C-Jun the Activator Protein 1 Transcription Factor T Cell Development by Δγ / Βα Regulation Of

Regulation of βα / δγ T Cell Development by the Activator Protein 1 Transcription Factor c-Jun

This information is current as Lluís Riera-Sans and Axel Behrens of September 26, 2021. J Immunol 2007; 178:5690-5700; ; doi: 10.4049/jimmunol.178.9.5690 http://www.jimmunol.org/content/178/9/5690 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Regulation of ␣␤/␥␦ T Cell Development by the Activator Protein 1 Transcription Factor c-Jun1

Lluı´s Riera-Sans2 and Axel Behrens3 c-Jun is a member of the AP-1 family of transcription factors, the activity of which is strongly augmented by TCR signaling. To elucidate the functions of c-Jun in mouse thymic lymphopoiesis, we conditionally inactivated c-Jun specifically during early T cell development. The loss of c-Jun resulted in enhanced generation of ␥␦ T cells, whereas ␣␤ T cell development was partially arrested at the double-negative 3 stage. The increased generation of ␥␦ T cells by loss of c-Jun was cell autonomous, because in a competitive reconstitution experiment the knockout-derived cells produced more ␥␦ T cells than did the control cells. C-jun- deficient immature T cells failed to efficiently repress transcription of IL-7R␣, resulting in augmented IL-7R␣ mRNA and surface levels. Chromatin immunoprecipitation assays revealed binding of c-Jun to AP-1 binding sites present in the IL-7R␣ promoter, ␣ ␣␤ ␥␦ indicating direct transcriptional regulation. Thus, c-Jun controls the transcription of IL-7R and is a novel regulator of the / Downloaded from T cell development. The Journal of Immunology, 2007, 178: 5690–5700.

ature T lymphocytes derive from lymphoid precursors not be the sole determinant of linage decision. If expression of an through several distinct cell fate specification events in-frame ␥␦ TCR invariably led to ␥␦ T cell development, one M (1). Common lymphoid precursors, which have al- might expect a complete block in ␣␤ T cell development in mice ready lost erythroid and myeloid potential, separate into B and T expressing a rearranged ␥␦ TCR transgene. This, however, is not http://www.jimmunol.org/ cell precursors in the thymus. Immature pro-T cells then develop the case: many ␥␦ TCR-transgenic mice contain significant into mature T cells characterized by surface expression of either numbers of ␣␤ lineage T cells (4, 5). Conversely, significant the CD4ϩ or the CD8ϩ coreceptor before leaving the thymus. numbers of ␥␦ T cells are present in mice expressing a rear- Thymocytes can be divided into four major subsets based on the ranged ␣␤ TCR transgene (6). Recent work suggests that quan- Ϫ Ϫ expression of CD4 and CD8. The CD4 CD8 double-negative titative differences in TCR signaling appear to be influencing (DN)4 cells are the most immature subset. The DN population can ␣␤/␥␦ lineage commitment (7, 8). be further divided into four subsets (DN1–DN4) based on their To make the developmental transition from DN3 to DN4, the differential expression of CD44 and CD25. DN thymocytes mature TCR chains must be assembled into the pre-TCR complex, which ϩ Ϫ ϩ ϩ in the order of CD44 CD25 (DN1), CD44 CD25 (DN2), consists of a TCR ␤-chain, the invariant pT ␣-chain, and CD3 by guest on September 26, 2021 Ϫ ϩ Ϫ Ϫ CD44 CD25 (DN3), and CD44 CD25 (DN4) (2). components. Only cells that have a functional pre-TCR survive the The decision between ␣␤ and ␥␦ T cells occurs at the DN stage, transition from DN3 to DN4, a process also known as ␤ selection. but the exact time point of specification remains to be determined In mice deficient for components of the pre-TCR developing ␣␤ (3). Likewise, whether the signaling from the ␣␤ or the ␥␦ TCR, lineage thymocytes are blocked at the DN3 stage and do not sur- respectively, is the instructive determinant of lineage choice or vive (9–14). The requirement for some pre-TCR components is whether ␣␤/␥␦ commitment occurs at least in part independently confined to developing ␣␤ T cells, because the absence of CD3␦ of TCR rearrangement is unclear. and CD3␨ has no effect on and pT␣ deficiency even increases ␥␦ Although it appears that TCR gene rearrangements influence the T cell number (9, 14, 15). ␣␤ vs ␥␦ lineage decision, there are also indications that this can- A number of signaling pathways in addition to the pre-TCR have been implicated in ␣␤/␥␦ lineage commitment, one of which is the IL-7/IL-7R pathway. IL-7R signaling is essential for the Mammalian Genetics Laboratory, Lincoln’s Inn Fields Laboratories, London Re- generation and maintenance of precursor cells committed to either search Institute, Cancer Research, London, United Kingdom the T or B cell lineage. In early thymocyte development, the loss Received for publication March 9, 2006. Accepted for publication February 22, 2007. of IL-7 or IL-7R␣ results in a substantial reduction in the number The costs of publication of this article were defrayed in part by the payment of page of total thymocytes and mature T cells (16, 17). IL-7R signaling charges. This article must therefore be hereby marked advertisement in accordance has an especially drastic effect on ␥␦ T cells, which are absent in with 18 U.S.C. Section 1734 solely to indicate this fact. mice lacking IL-7 or IL-7R␣ (18, 19). IL-7 treatment augments ␥␦ 1 The London Research Institute is funded by Cancer Research U.K. L.R.-S. acknowl- edges support from an European Union Marie-Curie fellowship. T cell number in ex vivo systems (20, 21), suggesting that IL-7 ␥␦ 2 Current address: Epithelial Homeostasis and Cancer, Department of Cell Differen- signaling can promote T cell development. tiation and Cancer, Center of Genomic Regulation, Doctor Aiguader 88, 08003 Bar- The transcription factor AP-1 consists of a variety of dimers celona, Spain. composed of members of the Fos and Jun families of proteins (22). 3 Address correspondence and reprint requests to Dr. Axel Behrens, Cancer Research Although the Fos proteins (c-Fos, FosB, Fra-1, Fra-2) can only U.K. London Research Institute, Lincoln’s Inn Fields Laboratories, Mammalian Genetics Laboratory, 44, Lincoln’s Inn Fields, London WC2A 3PX, U.K. E-mail heterodimerize with members of the Jun family, the Jun proteins address: [email protected] (c-Jun, JunB, JunD) can both homodimerize and heterodimerize 4 Abbreviations used in this paper: DN, double negative; BM, bone marrow; DAPI, with other Jun or Fos members to form transcriptionally active 4Ј,6Ј-diamidino-2-phenylindole; ChIP, chromatin immunoprecipitation; FTOC, fetal complexes (22–24). thymus organ culture. The activity of the AP-1 transcription factor is strongly induced Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 in response to numerous extracellular stimuli including TCR www.jimmunol.org The Journal of Immunology 5691 signaling. AP-1 stimulation is mediated, in part, by the phosphorylation of c-Jun by the JNKs (25). c-Jun N-terminal phosphorylation at serine residues 63 and 73 and threonine residues 91 and 93 within its transactivation domain is thought to increase transcription of target genes, one of which is the c-jun gene itself (26). The function of JNK signaling in T cells has been extensively studied and has revealed a multitude of JNK functions ranging from T cell development and proliferation to T cell differentiation (27–31). c-Jun is one of the main targets of JNK signaling, but its function in T cells is less well understood, in part due to the embryonic lethality of c-jun-deficient mice (32, 33). RAG2 complementation experiments with c-junϪ/Ϫ ES cells revealed reduced restoration of thymocytes but normal T cell proliferation and IL-2 production (34). The role of c-Jun and AP-1 in thymopoiesis has also been investigated using dominant-negative approaches in transgenic mice. B cell-activating transcription factor belongs to the AP-1 super- family of basic leucine zipper transcription factors and forms het- erodimers with Jun that possess minimal transcriptional activity. Downloaded from Overexpression of B cell-activating transcription factor using the lck promoter resulted in a specific defect in NKT cell development, and reduced thymocyte proliferation (35, 36). Moreover, transgenic overexpression of a dominant-negative version of c-Jun caused aberrant thymic organization accompanied by reduced T

