of IL-22–producing CD4+ T cells in the absence of functional Ikaros confers protection against mucosal pathogen infection.

FIGURE 2.

Aberrant IL-22 expression by Ikzf1ΔF4/ΔF4 T cells in vivo protects against C. rodentium infection. Littermate Rag1−/− mice were reconstituted with CD4+ T cells isolated from littermate mouse spleens of the indicated genotypes and infected with C. rodentium for 9 d. (AC) CD4+ T cells (CD4+CD3+TCRβ+) were isolated from gut lamina propria at day 9, counted, and stimulated by PMA/ionomycin/brefeldin A for 4 h, and cytokine expression was measured by intracellular staining and flow cytometry. Data in (A) are representative FACS plots of two independent experiments. (D) Fecal bacterial counts were measured at day 7 and normalized per gram of feces. (E and F) Midcolon sections were stained with H&E and then scored for disease severity (original magnification ×20). Data (B–D and F) show pooled data from two independent experiments (Ikzf1+/+ n = 4; Ikzf1ΔF4/ΔF4 n = 6). Mean ± SEM is shown. *p < 0.05, **p < 0.01.

FIGURE 2.

Aberrant IL-22 expression by Ikzf1ΔF4/ΔF4 T cells in vivo protects against C. rodentium infection. Littermate Rag1−/− mice were reconstituted with CD4+ T cells isolated from littermate mouse spleens of the indicated genotypes and infected with C. rodentium for 9 d. (AC) CD4+ T cells (CD4+CD3+TCRβ+) were isolated from gut lamina propria at day 9, counted, and stimulated by PMA/ionomycin/brefeldin A for 4 h, and cytokine expression was measured by intracellular staining and flow cytometry. Data in (A) are representative FACS plots of two independent experiments. (D) Fecal bacterial counts were measured at day 7 and normalized per gram of feces. (E and F) Midcolon sections were stained with H&E and then scored for disease severity (original magnification ×20). Data (B–D and F) show pooled data from two independent experiments (Ikzf1+/+ n = 4; Ikzf1ΔF4/ΔF4 n = 6). Mean ± SEM is shown. *p < 0.05, **p < 0.01.

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To further understand the cell-intrinsic mechanistic role of Ikaros in CD4+ T cell differentiation and/or cytokine regulation, we turned to an in vitro culture system. Considering our data demonstrating a marked decrease in Foxp3+Nrp-1 or Foxp3+Helios cells that were suggested to be pTregs and iTregs (43, 47, 48) (Fig. 1D, 1E), we first examined the iTregs under in vitro polarizing conditions. As expected, upon stimulation with TGF-β in vitro, both wild-type bulk CD4+ and naive CD4+ T cells (i.e., CD4+CD25CD62LhiCD44lo cells) were readily differentiated into iTregs, as measured by upregulation of Foxp3 (Fig. 3A, 3B, Supplemental Fig. 2A, 2B). Consistent with the in vivo defects in pTregs (Fig. 1D, 1E), Ikzf1ΔF4/ΔF4 CD4+ T cells had marked decreases in Foxp3 protein and mRNA expression compared with wild-type cells under iTreg conditions in vitro (i.e., TGF-β) (Fig. 3A, 3B, Supplemental Fig. 2A, 2B). Together, these data indicate that Ikaros is required for Foxp3 expression induced by TGF-β.

FIGURE 3.

Perturbed expression of Foxp3, IL-22, and IL-17 in Ikzf1ΔF4/ΔF4 CD4+ T cells in vitro. Bulk CD4+ T cells were purified from littermate mice of the indicated genotypes and activated by anti-CD3/CD28, with or without TGF-β and/or Ahr ligand FICZ for 96 h in culture. Foxp3 (A and B) and IL-22 and IL-17 (CE) protein expression was measured after 96 h by intracellular staining and flow cytometry. Data are representative of at least four independent experiments. Data (B, D, and E) are compiled from independent experiments (each symbol represents one mouse); mean ± SEM is shown. *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 3.

