HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Riera-Sans, L. Articles by Behrens, A. Search for Related Content PubMed PubMed Citation Articles by Riera-Sans, L. Articles by Behrens, A. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene

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

Mammalian Genetics Laboratory, Lincoln’s Inn Fields Laboratories, London Research Institute, Cancer Research, London, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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 / T cell development.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mature T lymphocytes derive from lymphoid precursors through several distinct cell fate specification events (1). Common lymphoid precursors, which have already lost erythroid and myeloid potential, separate into B and T cell precursors in the thymus. Immature pro-T cells then develop into mature T cells characterized by surface expression of either the CD4⁺ or the CD8⁺ coreceptor before leaving the thymus.

Thymocytes can be divided into four major subsets based on the expression of CD4 and CD8. The CD4^–CD8^– double-negative (DN)⁴ cells are the most immature subset. The DN population can be further divided into four subsets (DN1–DN4) based on their differential expression of CD44 and CD25. DN thymocytes mature in the order of CD44⁺CD25^– (DN1), CD44⁺CD25⁺ (DN2), CD44^–CD25⁺ (DN3), and CD44^–CD25^– (DN4) (2).

The decision between and T cells occurs at the DN stage, but the exact time point of specification remains to be determined (3). Likewise, whether the signaling from the or the TCR, respectively, is the instructive determinant of lineage choice or whether / commitment occurs at least in part independently of TCR rearrangement is unclear.

Although it appears that TCR gene rearrangements influence the vs lineage decision, there are also indications that this cannot be the sole determinant of linage decision. If expression of an in-frame TCR invariably led to T cell development, one might expect a complete block in T cell development in mice expressing a rearranged TCR transgene. This, however, is not the case: many TCR-transgenic mice contain significant numbers of lineage T cells (4, 5). Conversely, significant numbers of T cells are present in mice expressing a rearranged TCR transgene (6). Recent work suggests that quantitative differences in TCR signaling appear to be influencing / lineage commitment (7, 8).

To make the developmental transition from DN3 to DN4, the TCR chains must be assembled into the pre-TCR complex, which consists of a TCR -chain, the invariant pT -chain, and CD3 components. Only cells that have a functional pre-TCR survive the transition from DN3 to DN4, a process also known as selection. In mice deficient for components of the pre-TCR developing lineage thymocytes are blocked at the DN3 stage and do not survive (9, 10, 11, 12, 13, 14). The requirement for some pre-TCR components is confined to developing T cells, because the absence of CD3 and CD3 has no effect on and pT deficiency even increases T cell number (9, 14, 15).

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 generation and maintenance of precursor cells committed to either the T or B cell lineage. In early thymocyte development, the loss of IL-7 or IL-7R results in a substantial reduction in the number of total thymocytes and mature T cells (16, 17). IL-7R signaling has an especially drastic effect on T cells, which are absent in mice lacking IL-7 or IL-7R (18, 19). IL-7 treatment augments T cell number in ex vivo systems (20, 21), suggesting that IL-7 signaling can promote T cell development.

The transcription factor AP-1 consists of a variety of dimers composed of members of the Fos and Jun families of proteins (22). Although the Fos proteins (c-Fos, FosB, Fra-1, Fra-2) can only heterodimerize with members of the Jun family, the Jun proteins (c-Jun, JunB, JunD) can both homodimerize and heterodimerize with other Jun or Fos members to form transcriptionally active complexes (22, 23, 24).

The activity of the AP-1 transcription factor is strongly induced in response to numerous extracellular stimuli including TCR 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, 28, 29, 30, 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-jun^–/– 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 superfamily of basic leucine zipper transcription factors and forms heterodimers with Jun that possess minimal transcriptional activity. 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 also been investigated in jun^AA 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.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice

c-Jun^T mice were generated by crossing the c-jun^f/f mice (40) with mice carrying the Cre recombinase transgene under the control of the proximal 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 facility. The Home Office approved all experimental protocols used in this work.

Mixed bone marrow (BM) chimeras

Mixed BM chimeras were made according to standard protocols (42). rag2 knockout host mice were irradiated with two doses of 500 rads from a cesium source separated by 3 h. One hour after the second dose, 2 x 10⁶ mixed donor cells were injected i.v. The donor cells were a mix of BMs from C57BL/6 (harboring the CD45.1 allele) and c-jun^T mice. The BM cells were collected by flushing the bones from the hind legs. The donor 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 postinjection for the presence of thymic TCR/TCR T cells.