cell proliferation and IL-2 production (37). T cell development has http://www.jimmunol.org/ also been investigated in junAA mice, in which serines 63 and 73 in the c-Jun N terminus were replaced by alanines using a knock-in approach, but no defects were found (38, 39). To clarify the role of c-Jun in T cell development, we have used conditional mutagenesis to generate mice lacking c-Jun in the T cell lineage (c-jun⌬T mice; Refs. 40 and 41). In this study, we show that c-jun inactivation during early T cell development resulted not only in a severe developmental arrest of ␣␤ T cells at the DN3 stage but also in enhanced generation of ␥␦ T cells. by guest on September 26, 2021 Materials and Methods Mice FIGURE 1. Tissue-specific inactivation of c-jun gene during early T ⌬T c-junf/f c-Jun mice were generated by crossing the mice (40) with mice cell development. A, Scheme of floxed c-jun allele before and after cre carrying the Cre recombinase transgene under the control of the proximal recombination. Arrows indicate approximate position of PCR primers lck promoter (41). All mice used were between 6 and 8 wk old and always were bread and maintained in a clean individually ventilated cage animal (OL1, OL2, OL3) used to detect deletion. B, lck-cre transgene induces facility. The Home Office approved all experimental protocols used in c-jun deletion in thymus and spleen. PCR analysis of genomic DNA iso- ⌬ this work. lated from different tissues from c-junf/f or c-jun T mice. Th, Thymus; Sp, spleen; Li, liver; Hr, heart; Ki, kidney; Tl, tail. C, c-jun deletion is complete Mixed bone marrow (BM) chimeras ⌬ from DN4 onwards. PCR on genomic DNA from c-junf/f or c-jun T sorted Ϫ ϩ Mixed BM chimeras were made according to standard protocols (42). rag2 thymocytes on the bases of surface expression CD25 CD44 (DN1), knockout host mice were irradiated with two doses of 500 rads from a CD25ϩCD44ϩ (DN2), CD25ϩCD44Ϫ (DN3), CD25ϪCD44Ϫ (DN4), cesium source separated by 3 h. One hour after the second dose, 2 ϫ 106 CD4ϩCD8ϩ (DP), CD4ϩCD8Ϫ (CD4), CD4ϪCD8ϩ (CD8), and ␥␦TCRϩ mixed donor cells were injected i.v. The donor cells were a mix of BMs (TCR␥␦). D, c-Jun protein is undetectable in c-jun⌬T thymocytes and ⌬T from C57BL/6 (harboring the CD45.1 allele) and c-jun mice. The BM splenic CD4ϩ and CD8ϩ T cells. Protein extracts from total thymocytes or cells were collected by flushing the bones from the hind legs. The donor ϩ ϩ CD4 and CD8 T cells isolated from spleen with or without treatment cells were distinguished by the surface expression of CD45.1 (C57BL/6 ␮ ␮ wild type) and CD45.2 (c-jun⌬T). BM chimeras were analyzed 3 mo postin- with 20 g/ml PMA and 2 M ionomycin for 5 h were analyzed for c-Jun ␤ jection for the presence of thymic TCR␤/TCR␥␦ T cells. and -actin (loading control) expression.