Perturbed expression of Foxp3, IL-22, and IL-17 in Ikzf1ΔF4/ΔF4 CD4+ T cells in vitro. Bulk CD4+ T cells were purified from littermate mice of the indicated genotypes and activated by anti-CD3/CD28, with or without TGF-β and/or Ahr ligand FICZ for 96 h in culture. Foxp3 (A and B) and IL-22 and IL-17 (CE) protein expression was measured after 96 h by intracellular staining and flow cytometry. Data are representative of at least four independent experiments. Data (B, D, and E) are compiled from independent experiments (each symbol represents one mouse); mean ± SEM is shown. *p < 0.05, ***p < 0.001, ****p < 0.0001.

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Given the potential for a reciprocal balance between Treg and Th17 cell differentiation (15, 19), we next examined the Th17 cell–related cytokine expression (i.e., IL-17 and IL-22) in Ikzf1ΔF4/ΔF4 CD4+ T cells. Strikingly, total CD4+ T cells isolated from Ikzf1ΔF4/ΔF4 mice that contain both naive and memory T cells expressed high levels of IL-22 compared with wild-type CD4+ T cells without exogenous cytokine stimulation in the Th0 condition (i.e., only anti-CD3 and anti-CD28 stimulation) (Fig. 3C, 3D). In contrast, naive CD4+ T cells (CD4+CD25CD62LhiCD44lo) purified from Ikzf1ΔF4/ΔF4 mice did not appear to have upregulated expression of IL-22 in the Th0 condition (Supplemental Fig. 2C). Furthermore, naive CD4+ T cell (i.e., CD4+CD62LhiCD44lo) percentages in Ikzf1ΔF4/ΔF4 splenocytes were decreased and memory CD4+ T cell (i.e., CD4+CD62LloCD44hi) percentages were increased compared with wild-type splenocytes (Supplemental Fig. 2D, 2E). These data suggest a potential difference in the regulation of IL-22 by Ikaros in naive versus memory CD4+ T cells in the Th0 condition. Of note, lower cell viability of naive CD4+ T cells was observed in vitro in the Th0 conditions (data not shown). Thus, we cannot rule out the possibility that the lack of IL-22 expression by naive Ikzf1ΔF4/ΔF4 CD4+ T cells in the Th0 condition might be due to reduced general cell fitness. Nevertheless, even in the presence of TGF-β, a cytokine that was shown to inhibit IL-22 expression (1618), both bulk and naive Ikzf1ΔF4/ΔF4 CD4+ T cells aberrantly expressed IL-22 (Fig. 3C–E, Supplemental Fig. 2C). Expression of the key Th17 cell transcription factor RORγt also was upregulated in Ikzf1ΔF4/ΔF4 CD4+ T cells under Th0 and iTreg-polarizing conditions (Supplemental Fig. 2F). Together, these data suggest that Ikaros, in a zinc finger 4-dependent manner, is required to inhibit aberrant expression of Th17 cell–associated cytokine (especially IL-22) by CD4+ T cells in vitro.

Ahr activation by ligand FICZ was shown to enhance IL-17 and IL-22 production by CD4+ T cells (2224). Thus, we tested the effect of Ahr ligand on cytokine production by Ikzf1ΔF4/ΔF4 CD4+ T cells. Addition of FICZ to the cultures that contained TGF-β enhanced the upregulation of IL-22 and IL-17 in Ikzf1ΔF4/ΔF4 CD4+ T cells (Fig. 3C–E). It is worth mentioning that, under Th17 cell–polarizing conditions that allow optimal expression of both IL-17 and IL-22 (e.g., IL-6, TGF-β, and FICZ) (14), Ikzf1ΔF4/ΔF4 CD4+ T cells had no statistical differences in IL-17 production compared with wild-type CD4+ T cells (Supplemental Fig. 2G, 2H), consistent with our in vivo data during C. rodentium infection (Fig. 2B, 2C, middle panels). However, IL-22 production by Ikzf1ΔF4/ΔF4 CD4+ T cells was still markedly enhanced under Th17 cell–polarizing conditions, particularly by the IL-22 single-producing cells (Supplemental Fig. 2C, 2G, 2H). These data underscore the important negative and selective regulatory role of Ikaros in the regulation of IL-22 in CD4+ T cells and point to a differential regulation of IL-17 and IL-22.