Flow cytometry and cell sorting

FITC, PE, allophycocyanin, and biotin-conjugated Abs were obtained from BD Pharmingen. Tricolor-conjugated Abs or streptavidin were from Caltag Laboratories. The Fix & Perm kit from Caltag Laboratories was used for all the intracellular stainings following the manufacturer’s protocol. Stained cells were analyzed on a FACSCalibur (BD Biosciences), and the data were analyzed using FlowJo software (Tree Star). CD4^–CD8^– DN T cell subsets were analyzed for CD44 and CD25 surface expression by lineage exclusion of mature CD4⁺CD8⁺ double-positive and CD4⁺, CD8⁺ single-positive as well as non-T cell lineage cells using a mixture of biotinylated Abs (CD4, CD8, B220, Mac-1, pan-NK, Gr-1, and TCR) revealed with streptavidin-Tricolor and costained with CD25-FITC, CD44-PE, and Thy1.2-allophycocyanin. When only DN3 and DN4 were desired to be analyzed, CD44-biotin was included in the lineage mixture to exclude CD44⁺ DN1 and DN2. Thy1.2 was used in all the analysis as a T cell-specific marker. Specific cell subsets defined by their cell surface markers were sorted using a MoFlo cell sorter (DakoCytomation). Sorted cells were reanalyzed by FACS and were 95% pure.

Cell cycle and cell death analysis

Cellular DNA content was assayed on fixed cells using 4',6'-diamidino-2-phenylindole (DAPI) staining. Surface-stained thymocytes were fixed and permeabilized with the Fix & Perm kit from Caltag Laboratories following the manufacturer’s protocol. Stained cells were incubated with DAPI (10 µg/ml), and DNA content was analyzed using the LSRII cytometer (BD Biosciences) and using FlowJo software. Cell death was analyzed on nonfixed thymocytes by the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences) following the manufacturer’s protocol. Stained cells were analyzed for cell death assays with a FACSCalibur (BD Biosciences).

Genomic PCR to detect c-jun deletion

Genomic DNA from different mouse tissues or sorted cells were obtained following standard techniques and subjected to PCR using three primers to detect the floxed and the deleted c-jun alleles as described (40). PCR products were separated on a 2% agarose gel. In a single PCR, these primers generate a 350-bp fragment indicative of the c-jun^f allele and a 500-bp fragment for the c-jun allele.

Western blot analysis

Protein extracts and SDS-PAGE analysis were performed according to standard techniques (43). The blot was developed with the indicated Abs (c-Jun H-79 purchased from Santa Cruz; -actin from Sigma-Aldrich) and visualized by an ECL detection system (Amersham Pharmacia).

Semiquantitative and quantitative real-time PCR

Total RNA was isolated from sorted DN3 using RNAeasy (Qiagen). cDNA was made using the Ready-To-Go First-Strand Beads system (Amersham Biosciences) following the manufacturer’s protocol. For semiquantitative PCR, different amounts of cDNA were used and amplified using Qiagen Taq polymerase. The primers used to amplify GAPDH, Notch1, RBP-j, and IL-7R cDNAs are available upon request.

Real-time PCR was using a Chromo4Fluorescence machine (MJ Research), and the data were analyzed using the provided Opticon Monitor3 software. The reaction mixture consist on 2.5 µl of cDNA, 12.5 µl of 2x SYBR Green PCR master mixture (Roche Applied Science), 2 µl of 5 µM forward primer, and 2 µl of 5 µM reverse primer in a 25-µl reaction volume. The same primer pairs were used for real-time PCR and semiquantitative 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 annealing/extension at 60°C for 1 min. All real-time PCR data are expressed as fold change in mRNA levels with respect to control after normalizing to the levels of GAPDH.

Chromatin immunoprecipitation (ChIP)

Jurkat human leukemia T cells were maintained in log phase growth at 37°C under a humidified atmosphere with 5% CO₂ in RPMI 1640 supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin.

ChIP analysis was performed as described previously (43). Immunoprecipitations were conducted with c-Jun-specific or control rabbit IgG Abs. The oligonucleotide sequences used to amplify the DNA fragments are available upon request.

