BATF regulates the development and function of IL-17 producing iNKT cells
© Jordan-Williams et al.; licensee BioMed Central Ltd. 2013
Received: 28 September 2012
Accepted: 5 March 2013
Published: 27 March 2013
BATF plays important roles in the function of the immune system. Batf null mice are deficient in both CD4+ Th17 cells and T follicular helper cells and possess an intrinsic B cell defect that leads to the complete absence of class switched Ig. In this study, Tg mice overexpressing BATF in T cells were used together with Batf null mice to investigate how altering levels of BATF expression in T cells impacts the development and function of a recently characterized population of iNKT cells expressing IL-17 (iNKT-17).
BATF has a direct impact on IL-17 expression by iNKT cells. However, in contrast to the Th17 lineage where BATF activates IL-17 expression and leads to the expansion of the lineage, BATF overexpression restricts overall iNKT cell numbers while skewing the compartment in vivo and in vitro toward an iNKT-17 phenotype.
This work is the first to demonstrate that BATF joins RORγt as the molecular signature for all IL-17 producing cells in vivo and identifies BATF as a component of the nuclear protein network that could be targeted to regulate IL-17-mediated disease. Interestingly, these studies also reveal that while the Il17a gene is a common target for BATF regulation in Th17 and iNKT-17 cells, this regulation is accompanied by opposite effects on the growth and expansion of these two cell lineages.
KeywordsBATF Activator-protein-1 iNKT cells IL-17 Mouse models
BATF is a basic leucine zipper transcription factor that dimerizes with the JUN proteins to direct patterns of activator protein-1 (AP-1)-mediated gene expression in the immune system . The impact of disrupting BATF function in vivo has been examined by several groups [2–5]. Mice in which BATF is overexpressed using a T cell-specific promoter display a reduced number of iNKT cells , an increased number of CD4+ T cells expressing IL-17 (Th17)  and an altered cytokine environment that promotes the gross overproduction of class switched Ig by B cells . Batf null mice are viable, yet display a severe deficiency in Th17 and T follicular helper cells [2, 3, 5]. The T cell deficiencies are combined with an intrinsic B cell defect blocking the production of class switched Ig to impair the immune response of these animals to antigen challenge [2, 3]. The dramatic consequences of altering BATF expression in vivo provides evidence that BATF functions to coordinate immune system activities critical in autoimmunity, inflammation and the host response to pathogens.
The ability of BATF to promote the differentiation of naïve CD4+ T cells to the Th17 lineage has been shown to rely on the formation of IRF4/BATF protein complexes that bind and transactivate a number of genes, including Il17a/f. Interestingly, we have observed a negative influence of BATF on the development of iNKT cells [6, 9] and therefore sought to examine how these two opposing activities of BATF may influence the development of a recently identified subset of iNKT cells that expresses IL-17 [10–13]. Murine iNKT-17 cells are a CD4- NK1.1- population that is enriched in peripheral LN (PLN) and respond following stimulation by rapidly secreting IL-17. iNKT-17 cells express RORγt and additional markers that define them as a lineage distinct from classic iNKT cells. A role for iNKT-17 cells has been demonstrated in experimental models of airway disease, asthma and collagen-induced arthritis [12, 14, 15]. iNKT-17 cells are over-represented in NOD mice where their influence in the pancreas exacerbates the development of diabetes . In the present study, using mouse models of BATF overexpression (CD2-HA-BATF) and deficiency (Batf ΔZ/ΔZ ), we demonstrate the importance of BATF to the development of iNKT-17 cells. Despite the overall reduction in the number of iNKT cells in CD2-HA-BATF mice, the majority express IL-17. Likewise, while peripheral iNKT cell numbers are increased in Batf ΔZ/ΔZ mice, the cells are deficient in IL-17 production. These data are consistent with results in Th17 cells [5, 17] and suggest that BATF-containing protein complexes transactivate the Il17a/f gene in NKT cells as well. The novel finding is that the function of BATF as an IL-17 inducer is separate from its effect on cell growth since Th17 cell numbers are expanded in the presence of BATF, while iNKT cell numbers are reduced. To identify an in vitro system that would facilitate the study of BATF-mediated gene regulatory events relevant to the iNKT cell lineage, we describe features of the DN32.D3 hybridoma  that indicate similarity to the iNKT-17 lineage, including the BATF-dependent expression of Il17a mRNA. We conclude that BATF joins RORγt as the molecular signature for all IL-17 producing cells in vivo and represents an essential component of a nuclear protein network that could be targeted to regulate IL-17-mediated disease.