To determine whether this aberrant IL-22 upregulation was a loss-of-function or gain-of-function phenotype due to the expression of the mutant form of Ikaros lacking zinc finger 4, we conducted the same in vitro experiments on CD4+ T cells isolated from Ikzf1+/− mice, which had been fully backcrossed on a C57BL/6 background. Because of embryonic lethality of Ikaros-null mice on the C57BL/6 background (34), we used CD4+ T cells from the heterozygotes to examine Th17 cell–associated cytokine expression levels. Consistent with the data in Ikaros zinc finger 4–mutant cells, the expression of IL-22 also was elevated in Ikzf1+/− CD4+ T cells compared with wild-type littermate controls under Th0 or iTreg-polarizing conditions and was further enhanced upon the addition of FICZ (Supplemental Fig. 2I), suggesting a loss-of-function phenotype of Ikaros in Ikzf1ΔF4/ΔF4 mice.

Upregulation of IL-22 in Ikaros-mutant CD4+ T cells prompted us to examine the expression and activation of Ahr, a key regulator that cooperates with RORγt to enhance Il22 gene transcription (6, 51). Unexpectedly, Ahr expression induced by TGF-β, TGF-β plus FICZ, or TGF-β and IL-6 plus FICZ was greatly reduced in Ikzf1ΔF4/ΔF4 CD4+ T cells compared with control cells, despite the upregulation of Il22 (Fig. 4A, 4B). These data suggest that Ikaros may function as a positive regulator for Ahr transcription, consistent with the direct binding of Ikaros at the Ahr promoter (35). Consistent with the reduced expression of Ahr, Cyp1a1, a known target gene of Ahr, was downregulated in Ikzf1ΔF4/ΔF4 CD4+ T cells (Fig. 4C). Notably, the aberrant Il21 expression in Ikzf1ΔF4/ΔF4 CD4+ T cells is independent of Ahr (Fig. 4D).

FIGURE 4.

Regulation of Ahr expression and its role in aberrant IL-22 expression in Ikzf1ΔF4/ΔF4 CD4+ T cells. CD4+ T cells were purified from littermate mice of the indicated genotypes and activated by anti-CD3/CD28, with or without TGF-β, IL-6, and/or Ahr ligand FICZ. mRNA expression (AD) was measured by real-time RT-PCR after 48 h in culture, and protein expression (E) was measured after 96 h in culture by intracellular staining and flow cytometry. Mean ± SD (A–D) of experimental triplicates is shown. Data are representative of at least two independent experiments.

FIGURE 4.

Regulation of Ahr expression and its role in aberrant IL-22 expression in Ikzf1ΔF4/ΔF4 CD4+ T cells. CD4+ T cells were purified from littermate mice of the indicated genotypes and activated by anti-CD3/CD28, with or without TGF-β, IL-6, and/or Ahr ligand FICZ. mRNA expression (AD) was measured by real-time RT-PCR after 48 h in culture, and protein expression (E) was measured after 96 h in culture by intracellular staining and flow cytometry. Mean ± SD (A–D) of experimental triplicates is shown. Data are representative of at least two independent experiments.

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Considering the paradoxical upregulation of IL-22 with decreased expression of Ahr in Ikzf1ΔF4/ΔF4 CD4+ T cells, we questioned whether Ahr was required for the observed IL-22 expression in these cells. To this end, we generated Ahr−/−Ikzf1ΔF4/ΔF4 mice to genetically ablate Ahr expression in Ikaros-mutant mice. When these compound-mutant CD4+ T cells were cultured with TGF-β, TGF-β plus FICZ, or TGF-β and IL-6 plus FICZ, the aberrant production of IL-22 was markedly reduced (compare the IL-22 production by CD4+ T cells from Ahr+/−Ikzf1ΔF4/ΔF4 mice and from Ahr−/−Ikzf1ΔF4/ΔF4 mice) (Fig. 4B, 4E, Supplemental Fig. 3). Together, these data demonstrate that Ahr is essential for IL-22 upregulation also in Ikaros-mutant CD4+ T cells, as reported in wild-type cells. The expression level of Ahr correlated with Cyp1a1, but surprisingly not IL-22, expression in Ikaros-mutant CD4+ T cells, suggesting that Ikaros may regulate Ahr expression, as well as its activity, in a target gene–specific manner in CD4+ T cells.