FTOC

Thymi from c-jun^f/f or c-jun^T E15.5 embryos were dissected in single lobes, and each lobe was placed on the surface of polycarbonate filters (0.8-µm pore size; Nuclepore) that were supported on Gelfoam in 5 ml of 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 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 period, the T cell development was analyzed by flow cytometry.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Intrathymic deletion of c-jun by lck-cre

To assess the functions of c-jun in T cells, we deleted c-jun during early T cell development by crossing c-jun^f/f mice with lck-cre transgenics (c-jun^T mice; Refs. 40 and 41). lck-cre-mediated deletion of c-jun^f was efficient in thymus and detectable in spleen but could not be detected in nonlymphoid organs (Fig. 1, A and B). To further characterize deletion of the floxed c-jun allele during T cell development, genomic DNA was purified from total and sorted thymocyte subpopulations, and the deletion efficiency was determined by PCR analysis. c-jun^f deletion became detectable at the DN2 stage and reached completion at the DN4 stage. c-jun^f was also completely inactivated in CD4⁺CD8⁺ double-positive, CD4⁺CD8^– and CD4^–CD8⁺ single-positive T cells as well as in T cells (Fig. 1C). The loss of the c-Jun protein in c-jun^T T cells was also confirmed by Western blot analyses. c-Jun protein became detectable in total wild-type thymocytes only after treatment with PMA-ionomycin, but c-jun^T thymocytes lacked c-Jun protein. Likewise, low levels of c-Jun were present in control peripheral T cells, and protein levels were strongly induced by treatment with PMA-ionomycin, but neither basal nor induced c-Jun protein could be detected in c-jun^T peripheral T cells (Fig. 1D).

View larger version (43K): [in this window] [in a new window]   FIGURE 1. Tissue-specific inactivation of c-jun gene during early T cell development. A, Scheme of floxed c-jun allele before and after cre recombination. Arrows indicate approximate position of PCR primers (OL1, OL2, OL3) used to detect deletion. B, lck-cre transgene induces c-jun deletion in thymus and spleen. PCR analysis of genomic DNA isolated from different tissues from c-junf/f or c-junT mice. Th, Thymus; Sp, spleen; Li, liver; Hr, heart; Ki, kidney; Tl, tail. C, c-jun deletion is complete from DN4 onwards. PCR on genomic DNA from c-junf/f or c-junT sorted thymocytes on the bases of surface expression CD25–CD44+ (DN1), CD25+CD44+ (DN2), CD25+CD44– (DN3), CD25–CD44– (DN4), CD4+CD8+ (DP), CD4+CD8– (CD4), CD4–CD8+ (CD8), and TCR+ (TCR). D, c-Jun protein is undetectable in c-junT thymocytes and splenic CD4+ and CD8+ T cells. Protein extracts from total thymocytes or CD4+ and CD8+ T cells isolated from spleen with or without treatment with 20 µg/ml PMA and 2 µM ionomycin for 5 h were analyzed for c-Jun and -actin (loading control) expression.

At the age of 8 wk, the size of the thymus was reduced in c-jun^T mice relative to c-jun^f/f controls (Fig. 2A), but gross thymic architecture and histology was normal (Fig. 2B). Consistent with the reduced thymus size, the absolute number of c-jun^T total thymocytes was reduced 4- to 5-fold to 20–25% of that of control mice because of a decrease in all thymocyte subsets with the exception of the DN subpopulation (Fig. 2C). However, the relative proportions of T cell subsets were not drastically skewed except for an increase in DN cells (Fig. 2C). Further examination of the DN thymocyte population using CD44 vs CD25 FACS profiles revealed an accumulation of DN2/DN3 cells and an increase of CD25^bright/+ cells in c-jun^T mice, suggesting a problem at DN3 to DN4 transition of T cell development (Fig. 2D).

View larger version (71K): [in this window] [in a new window]   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-junT littermates illustrating reduced size of c-junT thymus. B, H&E staining of c-junf/f and c-junT thymus sections. C, c-junT deletion causes a partial block at DN stage of T cell development. Total thymocytes from c-junf/f and c-junT mice were analyzed for surface expression of CD4 and CD8 on Thy1.2+ 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-junT mice. Bars, Mean ± SD from eight mice each genotype. D, Accumulation of DN3 thymocytes in c-junT mice. A representative plot of CD44 vs CD25 on lineage–Thy1.2+ gated DN thymocytes from littermate c-junf/f and c-junT mice (upper plot). Values are the mean percentages from eight mice. Histogram overlay of CD25 expression on lineage–Thy1.2+ gated DN thymocytes comparing c-junf/f (shaded histogram) and c-junT (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 ± SD from eight mice each genotype. *, p < 0.05; **, p < 0.01; Student’s unpaired t test.