Flow cytometry and cell sorting CD44ϩ DN1 and DN2. Thy1.2 was used in all the analysis as a T cell- FITC, PE, allophycocyanin, and biotin-conjugated Abs were obtained from specific marker. Specific cell subsets defined by their cell surface markers BD Pharmingen. Tricolor-conjugated Abs or streptavidin were from Caltag were sorted using a MoFlo cell sorter (DakoCytomation). Sorted cells were Ն Laboratories. The Fix & Perm kit from Caltag Laboratories was used for all reanalyzed by FACS and were 95% pure. the intracellular stainings following the manufacturer’s protocol. Stained Cell cycle and cell death analysis cells were analyzed on a FACSCalibur (BD Biosciences), and the data were analyzed using FlowJo software (Tree Star). CD4ϪCD8Ϫ DN T cell Cellular DNA content was assayed on fixed cells using 4Ј,6Ј-diamidino- subsets were analyzed for CD44 and CD25 surface expression by lineage 2-phenylindole (DAPI) staining. Surface-stained thymocytes were fixed exclusion of mature CD4ϩCD8ϩ double-positive and CD4ϩ, CD8ϩ single- and permeabilized with the Fix & Perm kit from Caltag Laboratories fol- positive as well as non-T cell lineage cells using a mixture of biotinylated lowing the manufacturer’s protocol. Stained cells were incubated with Abs (CD4, CD8, B220, Mac-1, pan-NK, Gr-1, and TCR␥␦) revealed with DAPI (10 ␮g/ml), and DNA content was analyzed using the LSRII cy- streptavidin-Tricolor and costained with CD25-FITC, CD44-PE, and tometer (BD Biosciences) and using FlowJo software. Cell death was an- Thy1.2-allophycocyanin. When only DN3 and DN4 were desired to be alyzed on nonfixed thymocytes by the Annexin V-FITC Apoptosis Detec- analyzed, CD44-biotin was included in the lineage mixture to exclude tion Kit (BD Biosciences) following the manufacturer’s protocol. Stained 5692 REGULATION OF T CELL DEVELOPMENT BY c-Jun Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. c-jun deletion in early thymocyte development reduces thymus cellularity and causes a partial block at the DN3 stage of T cell development. A, Thymi from c-junf/f and c-jun⌬T littermates illustrating reduced size of c-jun⌬T thymus. B, H&E staining of c-junf/f and c-jun⌬T thymus sections. C, c-jun⌬T deletion causes a partial block at DN stage of T cell development. Total thymocytes from c-junf/f and c-jun⌬T mice were analyzed for surface expression of CD4 and CD8 on Thy1.2ϩ gated by ﬂow cytometry. Numbers within the plots indicate the mean of percentages from eight mice of each genotype. On the right are absolute cell numbers for total thymocytes and T cell subpopulations from c-junf/f and c-jun⌬T mice. Bars, Mean Ϯ SD from eight mice each genotype. D, Accumulation of DN3 thymocytes in c-jun⌬T mice. A representative plot of CD44 vs CD25 on lineageϪThy1.2ϩ gated DN thymocytes from littermate c-junf/f and c-jun⌬T mice (upper plot). Values are the mean percentages from eight mice. Histogram overlay of CD25 expression on lineageϪThy1.2ϩ gated DN thymocytes comparing c-junf/f (shaded histogram) and c-jun⌬T (solid line). The gate on the histogram deﬁnes CD25bright thymocytes. Absolute cell numbers for each DN subset and CD25bright thymocytes are shown. DP, Double positive; CD25hi, CD25high; bars, mean Ϯ SD .p Ͻ 0.01; Student’s unpaired t test ,ءء ;p Ͻ 0.05 ,ء .from eight mice each genotype

cells were analyzed for cell death assays with a FACSCalibur (BD generate a 350-bp fragment indicative of the c-junf allele and a 500-bp Biosciences). fragment for the c-jun⌬ allele. Genomic PCR to detect c-jun deletion Western blot analysis Genomic DNA from different mouse tissues or sorted cells were obtained Protein extracts and SDS-PAGE analysis were performed according to following standard techniques and subjected to PCR using three primers to standard techniques (43). The blot was developed with the indicated Abs detect the ﬂoxed and the deleted c-jun alleles as described (40). PCR prod- (c-Jun H-79 purchased from Santa Cruz; ␤-actin from Sigma-Aldrich) and ucts were separated on a 2% agarose gel. In a single PCR, these primers visualized by an ECL detection system (Amersham Pharmacia). The Journal of Immunology 5693