Both IL-6 and IL-21 were shown to promote Th17 cell differentiation and to inhibit iTreg differentiation in vitro (52). Thus, we investigated whether these cytokines are responsible for the aberrant upregulation of IL-17 and/or IL-22 in Ikzf1ΔF4/ΔF4 CD4+ T cells. We did not detect IL-6 secreted by Ikzf1ΔF4/ΔF4 CD4+ T cells (data not shown), consistent with the production of IL-6 by APCs but not by T cells (14). In wild-type CD4+ T cells, IL-21 can be induced under Th17 cell–polarizing condition (i.e., IL-6 plus TGF-β). In contrast, IL-21 was markedly increased in Ikzf1ΔF4/ΔF4 CD4+ T cells, even in the absence of any exogenous cytokines (Fig. 5A, 5B). This is consistent with the crucial role of IL-21 in promoting IL-17 and IL-22 expression by CD4+ T cells in an autocrine manner (11, 12, 19). Interestingly, CD4+ T cells directly isolated ex vivo from the spleen of Ikzf1ΔF4/ΔF4 mice expressed slightly higher levels of IL-21 than did wild-type cells (Fig. 5B); however, stimulation with anti-CD3 and anti-CD28 in vitro (i.e., Th0 condition) substantially increased the production of IL-21 by Ikzf1ΔF4/ΔF4 CD4+ T cells (Fig. 5B), suggesting that aberrant upregulation of IL-21 in Ikzf1ΔF4/ΔF4 CD4+ T cells is dependent on the TCR-signaling pathway.

FIGURE 5.

Aberrant cytokine expression by Ikzf1ΔF4/ΔF4 mice requires TCR activation. CD4+ T cells were purified from Ikzf1ΔF4/ΔF4 or wild-type littermate mice, activated by anti-CD3/CD28, and cultured with the indicated cytokines. (A) IL-21 protein expression was measured by ELISA after 96 h. Mean ± SD of experimental triplicates are shown. (B) CD4+ T cells were purified from littermate mice of the indicated genotypes, and mRNA was measured either directly ex vivo or after 12 h of in vitro activation by anti-CD3/CD28 (Th0); mean ± SD of experimental triplicates are shown. CD4+ T cells were purified from littermate mice of the indicated genotypes and activated by anti-CD28 (at constant concentration of 1 μg/ml) and varying concentrations of anti-CD3, with or without TGF-β and with or without Ahr ligand FICZ. FSC (C) and cytokine expression (D) were measured after 96 h by flow cytometry and/or intracellular staining. (E) CD4+ T cells were purified from Ikzf1ΔF4/ΔF4 or wild-type littermate mice, activated by anti-CD3/CD28, cultured with or without TGF-β for 24 h, and H3K4me3 chromatin marks at the Il21 promoter were measured by ChIP (primer sequences given in Supplemental Table I). Mean ± SD of experimental triplicates is shown. Data are representative of three independent experiments.

FIGURE 5.

Aberrant cytokine expression by Ikzf1ΔF4/ΔF4 mice requires TCR activation. CD4+ T cells were purified from Ikzf1ΔF4/ΔF4 or wild-type littermate mice, activated by anti-CD3/CD28, and cultured with the indicated cytokines. (A) IL-21 protein expression was measured by ELISA after 96 h. Mean ± SD of experimental triplicates are shown. (B) CD4+ T cells were purified from littermate mice of the indicated genotypes, and mRNA was measured either directly ex vivo or after 12 h of in vitro activation by anti-CD3/CD28 (Th0); mean ± SD of experimental triplicates are shown. CD4+ T cells were purified from littermate mice of the indicated genotypes and activated by anti-CD28 (at constant concentration of 1 μg/ml) and varying concentrations of anti-CD3, with or without TGF-β and with or without Ahr ligand FICZ. FSC (C) and cytokine expression (D) were measured after 96 h by flow cytometry and/or intracellular staining. (E) CD4+ T cells were purified from Ikzf1ΔF4/ΔF4 or wild-type littermate mice, activated by anti-CD3/CD28, cultured with or without TGF-β for 24 h, and H3K4me3 chromatin marks at the Il21 promoter were measured by ChIP (primer sequences given in Supplemental Table I). Mean ± SD of experimental triplicates is shown. Data are representative of three independent experiments.