T

T

T

T

View larger version (58K): [in this window] [in a new window]   FIGURE 3. Increased numbers of thymic and splenic T cells in c-junT mice. A, Increased numbers of thymic T cells in c-junT mice. Representative 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. Thy1.2+ splenocytes represented by TCR vs TCR surface expression and absolute cell numbers from eight mice. C, Increase in TCR+CD8– and TCR+CD8+ cells in c-jun mutant mice. A representative plot of CD8 vs TCR on Thy1.2+ splenocytes and absolute cell numbers from eight mice. **, p < 0.01; Student’s unpaired t test.

T

View larger version (38K): [in this window] [in a new window]   FIGURE 4. Unaffected cell cycle progression but different cell death behavior in c-junT mice. A, Normal cell cycle profile in DN3, DN4, and TCR c-junT cells. Representative histograms of DNA content (left) of lineage–Thy1.2+ gated DN3, DN4 cells and Thy1.2+ gated TCR of total thymocytes from c-junf/f or c-junT 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-junT DN3/DN4 cells. Lineage negative (CD4, CD8, CD44, B220, PanNK, Mac1, Gr1, TCR), Thy1.2+ 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 V+ cells in DN3 and DN4 subpopulations. Mean ± SD from four different mice of each genotype.

View larger version (47K): [in this window] [in a new window]   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 Thy1.2+ 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 ± SD from six mice of each genotype. B, Representative plot of CD25 vs CD44 on lineage–Thy1.2+ DN thymocytes. Histogram overlay of CD25 expression on lineage–Thy1.2+ 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 ± SD from six mice each genotype. C, Plot analysis of TCR vs TCR surface expression on Thy1.2+ gated thymocytes from wild-type and lck-cre transgenic and absolute cell number of T cells. Bars, Mean ± SD from six mice of each genotype. *, p < 0.05; Student’s unpaired t test.

Deregulation of and T cell precursors in DN4 thymocytes

The increase in lineage T cells in c-jun conditional knockout mice could be due to the partial block in development of lineage cells, allowing for expansion of T cells in the thymus. To address this problem, we generated mixed BM chimeric mice by coinjecting BM cells from c-jun^T and wild-type control mice into lethally irradiated rag2^–/– hosts. The analysis of reconstituted mice revealed that c-jun^T cells were slightly less efficient in generating T cells, but they showed markedly increased T cell development (Table I), suggesting that the effect on T cells by c-jun deletion is cell autonomous.

View this table: [in this window] [in a new window]   Table I. Absence of c-jun increases generation of T cells in competitive reconstitution experimentsa

T

T

T

T

View larger version (24K): [in this window] [in a new window]   FIGURE 6. Abnormal intracellular expression of TCR and TCR in c-junT DN4 thymocytes. A, Normal intracellular CD3 expression in DN3 and DN4 c-junT thymocytes. Histogram representation of intracellular staining for CD3 on DN3 and DN4 c-junf/f and c-junT thymocytes (filled histogram) and isotype control (dotted line). B, Reduced percentage of c-junT mice DN4 cells expressing intracellular TCR. Histogram representation of intracellular staining for TCR in DN3 and DN4 c-junf/f, lck-cre, and c-junT thymocytes. The values represent the percentage of icTCR+, mean ± SD of four mice of each genotype. C, Increased percentage of c-junT mice DN4 cells expressing intracellular TCR. Intracellular TCR expression on DN3 and DN4 cells is shown on Thy1.2+ c-junf/f, lck-cre, and c-junT thymocytes, the value represents the percentage of icTCR cells. In B and C, the values are mean ± SD.