FIGURE 3. Increased numbers of thymic and splenic ␥␦ T cells in c-jun⌬T mice. A, Increased numbers of thymic ␥␦ T cells in c-jun⌬T mice. Represen- tative plot of TCR␤ vs TCR␥␦ surface expression on Thy1.2 gated thymocytes and absolute cell numbers from six mice. B, c-junf/f deletion increases ␥␦ T cell number and reduction of mature ␣␤ T cells in spleen. Thy1.2ϩ splenocytes represented by TCR␤ vs TCR␥␦ surface expression and abso- Downloaded from lute cell numbers from eight mice. C, Increase in TCR␥␦ϩCD8␣Ϫ and TCR␥␦ϩCD8␣ϩ cells in c-jun mutant mice. A representative plot of CD8␣ vs TCR␥␦ on Thy1.2ϩ splenocytes and absolute cell numbers from eight Ͻ ءء mice. , p 0.01; Student’s un- http://www.jimmunol.org/ paired t test. by guest on September 26, 2021

FIGURE 4. Unaffected cell cycle progression but different cell death behavior in c-jun⌬T mice. A, Normal cell cycle proﬁle in DN3, DN4, and TCR␥␦ c-jun⌬T cells. Representative histograms of DNA content (left)of lineageϪThy1.2ϩ gated DN3, DN4 cells and Thy1.2ϩ gated TCR␥␦ of total thymocytes from c-junf/f or c-jun⌬T mice. The cell cycle analysis (right) has been done on FlowJo by the Dean/Jett/Fox model; results are the proportion of cells in each phase of the cell cycle. Data are derived from four mice of each genotype. B, Increased cell death in c-jun⌬T DN3/ DN4 cells. Lineage negative (CD4, CD8, CD44, B220, PanNK, Mac1, Gr1, TCR␥␦), Thy1.2ϩ thymocytes are represented by CD25 vs annexin V surface expression to analyze cell death on DN3 and DN4 cells. Shown is a representative plot with the mean of the percentages in each quadrant. Values are the percentages of annexin Vϩ cells in DN3 and DN4 subpopulations. Mean Ϯ SD from four different mice of each genotype. 5694 REGULATION OF T CELL DEVELOPMENT BY c-Jun Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. lck-cre transgene interferes with normal T cell development. A, Total thymocytes from wild-type (Wt) and lck-cre transgenic mice were analyzed for surface expression on CD4 and CD8 on Thy1.2ϩ cells gated by ﬂow cytometry. Numbers within the plots indicate the mean of percentages from six mice of each genotype. Right, Absolute cell numbers for total thymocytes and T cell subpopulations mean Ϯ SD from six mice of each genotype. B, Representative plot of CD25 vs CD44 on lineageϪThy1.2ϩ DN thymocytes. Histogram overlay of CD25 expression on lineageϪThy1.2ϩ DN thymocytes comparing wild-type (Wt) (shaded histogram) and lck-cre transgenic (solid line). The gate in the histogram deﬁnes CD25high DN gated thymocytes. Absolute cell numbers for each DN subset and CD25high thymocytes are shown mean Ϯ SD from six mice each genotype. C, Plot analysis of TCR␤ vs TCR␥␦ surface expression on Thy1.2ϩ gated thymocytes from wild-type and lck-cre transgenic and absolute cell number of ␥␦ T cells. Bars, Mean Ϯ SD .p Ͻ 0.05; Student’s unpaired t test ,ء .from six mice of each genotype

Semiquantitative and quantitative real-time PCR quantitative PCR. The PCR protocol consisted of one 10-min denaturation cycle at 95°C followed by 40 cycles of denaturation at 95°C for 15 s and Total RNA was isolated from sorted DN3 using RNAeasy (Qiagen). cDNA annealing/extension at 60°C for 1 min. All real-time PCR data are ex- was made using the Ready-To-Go First-Strand Beads system (Amersham pressed as fold change in mRNA levels with respect to control after nor- Biosciences) following the manufacturer’s protocol. For semiquantitative malizing to the levels of GAPDH. PCR, different amounts of cDNA were used and amplified using Qiagen Taq polymerase. The primers used to amplify GAPDH, Notch1, RBP-␬j, Chromatin immunoprecipitation (ChIP) and IL-7R␣ cDNAs are available upon request. Real-time PCR was using a Chromo4Fluorescence machine (MJ Re- Jurkat human leukemia T cells were maintained in log phase growth at search), and the data were analyzed using the provided Opticon Monitor3 37°C under a humidified atmosphere with 5% CO2 in RPMI 1640 supple- software. The reaction mixture consist on 2.5 ␮l of cDNA, 12.5 ␮lof2ϫ mented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/ml penicillin, and SYBR Green PCR master mixture (Roche Applied Science), 2 ␮lof5␮M 100 mg/ml streptomycin. forward primer, and 2 ␮lof5␮M reverse primer in a 25-␮l reaction ChIP analysis was performed as described previously (43). Immunopre- volume. The same primer pairs were used for real-time PCR and semi- cipitations were conducted with c-Jun-specific or control rabbit IgG Abs. The Journal of Immunology 5695