Close modal

To determine the Ikaros zinc finger 4 mutant CD4+ T cells’ response to TCR activation, we performed TCR titrations on in vitro–cultured cells and measured cytokine expression. We used FSC as surrogate readout for T cell activation. Consistent with the literature on Ikaros-null CD4+ T cells (53), we observed that Ikzf1ΔF4/ΔF4 CD4+ T cells had a decreased threshold for TCR activation (Fig. 5C) and produced elevated levels of cytokines (i.e., IL-17 and IL-22) at low concentrations of anti-CD3 (Fig. 5D). We also observed increased histone H3 K4 trimethylation marks (H3K4me3) at the promoter of the Il21 locus in Ikzf1ΔF4/ΔF4 CD4+ T cells in the Th0 (i.e., none) and iTreg (i.e., TGF-β)–polarizing conditions, consistent with an open chromatin and enhanced expression of Il21 in the absence of Ikaros (Fig. 5A, 5B, 5E). Together, these data indicate that Ikaros negatively regulates IL-21 expression in CD4+ T cells.

To test whether the aberrant IL-21 expression in Ikzf1ΔF4/ΔF4 CD4+ T cells was involved in and/or required for the observed aberrant IL-22 expression, we blocked IL-21 with neutralizing Abs. Blocking IL-21 in the cultures greatly reduced the polarization of Ikzf1ΔF4/ΔF4 CD4+ T cells toward a Th17 cell–like phenotype by suppressing the expression of both IL-17 and IL-22 (Fig. 6A). In contrast, neutralizing Abs against IL-6 had no effect (data not shown). Neutralizing IL-21 completely relieved the inhibitory effect of exogenously added IL-21 on TGF-β–induced Foxp3 in wild-type cells, and it partially restored Foxp3 expression in Ikzf1ΔF4/ΔF4 CD4+ T cells (Fig. 6B), suggesting that one potential mechanism underlying Foxp3 downregulation in Ikzf1ΔF4/ΔF4 CD4+ T cells is due, in part, to aberrant expression of IL-21. IL-21 was shown to promote Th17 cell differentiation by activation of the Stat3 pathway (10, 54). Indeed, blocking Stat3 in CD4+ T cells by the pharmacological agent STA-21 (55) (Fig. 6C) or by genetic deletion (Fig. 6D) eliminated the aberrant IL-17 and IL-22 expression in Ikzf1ΔF4/ΔF4 CD4+ T cells, but it only partially restored TGF-β–induced Foxp3 expression (data not shown). Together, these data suggest that Ikaros, through zinc finger 4, regulates Th17 cell–related cytokine production and TGF-β–induced Foxp3 expression via inhibition of IL-21– and Stat3-dependent pathways.

FIGURE 6.

Aberrant expression of IL-21 in Ikzf1ΔF4/ΔF4 CD4+ T cells promotes Th17 cell–like phenotype via Stat3 pathway. In vitro neutralization assays for IL-21 (A and B) and Stat3 inhibition (C) or genetic deletion (D) were conducted on CD4+ T cells purified from littermate mice of the indicated genotypes, activated by anti-CD3/CD28, and cultured or not with TGF-β, Ahr ligand FICZ, and neutralizing Abs (A and B) or Stat3 inhibitor STA-21 (C) for 96 h. IL-17 and IL-22 expression (A, C, and D) or Foxp3 (B) expression was measured by intracellular staining and flow cytometry. Data are representative of at least two independent experiments.

FIGURE 6.

Aberrant expression of IL-21 in Ikzf1ΔF4/ΔF4 CD4+ T cells promotes Th17 cell–like phenotype via Stat3 pathway. In vitro neutralization assays for IL-21 (A and B) and Stat3 inhibition (C) or genetic deletion (D) were conducted on CD4+ T cells purified from littermate mice of the indicated genotypes, activated by anti-CD3/CD28, and cultured or not with TGF-β, Ahr ligand FICZ, and neutralizing Abs (A and B) or Stat3 inhibitor STA-21 (C) for 96 h. IL-17 and IL-22 expression (A, C, and D) or Foxp3 (B) expression was measured by intracellular staining and flow cytometry. Data are representative of at least two independent experiments.