c-Jun N-terminal phosphorylation is dispensable for / T cell lineage decision

c-Jun, like other transcription factors, can both activate and repress gene expression (46). Phosphorylation of serines 63 and 73 within the c-Jun transactivation domain by the JNKs greatly augments the activity of c-Jun (25). jun^AA mice, in which serines 63 and 73 in the c-Jun N terminus were replaced by alanines using a knock-in approach, were previously shown to have normal T cell development (38, 39). To determine whether target gene activation by c-Jun mediated through c-Jun N-terminal phosphorylation plays a role in T cell lineage decision, we investigated T cell development in jun^AA mice. jun^AA homozygous thymocytes had normal levels of TCR surface expression and the absolute number of T cells from thymi of jun^AA mice was unaltered (Fig. 7A). Similarly, the number of splenic T cells, of both the CD8⁺ and CD8^– subset, was comparable between jun^AA and control mice (Fig. 7, B and C). Therefore, stimulation of c-Jun function by N-terminal phosphorylation appears to be dispensable for / T cell lineage decision, suggesting that T cell development may be controlled by c-Jun-mediated target gene repression, rather than target gene activation.

View larger version (49K): [in this window] [in a new window]   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 Thy1.2+ gated thymocytes and absolute cell numbers from four mice of each genotype. B, Normal levels of T cells in junAA spleen. Thy1.2+ splenocytes represented by TCR vs TCR surface expression and absolute cell numbers from four mice. C, Equal levels of TCR:CD8– and TCR:CD8+ cells in junAA mice. CD8 vs TCR plot on Thy1.2+ splenocytes and absolute cell number from four mice.

Deregulated expression of IL-7R in c-jun ^T pro-T cells

To gain insight into how c-jun controls T cell development, we tested the transcriptional regulation of molecules known to be involved in / lineage decision. We first investigated the expression of components of the TCR complex, which are key regulators of early T cell development (9, 10, 11, 12, 13, 14). However, the expression levels of pT, TCR, and of the CD3 components of the pre-TCR, CD3, CD3, CD3, and CD3 were not affected in the absence of c-jun. There was also no alteration in the mRNA levels of RAG1 and RAG2, which are required for TCR rearrangement (data not shown).

In addition to the TCR, several other signaling pathways are known to regulate / T cell development, including Notch and IL-7 signaling (18, 19, 47, 48). Although the expression of the Notch1 and its transcriptional partner RBP-j was unaffected, mRNA levels of IL-7R were increased in c-jun^T DN3 cells, as detected by both conventional and quantitative PCR (Fig. 8A). As a consequence, IL-7R protein surface levels were also higher in c-jun^T DN3 and DN4 cells as well as in c-jun^T T cells compared with c-jun^f/f and lck-cre transgenic controls (Fig. 8, B and C).

View larger version (61K): [in this window] [in a new window]   FIGURE 8. Increased expression of IL-7R in c-junT thymocytes. A, Abnormal IL-7R expression in c-junT 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-junT 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-junT (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 Thy1.2+ 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.

Increased T cell development of c-jun-deficient thymocytes in vitro

To directly investigate the relevance of IL-7R regulation by c-jun, the development of thymocytes isolated from control and c-jun^T embryos was studied in FTOCs. Abnormal / T cell development of c-jun^T mice was reproduced in FTOC because both DP and CD4⁺ and CD8⁺ SP T cells were reduced in number, and there was a substantial increase in T cells in c-jun^T cultures (Fig. 9). IL-7 treatment of control FTOC induced T cell development, which, however, did not reach the numbers observed in untreated c-jun-deficient cultures. The number of T cells in c-jun^T cultures was further augmented by IL-7, at the expense of DP T cells whose number was drastically reduced (Fig. 9). IL-7 treatment had only a marginal effect on control T cells (Fig. 9). Therefore, deregulation of IL-7 signaling may contribute to the defect in T cell development in the absence of c-jun.

View larger version (42K): [in this window] [in a new window]   FIGURE 9. Increased development of c-junT T cells in FTOC. A, FACS analysis of FTOC of E15.5 thymus (Thym) from c-junT 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 DAPI–Thy1.2+ gated cells, the dot plot represents CD4 vs CD8 on TCR– cells from the histogram (arrow). B, Absolute cell numbers of one representative FTOC experiment. The values are the mean ± SD.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

c-Jun is an important target of the JNK signaling pathway, but c-Jun N-terminal phosphorylation appears to be dispensable for c-Jun function in thymocytes. T cell development proceeds normally in the absence of both jnk1 and jnk2, which are likely to encode all JNK isoenzymes present in thymus (29). Similarly, there were no detectable abnormalities in / lineage commitment or selection in homozygous jun^AA mice (Fig. 7 and data not shown). As c-Jun N-terminal phosphorylation is believed to be a key modification required for target gene activation, these data are in agreement with the notion that c-Jun regulates the / lineage decision by repressing, rather than activating, essential target genes.