Results and discussion
BATF controls CD4+ Th17 cell differentiation
Levels of BATF correlate with altered iNKT cell numbers in vivo
Batf regulates the production of IL-17 by iNKT cells
DN32.D3 cells model IL-17 production by iNKT cells
IRF4/BATF protein complexes bind to DNA within the mouse Il17a/f locus and are essential for the transactivation of the Il17a/f gene in Th17 cells . However, the mechanism by which BATF functions to regulate gene expression in different cellular contexts, including iNKT-17 cells where IRF4 does not play a transcriptional role , continues to be investigated. Additionally, BATF and its interaction partner proteins such as JUNB and the IRF4 and 8 proteins [8, 17], are expressed independently of IL-17 status in other T cell lineages [1, 9] and BATF-containing protein complexes bind directly to genes where the transcriptional outcome is the inhibition of gene expression [1, 4, 19, 20]. These facts complicate proposing a unified model to explain how BATF regulates its target genes. Therefore, to address the role of BATF in regulating Il17a and other target genes in iNKT cells, we sought to identify an in vitro system that could be used for this purpose.
Toward that goal, DN32.D3 cells were treated with Batf siRNA or control siRNA, stimulated with αGalCer loaded dimers and after 2 hr, RNA was analyzed by qRT-PCR. Batf siRNA resulted in a 50% reduction in Batf mRNA expression as well as a corresponding reduction in the level of Il17a mRNA (Figure 5C). These data support a critical role for BATF in Il17a gene regulation while demonstrating that signaling through the iNKT cell TCR expressed by DN32.D3 cells triggers cooperating molecular events that are required for efficient IL-17 induction. We conclude that the DN32.D3 iNKT cell line is a convenient in vitro model system in which the details of these molecular events can be investigated further.
Our studies have identified BATF as a common regulator of lineage decisions in the murine immune system that involve the expression of IL-17. BATF joins RORγt in the transcription factor network that promotes the differentiation of IL-17 expressing cells downstream of pathways triggered by TGFβ and IL-6 in T cells  and by a pathway dependent on TGFβ, but not IL-6, in iNKT cells [11, 26]. These studies have characterized the DN32.D3 iNKT cell line as a model in which the regulation of Il17a gene and protein expression by this lineage can be investigated further. As the molecular details controlling IL-17 production in vivo continue to emerge, new approaches to control autoimmunity, inflammation and infectious disease will become a reality.
Batf ΔZ/ΔZ and CD2-HA-BATF mice expressing human, HA-tagged BATF were described previously [2, 7] and were maintained by breeding to C57Bl/6 mice (Harlan, Indianapolis, IN). Experiments were performed using sex-matched littermates between 6 and 12 wk of age. Mice were housed in a specific, pathogen-free facility. All protocols were approved by the Purdue University Animal Care and Use Committee.
Antibodies and reagents
All antibodies, cytokines and the mouse CD1d dimers were obtained from BD Biosciences unless otherwise specified. αGalCer was obtained from Axxora (Farmingdale, NY). PE-labeled, CD1d tetramers, empty or loaded with PBS-57, were obtained from the NIH tetramer core facility.
Cell culture and the analysis of gene expression
CD4+ T cells were isolated using MACS (Miltenyi Biotec, Auburn, CA). Cells were stimulated with anti-CD3 and anti-CD28 Ab for 48 h as described . For in vitro differentiation assays, naïve CD4+ T cells were isolated using MACS and cultured for 5 d under Th1 conditions  or under the following conditions for Th17: 5 μg/ml anti-CD3ε, 2 μg/ml anti-CD28, 5 ng/ml rh TGFβ, 100 ng/ml rm IL-6 and 10 μg/ml anti-IL-4 and 10 μg/ml anti-IFNγ (BioXCell, West Lebanon, NH) neutralizing antibodies. Cells were re-stimulated for 6 hr with 2 μg/ml anti-CD3ε prior to analysis. DN32.D3 cells were cultured as described  and stimulated for 24 h with plate-bound CD1d dimers +/− glycolipid prior to analysis.