Close modal

RORγt is a key transcription factor that promotes transcription of both IL-17 and IL-22 in wild-type CD4+ T cells (13). To investigate the potential role of RORγt in aberrant production of Th17 cell–associated cytokines by Ikzf1ΔF4/ΔF4 CD4+ T cells, we cultured CD4+ T cells with TGF-β or TGF-β plus FICZ, with or without digoxin, a pharmacological agent that antagonizes RORγt activity and is known to suppress Th17 cell differentiation (56, 57). Intriguingly, inhibition of RORγt activity greatly diminished the aberrant IL-17 expression, but it had minimal effect on IL-22 expression, in Ikzf1ΔF4/ΔF4 CD4+ T cells and especially IL-22+IL-17 cells were not reduced by digoxin-mediated RORγt activity blockade (Fig. 7A).

FIGURE 7.

RORγt differentially regulates and Foxp3 suppresses aberrant Th17-associated cytokines in Ikzf1ΔF4/ΔF4 CD4+ T cells. CD4+ T cells were purified from littermate mice of the indicated genotypes and activated by anti-CD3/CD28, with or without TGF-β (A and B) and with or without Ahr ligand FICZ (A), together with or without RORγt inhibitor digoxin or DMSO as a control (A). (A and B) Cytokine expression was measured after 96 h by intracellular staining and flow cytometry. (C) CD4+ T cells were purified from littermate mice of the indicated genotypes, activated by anti-CD3/CD28, transduced with empty vector MIG plasmid or Foxp3-expressing retroviral plasmid, and skewed with TGF-β for 96 h. (C) IL-17, IL-22, and GFP expression was measured by intracellular staining and flow cytometry. Data are representative of at least three independent experiments.

FIGURE 7.

RORγt differentially regulates and Foxp3 suppresses aberrant Th17-associated cytokines in Ikzf1ΔF4/ΔF4 CD4+ T cells. CD4+ T cells were purified from littermate mice of the indicated genotypes and activated by anti-CD3/CD28, with or without TGF-β (A and B) and with or without Ahr ligand FICZ (A), together with or without RORγt inhibitor digoxin or DMSO as a control (A). (A and B) Cytokine expression was measured after 96 h by intracellular staining and flow cytometry. (C) CD4+ T cells were purified from littermate mice of the indicated genotypes, activated by anti-CD3/CD28, transduced with empty vector MIG plasmid or Foxp3-expressing retroviral plasmid, and skewed with TGF-β for 96 h. (C) IL-17, IL-22, and GFP expression was measured by intracellular staining and flow cytometry. Data are representative of at least three independent experiments.

Close modal

To test whether the residual activity of RORγt in the presence of digoxin was sufficient to support the observed IL-22 expression in Ikaros-mutant CD4+ T cells, we genetically ablated RORγt expression in Ikaros-mutant mice by generating Rorcgfp/gfpIkzf1ΔF4/ΔF4 mice. Consistent with the data using the RORγt pharmacological inhibitor (Fig. 7A), when CD4+ T cells were cultured with or without TGF-β, the production of IL-22 in Ikaros-mutant mice was still aberrantly upregulated when RORγt was genetically deleted (compare IL-22 production by CD4+ T cells from Rorcgfp/+Ikzf1ΔF4/ΔF4 mice and Rorcgfp/gfpIkzf1ΔF4/ΔF4 mice) (Fig. 7B). Of note, IL-17, but not IL-22, production was markedly reduced in the Ikaros-mutant CD4+ T cells on RORγt heterozygous background compared with wild-type background (i.e., Rorcgfp/+Ikzf1ΔF4/ΔF4 versus Ikzf1ΔF4/ΔF4) (Figs. 3C, 7B), consistent with the dependency of IL-17 on RORγt (13). Together, these data indicate a selective requirement for RORγt in upregulation of IL-17, but not IL-22, in Ikaros-mutant CD4+ T cells and underscore the differential regulation of the IL-22 single-producing cells versus IL-17 and IL-22 double-producing cells (e.g., Th17 cells).