The block of T cell development in c-jun^T mice is incomplete, because CD4^– and CD8⁺ peripheral T cells are present, although reduced in number. It is possible that the T cells developing in c-jun^T mice are derived from thymocytes that have escaped lck-cre-mediated deletion of c-jun^f. If this was the case, inactivation of c-jun at even earlier stages of T cell development should prevent the generation of any mature T cell. However, RAG2 complementation experiments showed that c-jun^–/– ES cells can give rise to T cells, although with reduced efficiency, suggesting that there is no absolute requirement for c-jun in T cell lymphopoiesis, while T cells were not analyzed (34).

Although the details of the underlying molecular mechanism are only incompletely understood, it is clear that several signaling pathways can influence / lineage decision (53). The IL-7R is required for T cell development (18), and there is evidence that increased IL-7R signaling correlates with T cell generation (20, 21). Expression of IL-7R is down-regulated during progression from DN2 to DN3 (21, 54) and microarray analysis of various stages of thymocyte development has identified c-jun as being specifically up-regulated at the DN3 stage (55). This inverse correlation suggests that c-Jun may be involved in the repression of IL-7R expression at the DN2/DN3 transition (Fig. 8).

The relative strength of TCR signaling has been proposed to be a determinant of the / lineage decision. According to this model, T cell development would be favored by strong TCR signaling, and T cell development would be resulting from weaker TCR signals (7, 8).

IL-7R expression is heterogeneous in DN1 thymocytes, and at this stage high level of IL-7R expression does not indicate increased T cell developmental potential (56, 57). However, at the DN2 stage increased IL-7R expression correlates with increased generation of T cells (21), indicating that IL-7R signal strength may favor linage choice, possibly by promoting rearrangement and/or expression of the TCR locus (58, 59, 60, 61). The relationship between TCR and IL-7 signaling is controversial (53), and it is unclear whether TCR and IL-7R act independently or whether IL-7R might, using an unknown mechanism, positively modulate or mimic some aspects of TCR signaling, thereby increasing the strength of the T cell-inducing signal.

This study has identified c-Jun as a novel regulator of T cell lineage decision and development. The molecular mechanisms governing / lineage decision are not well understood, and it is likely that several pathways contribute to the molecular control of this biological process. Therefore, by regulating IL-7R expression (and probably other as yet unidentified target genes), c-Jun may be part of a signaling network that regulates thymic T cell development.

	Acknowledgments

 
We thank A. Jandke, N. Kanu, K. Lightfood, K. Nagakawa, and H. Hinton for technical help and advice; Y. Westermarck for c-Jun small hairpin RNA plasmids; and Y. Carrasco, D. Pennington, and C. Reis Sousa for critical reading of the manuscript.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ The London Research Institute is funded by Cancer Research U.K. L.R.-S. acknowledges support from an European Union Marie-Curie fellowship.

² Current address: Epithelial Homeostasis and Cancer, Department of Cell Differentiation and Cancer, Center of Genomic Regulation, Doctor Aiguader 88, 08003 Barcelona, Spain.

³ Address correspondence and reprint requests to Dr. Axel Behrens, Cancer Research 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 address: axel.behrens{at}cancer.org.uk

⁴ Abbreviations used in this paper: DN, double negative; BM, bone marrow; DAPI, 4',6'-diamidino-2-phenylindole; ChIP, chromatin immunoprecipitation; FTOC, fetal thymus organ culture.