Table I. Absence of c-jun increases generation of ␥␦ T cells in competitive reconstitution experimentsa

Thy1.2ϩ

Thy1.2ϩ TCR␥␦ϩ TCR␤ϩ

Mice %CD45.1 %CD45.2 %CD45.1 %CD45.2 %CD45.1 %CD45.2

1⌬Tb:1wtc 1 48.1 31.3 21.6 58.1 64.5 22.5 2 40.2 39.4 13.6 67.2 43.2 37.6 3 12.6 72.9 2.74 80.8 18.4 71.0 4 31.7 46.3 14.1 65.2 40.3 41.6 3⌬Tb:1wtc 5 9.47 78.4 1.77 79.0 22.1 66.5 6 3.61 89.0 0.59 86.2 6.11 87.7 7 23.8 56.3 7.48 78.2 30.9 53.2

a A small percentage of cells did not show unambiguous staining for a single CD45 marker and was excluded from the analysis. b ⌬T BM donor cells are CD45.2. c Wild-type (wt) BM donor cells are CD45.1. Downloaded from http://www.jimmunol.org/

FIGURE 6. Abnormal intracellular expression of TCR␤ and TCR␥␦ in c-jun⌬T DN4 thymocytes. A, Nor- mal intracellular CD3␧ expression in by guest on September 26, 2021 DN3 and DN4 c-jun⌬T thymocytes. Histogram representation of intracellular staining for CD3␧ on DN3 and DN4 c-junf/f and c-jun⌬T thymocytes (ﬁlled histogram) and isotype control (dotted line). B, Reduced percentage of c-jun⌬T mice DN4 cells expressing intracellular TCR␤. Histogram representation of intracellular staining for TCR␤ in DN3 and DN4 c-junf/f, lck- cre, and c-jun⌬T thymocytes. The values represent the percentage of icTCR␤ϩ, mean Ϯ SD of four mice of each genotype. C, Increased percentage of c-jun⌬T mice DN4 cells expressing intracellular TCR␥␦. Intra- cellular TCR␥␦ expression on DN3 and DN4 cells is shown on Thy1.2ϩ c-junf/f, lck-cre, and c-jun⌬T thymocytes, the value represents the percentage of icTCR␥␦ cells. In B and C, the values are mean Ϯ SD. 5696 REGULATION OF T CELL DEVELOPMENT BY c-Jun

FIGURE 7. Normal development of ␥␦ T cells in junAA homozygous mice. A, Normal numbers of thymic ␥␦ T cells in junAA mice. Representative plot of TCR␤ vs TCR␥␦ surface expression on Thy1.2ϩ gated thymocytes and absolute cell numbers from four mice of each genotype. B, Normal levels of ␥␦ T cells in junAA spleen. Thy1.2ϩ splenocytes represented by TCR␤ vs TCR␥␦ surface expression and absolute cell numbers Downloaded from from four mice. C, Equal levels of TCR␥␦: CD8␣Ϫ and TCR␥␦:CD8␣ϩ cells in junAA mice. CD8␣ vs TCR␥␦ plot on Thy1.2ϩ splenocytes and absolute cell number from four mice. http://www.jimmunol.org/ by guest on September 26, 2021