Foxp3 was shown to inhibit Th17-associated cytokine expression by antagonizing RORγt activity (19). Given that Foxp3 expression is decreased in TGF-β–skewed Ikzf1ΔF4/ΔF4 CD4+ T cells, we next forced expression of Foxp3 in Ikzf1ΔF4/ΔF4 CD4+ T cells by retroviral transduction. Indeed, ectopic expression of Foxp3 in Ikzf1ΔF4/ΔF4 CD4+ T cells suppressed the aberrant expression of Th17 cell–associated cytokines (especially IL-22 cytokine production) (Fig. 7C). Notably, downregulation of IL-22 also was observed in the nontransduced cells (e.g., GFP cells) when Foxp3 was expressed in Ikzf1ΔF4/ΔF4 CD4+ T cells, suggesting a bystander effect on inhibition of IL-22 expression. Together, these data indicate that lack of Foxp3 induction is responsible, at least in part, for aberrant upregulation of proinflammatory cytokine production (i.e., IL-17 and IL-22) by Ikzf1ΔF4/ΔF4 CD4+ T cells.

The role of Ikaros and its family members in immune cell development has been increasingly recognized over the past decade. In CD4+ T cells, Ikaros has been identified as a key regulator for Th1, Th2, and Th17 cell subsets (29, 35, 58, 59), and family members Helios, Eos, and Aiolos were shown to play important roles in the Treg compartments (35, 4244, 60). Our studies uncover a new role for Ikaros in the differential regulation of various Treg compartments (e.g., tTregs, pTregs, and iTregs). We further demonstrate a unique role for Ikaros in inhibiting IL-22–producing CD4+ T cells (especially IL-22+IL-17 CD4+ T cells) in vitro and in vivo in mucosal immunity during both the steady-state and bacterial infection.

In contrast to our study, a recent report showed that Ikaros positively regulates Th17 cell differentiation (e.g., IL-17 and IL-22 production) (35). The precise reasons underlying this discrepancy are unknown and most likely are due to differences in the in vitro culturing conditions, mouse genetic backgrounds, and/or organ(s) assessed in vivo. For example, using Ikaros-null mice on a mixed genetic background will most likely confound the data, consistent with a reduced Ahr affinity for ligand in certain mouse strains (i.e., DBA/2J and 129/SvJ) (61). In addition, Th17 cells (e.g., CD4+ T cells producing IL-17) in Ikaros-null mice were only examined in the thymus (35) and not in other organs, such as the gut, where most in vivo Th17 cells are located (13). Finally, differential regulation of IL-17 and IL-22 production on a per-cell basis by Ikaros was not assessed in the previous study (35). Nevertheless, these studies [(35) and our findings] underscore the complex role of Ikaros in regulating Th17 cell–associated cytokine expression.

Previous data showed that of the four N-terminal DNA-binding zinc fingers, fingers 2 and 3 are essential for the protein–DNA binding process, and fingers 1 and 4 may modulate the binding of Ikaros to DNA (33, 34, 62). Our data showed that the absence of the fourth DNA-binding zinc finger of Ikaros (i.e., Ikzf1ΔF4/ΔF4) led to impaired induction of Foxp3 (i.e., iTregs) and aberrant upregulation of IL-17 and IL-22 expression upon TCR activation and TGF-β stimulation in T cells. Our data further showed that, under bona fide Th17 cell–polarizing conditions (i.e., IL-6, TGF-β, and/or FICZ), Ikzf1ΔF4/ΔF4 CD4+ T cells still produced more IL-22 but not IL-17. The precise mechanism that controls IL-17 expression by Ikzf1ΔF4/ΔF4 CD4+ T cells in different differentiation conditions (i.e., Th17 condition versus Th0 and iTreg conditions) remains to be determined.