RNA was isolated using Trizol and analyzed by qRT-PCR using SYBR green (Roche Diagnostics, Indianapolis, IN) and an ABI 7300 real-time PCR system. Data were normalized to Hprt expression and relative mRNA levels calculated using ΔΔCt values. The primers for Hprt, Actin, Batf, Il4, Il23R, Il17a and Il21 have been described . Il10 primers were from Qiagen (Gaithersburg, MD) and additional primers (5’-3’) were
Rorγt: For TGTCCTGGGCTACCCTACTG, Rev GTGCAGGAGTAGGCCACATT;
Ifnγ: For GGATGCATTCATGAGTATTGC, Rev CCTTTTCCGCTTCCTGAGG;
cJun: For CAGTCCAGCAATGGGCACATCA, Rev GGAAGCGTGTTCTGGCTATGCA;
JunB: For GACCTGCACAAGATGAACCACG, Rev ACTGCTGAGGTTGGTGTAGACG;
JunD: For ACCTGCACAAGCAAAGCCAGCT, Rev CGAAACTGCTCAGGTTGGCGTA;
Zbtb16: For CCCAGTTCTCAAAGGAGGATG, Rev TTCCCACACAGCAGACAGAAG;
Nrp1: For GCCTGCTTTCTTCTCTTGGTTTCA, Rev GCTCTGGGCACTGGGCTACA;
Erg1: For GAGGAGATGATGCTGCTGAG, Rev TGCTGCTGCTGCTATTACC;
Erg2: For CCTCCACTCACGCCACTCTC, Rev CACCACCTCCACTTGCTCCTG;
Il13: For GCTTATTGAGGAGCTGAGCAACA, Rev GGCCAGGTCCACACTCCATA
For BATF knock-down, DN32.D3 cells were cultured for 24 h in siRNA delivery media containing 1 μM BATF targeting (Accell siRNA smart pool) or non-targeting siRNA (Accell non-targeting siRNA 1) (Dharmacon/Thermo Fisher Scientific, Waltham, MA). Cells were washed and stimulated for 2 h prior to analysis as described above.
Splenocytes at a density of 5×106 cells/ml were cultured with 100 ng/ml αGalCer for 72 h. Supernatants were analyzed for secreted IL-4 (BD) or IL-17A (eBiosciences, San Diego, CA) using manufacturers’ protocols.
Axillary, brachial, inguinal, and popliteal lymph nodes were pooled to generate PLN cultures. iNKT cells were enriched from PLN using CD8 (Ly-2) and CD19 microbeads (Miltenyi) as described . Cells were surface stained after blocking with FcBlock (BD Biosciences, Franklin Lakes, NJ) using APC-anti- TCRβ (eBioscience), FITC-anti-B220, PE-Cy5-anti-B220, PBS-57-PE-CD1d tetramers and PE-Cy7-anti-NK1.1. Intracellular staining was performed as described  using FITC-anti-IL17A (eBioscience) after stimulation for 4 h with 5 ng/ml PMA and 500 ng/ml ionomycin in the presence of Golgi-plug (BD). Data were collected on a Beckman Coulter FC500 and analyzed using FCS Express V3 software (DeNovo, Los Angeles, CA).
Peripheral Lymph Node
quantitative Reverse Transcriptase Polymerase Chain Reaction
The Flow Cytometry, DNA Sequencing and Transgenic Mouse Shared Resources of the Purdue University Center for Cancer Research played key roles in generating raw data for this study. The authors thank A. Kaufmann, J. Skiba, M. Lawson and K. Oates for routine care of mice. This work was supported by NIH grants CA782464 and CA114381 (EJT). Publication costs were covered by funds awarded to EJT by the Purdue University Center for Cancer Research. KLJ-W was supported by NIH T32 GM08298.
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