The oligonucleotide sequences used to amplify the DNA fragments are came detectable in total wild-type thymocytes only after treatment available upon request. with PMA-ionomycin, but c-jun⌬T thymocytes lacked c-Jun protein. FTOC Likewise, low levels of c-Jun were present in control peripheral T cells, and protein levels were strongly induced by treatment with f/f ⌬T Thymi from c-jun or c-jun E15.5 embryos were dissected in single PMA-ionomycin, but neither basal nor induced c-Jun protein could be lobes, and each lobe was placed on the surface of polycarbonate filters ⌬T (0.8-␮m pore size; Nuclepore) that were supported on Gelfoam in 5 ml of detected in c-jun peripheral T cells (Fig. 1D). DMEM medium supplemented with L-glutamine, 25 mM HEPES, 10% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cultures were grown in c-jun is required for T cell development humidified atmosphere in 5% CO2 at 37°C for 11 days with or without mouse rIL-7 (R&D Systems), changing the medium every 3 days. After the culture At the age of 8 wk, the size of the thymus was reduced in c-jun⌬T period, the T cell development was analyzed by flow cytometry. mice relative to c-junf/f controls (Fig. 2A), but gross thymic archi- tecture and histology was normal (Fig. 2B). Consistent with the Results reduced thymus size, the absolute number of c-jun⌬T total thymo- Intrathymic deletion of c-jun by lck-cre cytes was reduced 4- to 5-fold to 20–25% of that of control mice To assess the functions of c-jun in T cells, we deleted c-jun during because of a decrease in all thymocyte subsets with the exception early T cell development by crossing c-junf/f mice with lck-cre of the DN subpopulation (Fig. 2C). However, the relative propor- transgenics (c-jun⌬T mice; Refs. 40 and 41). lck-cre-mediated de- tions of ␣␤ T cell subsets were not drastically skewed except for letion of c-junf was efficient in thymus and detectable in spleen but an increase in DN cells (Fig. 2C). Further examination of the DN could not be detected in nonlymphoid organs (Fig. 1, A and B). To thymocyte population using CD44 vs CD25 FACS profiles re- further characterize deletion of the floxed c-jun allele during T cell vealed an accumulation of DN2/DN3 cells and an increase of development, genomic DNA was purified from total and sorted CD25bright/ϩ cells in c-jun⌬T mice, suggesting a problem at DN3 to thymocyte subpopulations, and the deletion efficiency was deter- DN4 transition of T cell development (Fig. 2D). mined by PCR analysis. c-junf deletion became detectable at the To verify whether c-jun also plays a role in ␥␦ T cell develop- DN2 stage and reached completion at the DN4 stage. c-junf was ment, thymocytes from c-jun⌬T mice were stained for surface ex- also completely inactivated in CD4ϩCD8ϩ double-positive, pression of ␥␦ TCR. The absolute number of ␥␦ T cells from thymi CD4ϩCD8Ϫ and CD4ϪCD8ϩ single-positive ␣␤ T cells as well as of c-jun⌬T mice was increased ϳ2–3 times compared with that in ␥␦ T cells (Fig. 1C). The loss of the c-Jun protein in c-jun⌬T T from controls (Fig. 3A). The number of peripheral ␥␦ T cells was cells was also confirmed by Western blot analyses. c-Jun protein be- also higher in the spleens of c-jun⌬T mice, whereas the percentages The Journal of Immunology 5697 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 8. Increased expression of IL-7R␣ in c-jun⌬T thymocytes. A, Abnormal IL-7R␣ expression in c-jun⌬T DN3 cells. Semiquantitative (left panel) and real-time (right panel) RT-PCR on total RNA from sorted DN3 and DN4 cells. B, Increased surface expression of IL-7R␣ in c-jun⌬T thymocytes mice. A representative histogram overlay of surface IL-7R␣ expression (three independent experiments done with three to four mice each) on gated DN3 (top), DN4 (middle), and ␥␦ T cells (bottom) comparing c-junf/f (shaded histogram) and c-jun⌬T (black line). C, No effect of lck-cre transgene on IL-7R␣ surface levels. A representative histogram overlay of surface IL-7R␣ expression on gated DN3 (top), DN4 (middle) and Thy1.2ϩ cells (bottom) comparing wild-type (Wt; shaded histogram) and lck-cre (black line). D, c-Jun binds to IL-7R␣ promoter in Jurkat T cells. Schematic representation of AP-1 binding sites (gray ovals) or AP-1-like sites (black ovals) on the human IL-7R␣ promoter (top). ChIP using Jurkat T cells were performed with a c-Jun polyclonal Ab (␣-c-Jun), and puriﬁed rabbit IgG (Rb IgG) or without any Ab as controls. PCR was performed on input and immunoprecipitated DNA using primer pairs spanning AP-1 bindings sites in human IL-7R␣ promoter as indicated. The c-Jun target genes c-jun and cdc2 served as positive and gapdh as negative controls for c-Jun ChIP. The approximate positions of PCR primers (OL1–4) are indicated.

and numbers of ␣␤ T cells was reduced (Fig. 3B). There was a and B) than c-Jun deletion (Fig. 2) and induced only a marginal significant increase in both CD8␣ϩ and CD8␣Ϫ splenic ␥␦ T cells increase of ␥␦ T cell production (Fig. 5C). in c-jun⌬T mice (Fig. 3C). ␣␤ ␥␦ To investigate the reason underlying the defects in T cell de- Deregulation of and T cell precursors in DN4 thymocytes velopment in the absence of c-jun, we analyzed cell cycle progres- The increase in ␥␦ lineage T cells in c-jun conditional knockout sion and cell death. The percentages of DN3, DN4, and ␥␦ T cells mice could be due to the partial block in development of ␣␤ lin- ␥␦ in G1, S, and G2-M phases of the cell cycle was comparable be- eage cells, allowing for expansion of T cells in the thymus. To tween in c-jun⌬T and control mice, indicating that the c-jun is not address this problem, we generated mixed BM chimeric mice by required for thymocyte cell cycle progression (Fig. 4A). In con- coinjecting BM cells from c-jun⌬T and wild-type control mice into trast, there were differences in the extent of cell death. The per- lethally irradiated rag2Ϫ/Ϫ hosts. The analysis of reconstituted centage of annexin V-positive cells was increased in c-jun-mutant mice revealed that c-jun⌬T cells were slightly less efficient in gen- DN3 and DN4 thymocytes, suggesting a role for c-Jun in thymo- erating ␣␤ T cells, but they showed markedly increased ␥␦ T cell cyte survival (Fig. 4B). development (Table I), suggesting that the effect on ␥␦ T cells by Although our experiments indicated a role for c-jun in T cell c-jun deletion is cell autonomous. development, we detected as well an effect of the lck-cre transgene We next tested whether c-jun has a function in the early stages itself on T cell development, as published before (44). However, of ␥␦ T cell development. Precursors of ␣␤ and ␥␦ T cells can be this effect was less severe in the ␣␤ linage development (Fig. 5, A identified in the DN4 population by intracellular expression of 5698 REGULATION OF T CELL DEVELOPMENT BY c-Jun Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 9. Increased development of c-jun⌬T ␥␦ T cells in FTOC. A, FACS analysis of FTOC of E15.5 thymus (Thym) from c-jun⌬T or c-junf/f embryos cultured for 11 days with or without 2 ng/ml recombinant murine IL-7. A representative histogram and dot plot from two independent experiments with at least four animals per genotype per experiment is shown. The histograms represents surface expression of TCR␥␦ on DAPIϪThy1.2ϩ gated cells, the dot plot represents CD4 vs CD8 on TCR␥␦Ϫ cells from the histogram (arrow). B, Absolute cell numbers of one representative FTOC experiment. The values are the mean Ϯ SD.