We favor a model that the absence of wild-type Ikaros resulted in unrestricted or derepressed IL-21 expression that, under Th0 condition (i.e., anti-CD3/CD28), promoted the production of IL-22 by CD4+ T cells and, together with the exogenous addition of TGF-β, skewed the cells into a Th17 cell–like state (i.e., production of IL-17 and IL-22). Consistent with this notion, neutralizing IL-21 or blocking the Stat3 pathway that is activated by IL-21 efficiently inhibited the aberrant cytokine production (i.e., IL-22 and IL-17) in Ikzf1ΔF4/ΔF4 cells. Furthermore, severely impaired induction of Foxp3 at least partially accounted for the aberrant upregulation of IL-17 and IL-22 cytokine production by Ikzf1ΔF4/ΔF4 CD4+ T cells upon TGF-β treatment. Indeed, forced expression of Foxp3 can completely block aberrant expression of IL-17 and IL-22, consistent with a negative role for Foxp3 in the regulation of RORγt and/or Ahr activity (19). c-Maf was suggested to mediate the TGF-β–dependent suppression of IL-22 production by CD4+ T cells (16); however, we found no reduction in the expression of this transcription factor in Ikzf1ΔF4/ΔF4 CD4+ T cells compared with wild-type cells (data not shown).

The precise mechanisms underlying the differential regulation of distinct Treg compartments remain to be determined. Intriguingly, although the pTregs and iTregs were decreased, the tTregs were enhanced in Ikzf1ΔF4/ΔF4 mice. These findings are consistent with the requirement for stronger TCR activation during tTreg development (63) and with the reduced TCR activation threshold observed in Ikzf1ΔF4/ΔF4 mice, in agreement with previously reported findings in Ikaros-null CD4+ T cells (53). Blocking IL-21 in Ikzf1ΔF4/ΔF4 CD4+ T cells only partially restored the Foxp3 expression induced by TGF-β (i.e., iTregs), suggesting the contribution of additional mechanism(s) to the defects in iTreg differentiation in the absence of wild-type Ikaros. In contrast to tTregs, iTreg differentiation was shown to require suboptimal TCR signaling (64). Thus, in addition to aberrant expression of IL-21, the enhanced TCR strength may further contribute to the iTreg defects observed in Ikzf1ΔF4/ΔF4 mice.

Our previous findings showed that optimal expression of IL-22 in wild-type CD4+ T cells requires the presence of both transcription factors RORγt and Ahr that work in a cooperative manner (6). Intriguingly, our data showed that the aberrant production of IL-17, but not IL-22, by Ikzf1ΔF4/ΔF4 CD4+ T cells was dependent on RORγt. Although the precise mechanisms underlying the bypass of a requirement for RORγt in the regulation of Il22 transcription in Ikzf1ΔF4/ΔF4 CD4+ T cells remain to be determined, our data support a model of differential regulation of Th17 cell–associated cytokine transcription by Ikaros.

We thank the entire Zhou laboratory for help and suggestions, the Mouse Histology and Phenotyping Laboratory (Northwestern University) for services and assistance, Drs. S. Swaminathan and C. Goolsby (Flow Cytometry Facility, Northwestern University) for cell sorting support, Dr. L. Molinero for advice on ChIP analysis, and Dr. C. Mullighan and Dr. O. Cen for mice.

This work was supported by the National Institutes of Health (Grants AI089954 and AI091962 to L.Z. and R01 DK43726 to S.T.S.), a Cancer Research Institute Investigator Award, and a Skin Disease Research Center (Northwestern University) Pilot and Feasibility Award (both to L.Z.). L.Z. is a Pew Scholar in Biomedical Sciences, supported by the Pew Charitable Trusts, and an Investigator in the Pathogenesis of Infectious Disease, supported by the Burroughs Wellcome Fund. J.J.H. is supported by National Institutes of Health Training Grant T32 NIH T32 GM08061.

The online version of this article contains supplemental material.

Abbreviations used in this article:

Ahr

aryl hydrocarbon receptor

ChIP

chromatin immunoprecipitation

FICZ

6-formylindolo[3,2-b]carbazole

FSC

forward scatter

iTreg

inducible Treg

Nrp-1

neuropilin-1

pTreg

peripherally derived Treg

RORγt

RAR-related orphan receptor gamma t

Treg

regulatory T cell

tTreg

thymic Treg.

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The authors have no financial conflicts of interest.

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