TCR␤ and TCR␥␦ proteins, respectively (45). All DN3 and DN4 opment (38, 39). To determine whether target gene activation by thymocytes of c-jun⌬T mice as well as c-junf/f controls were pos- c-Jun mediated through c-Jun N-terminal phosphorylation plays a itive for intracellular staining for CD3␧ (icCD3␧), conﬁrming their role in T cell lineage decision, we investigated ␥␦ T cell develop- T cell identity (Fig. 6A). In contrast, the percentages of icTCR␤- ment in junAA mice. junAA homozygous thymocytes had normal expressing cells were normal in DN3 cells but reduced at the DN4 levels of ␥␦ TCR surface expression and the absolute number of stage in c-jun⌬T mice (Fig. 6B). lck-cre-transgenic mice showed a ␥␦ T cells from thymi of junAA mice was unaltered (Fig. 7A). smaller increase in icTCR␤ϩ cells, which was intermediate be- Similarly, the number of splenic ␥␦ T cells, of both the CD8␣ϩ tween c-jun⌬T and c-junf/f mice (Fig. 6B). The increased numbers and CD8␣Ϫ subset, was comparable between junAA and control of mature ␥␦ T cells were probably due to increased generation of mice (Fig. 7, B and C). Therefore, stimulation of c-Jun function by immature ␥␦ T cell precursors, given that there was an ϳ2- to N-terminal phosphorylation appears to be dispensable for ␣␤/␥␦ T 3-fold increase in the percentage of icTCR␥␦ϩ DN4 cells in cell lineage decision, suggesting that T cell development may be c-jun⌬T mice compared with c-junf/f and lck-cre-transgenic con- controlled by c-Jun-mediated target gene repression, rather than trols (Fig. 6C). Therefore, the alterations in the numbers of mature target gene activation. ␣␤ and ␥␦ T cells correlate with abnormal generation of their respective precursor cells. Deregulated expression of IL-7R␣ in c-jun⌬T pro-T cells c-Jun N-terminal phosphorylation is dispensable for ␣␤/␥␦ To gain insight into how c-jun controls T cell development, we T cell lineage decision tested the transcriptional regulation of molecules known to be involved in ␣␤/␥␦ lineage decision. We ﬁrst investigated the expres- c-Jun, like other transcription factors, can both activate and repress sion of components of the TCR complex, which are key regulators gene expression (46). Phosphorylation of serines 63 and 73 within of early T cell development (9–14). However, the expression levels of the c-Jun transactivation domain by the JNKs greatly augments the pT␣, TCR␤, and of the CD3 components of the pre-TCR, CD3␥, activity of c-Jun (25). junAA mice, in which serines 63 and 73 in the CD3␦, CD3␧, and CD3␨ were not affected in the absence of c-jun. c-Jun N terminus were replaced by alanines using a knock-in ap- There was also no alteration in the mRNA levels of RAG1 and proach, were previously shown to have normal ␣␤ T cell devel- RAG2, which are required for TCR rearrangement (data not shown). The Journal of Immunology 5699

In addition to the TCR, several other signaling pathways are cells can give rise to ␣␤ T cells, although with reduced efficiency, known to regulate ␣␤/␥␦ T cell development, including Notch and suggesting that there is no absolute requirement for c-jun in ␣␤ T IL-7 signaling (18, 19, 47, 48). Although the expression of the cell lymphopoiesis, while ␥␦ T cells were not analyzed (34). Notch1 and its transcriptional partner RBP-j␬ was unaffected, Although the details of the underlying molecular mechanism are mRNA levels of IL-7R␣ were increased in c-jun⌬T DN3 cells, as only incompletely understood, it is clear that several signaling detected by both conventional and quantitative PCR (Fig. 8A). As a pathways can influence ␣␤/␥␦ lineage decision (53). The IL-7R␣ consequence, IL-7R␣ protein surface levels were also higher in is required for ␥␦ T cell development (18), and there is evidence c-jun⌬T DN3 and DN4 cells as well as in c-jun⌬T ␥␦ T cells compared that increased IL-7R␣ signaling correlates with ␥␦ T cell genera- with c-junf/f and lck-cre transgenic controls (Fig. 8, B and C). tion (20, 21). Expression of IL-7R␣ is down-regulated during pro- Next, we examined the IL-7R␣ promoter for the presence of gression from DN2 to DN3 (21, 54) and microarray analysis of AP-1 sites. Within 3.2 kb upstream of the ATG initiation codon, various stages of thymocyte development has identified c-jun as several bona fide AP-1 and AP-1-like sites were found (Fig. 8D). being specifically up-regulated at the DN3 stage (55). This inverse ChIP assays revealed efficient binding of c-Jun to the jun1/2 sites correlation suggests that c-Jun may be involved in the repression of the c-jun promoter and to the cdc2 promoter, both established of IL-7R␣ expression at the DN2/DN3 transition (Fig. 8). c-Jun target genes (43, 49), but no binding to the gapdh promoter, The relative strength of TCR signaling has been proposed to be used as negative control (Fig. 8D). Strikingly, two regions of the a determinant of the ␣␤/␥␦ lineage decision. According to this IL-7R␣ promoter harboring multiple predicted c-Jun binding sites model, ␥␦ T cell development would be favored by strong TCR were detectable in c-Jun ChIP (Fig. 8D). Therefore, c-Jun binds to signaling, and ␣␤ T cell development would be resulting from the IL-7R␣ promoter and appears to directly regulate IL-7R␣. weaker TCR signals (7, 8). Downloaded from IL-7R␣ expression is heterogeneous in DN1 thymocytes, and at ␥␦ Increased T cell development of c-jun-deficient thymocytes this stage high level of IL-7R␣ expression does not indicate in- in vitro creased T cell developmental potential (56, 57). However, at the To directly investigate the relevance of IL-7R␣ regulation by c- DN2 stage increased IL-7R␣ expression correlates with increased jun, the development of thymocytes isolated from control and generation of ␥␦ T cells (21), indicating that IL-7R signal strength ⌬T c-jun embryos was studied in FTOCs. Abnormal ␣␤/␥␦ T cell may favor ␥␦ linage choice, possibly by promoting rearrangement http://www.jimmunol.org/ development of c-jun⌬T mice was reproduced in FTOC because and/or expression of the TCR␥ locus (58–61). The relationship both DP and CD4ϩ and CD8ϩ SP ␣␤ T cells were reduced in between TCR and IL-7 signaling is controversial (53), and it is number, and there was a substantial increase in ␥␦ T cells in unclear whether TCR and IL-7R act independently or whether c-jun⌬T cultures (Fig. 9). IL-7 treatment of control FTOC induced IL-7R might, using an unknown mechanism, positively modulate ␥␦ T cell development, which, however, did not reach the numbers or mimic some aspects of TCR signaling, thereby increasing the observed in untreated c-jun-deficient cultures. The number of ␥␦ T strength of the ␥␦ T cell-inducing signal. cells in c-jun⌬T cultures was further augmented by IL-7, at the ex- This study has identified c-Jun as a novel regulator of T cell lineage pense of DP ␣␤ T cells whose number was drastically reduced (Fig. decision and development. The molecular mechanisms governing 9). IL-7 treatment had only a marginal effect on control ␣␤ T cells ␣␤/␥␦ lineage decision are not well understood, and it is likely that by guest on September 26, 2021 (Fig. 9). Therefore, deregulation of IL-7 signaling may contribute to several pathways contribute to the molecular control of this biological the defect in T cell development in the absence of c-jun. process. Therefore, by regulating IL-7R␣ expression (and probably other as yet unidentified target genes), c-Jun may be part of a signal- Discussion ing network that regulates thymic T cell development. In this study, we have identified a role for c-jun in thymic T cell development. The important function for c-jun in T cell develop- Acknowledgments ment is unique among the AP-1 transcription factors analyzed to We thank A. Jandke, N. Kanu, K. Lightfood, K. Nagakawa, and H. Hinton date, because loss-of-function mutations in other AP-1 family for technical help and advice; Y. Westermarck for c-Jun small hairpin RNA member proteins, including the two other members of the Jun fam- plasmids; and Y. Carrasco, D. Pennington, and C. Reis Sousa for critical ily, JunB and JunD, and the c-fos proto-oncogene, have no signif- reading of the manuscript. icant effect on early thymic T cell development (50–52). c-Jun is an important target of the JNK signaling pathway, but Disclosures c-Jun N-terminal phosphorylation appears to be dispensable for The authors have no financial conflict of interest. c-Jun function in thymocytes. T cell development proceeds nor- References mally in the absence of both jnk1 and jnk2, which are likely to 1. Warren, L. A., and E. V. Rothenberg. 2003. Regulatory coding of lymphoid lineage encode all JNK isoenzymes present in thymus (29). Similarly, choice by hematopoietic transcription factors. Curr. Opin. Immunol. 15: 166–175. there were no detectable abnormalities in ␣␤/␥␦ lineage commit- 2. Rodewald, H. R., and H. J. Fehling. 1998. 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