Received for publication March 9, 2006. Accepted for publication February 22, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Warren, L. A., E. V. Rothenberg. 2003. Regulatory coding of lymphoid lineage choice by hematopoietic transcription factors. Curr. Opin. Immunol. 15: 166-175. [Medline]
2. Rodewald, H. R., H. J. Fehling. 1998. Molecular and cellular events in early thymocyte development. Adv. Immunol. 69: 1-112. [Medline]
3. Berg, L. J., J. Kang. 2001. Molecular determinants of TCR expression and selection. Curr. Opin. Immunol. 13: 232-241. [Medline]
4. Sim, G. K., C. Olsson, A. Augustin. 1995. Commitment and maintenance of the and T cell lineages. J. Immunol. 154: 5821-5831. [Abstract]
5. Schweighoffer, E., B. J. Fowlkes. 1996. Positive selection is not required for thymic maturation of transgenic T cells. J. Exp. Med. 183: 2033-2041. [Abstract/Free Full Text]
6. Terrence, K., C. P. Pavlovich, E. O. Matechak, B. J. Fowlkes. 2000. Premature expression of T cell receptor (TCR) suppresses TCR gene rearrangement but permits development of lineage T cells. J. Exp. Med. 192: 537-548. [Abstract/Free Full Text]
7. Haks, M. C., J. M. Lefebvre, J. P. Lauritsen, M. Carleton, M. Rhodes, T. Miyazaki, D. J. Kappes, D. L. Wiest. 2005. Attenuation of TCR signaling efficiently diverts thymocytes to the lineage. Immunity 22: 595-606. [Medline]
8. Hayes, S. M., L. Li, P. E. Love. 2005. TCR signal strength influences / lineage fate. Immunity 22: 583-593. [Medline]
9. Fehling, H. J., A. Krotkova, C. Saint-Ruf, H. von Boehmer. 1995. Crucial role of the pre-T-cell receptor gene in development of but not T cells. Nature 375: 795-798. [Medline]
10. Malissen, M., A. Gillet, L. Ardouin, G. Bouvier, J. Trucy, P. Ferrier, E. Vivier, B. Malissen. 1995. Altered T cell development in mice with a targeted mutation of the CD3- gene. EMBO J. 14: 4641-4653. [Medline]
11. Shinkai, Y., G. Rathbun, K. P. Lam, E. M. Oltz, V. Stewart, M. Mendelsohn, J. Charron, M. Datta, F. Young, A. M. Stall, et al 1992. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68: 855-867. [Medline]
12. Mombaerts, P., A. R. Clarke, M. A. Rudnicki, J. Iacomini, S. Itohara, J. J. Lafaille, L. Wang, Y. Ichikawa, R. Jaenisch, M. L. Hooper, et al 1992. Mutations in T-cell antigen receptor genes and block thymocyte development at different stages. Nature 360: 225-231. [Medline]
13. Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa, V. E. Papaioannou. 1992. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68: 869-877. [Medline]
14. Haks, M. C., P. Krimpenfort, J. Borst, A. M. Kruisbeek. 1998. The CD3 chain is essential for development of both the TCR and TCR lineages. EMBO J. 17: 1871-1882. [Medline]
15. Dave, V. P., Z. Cao, C. Browne, B. Alarcon, G. Fernandez-Miguel, J. Lafaille, A. de la Hera, S. Tonegawa, D. J. Kappes. 1997. CD3 deficiency arrests development of the but not the T cell lineage. EMBO J. 16: 1360-1370. [Medline]
16. Peschon, J. J., P. J. Morrissey, K. H. Grabstein, F. J. Ramsdell, E. Maraskovsky, B. C. Gliniak, L. S. Park, S. F. Ziegler, D. E. Williams, C. B. Ware, et al 1994. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180: 1955-1960. [Abstract/Free Full Text]
17. von Freeden-Jeffry, U., P. Vieira, L. A. Lucian, T. McNeil, S. E. Burdach, R. Murray. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181: 1519-1526. [Abstract/Free Full Text]
18. Maki, K., S. Sunaga, Y. Komagata, Y. Kodaira, A. Mabuchi, H. Karasuyama, K. Yokomuro, J. I. Miyazaki, K. Ikuta. 1996. Interleukin 7 receptor-deficient mice lack T cells. Proc. Natl. Acad. Sci. USA 93: 7172-7177. [Abstract/Free Full Text]
19. Moore, T. A., U. von Freeden-Jeffry, R. Murray, A. Zlotnik. 1996. Inhibition of T cell development and early thymocyte maturation in IL-7^–/– mice. J. Immunol. 157: 2366-2373. [Abstract]
20. Watanabe, Y., T. Sudo, N. Minato, A. Ohnishi, Y. Katsura. 1991. Interleukin 7 preferentially supports the growth of T cell receptor-bearing T cells from fetal thymocytes in vitro. Int. Immunol. 3: 1067-1075. [Abstract/Free Full Text]
21. Kang, J., A. Volkmann, D. H. Raulet. 2001. Evidence that versus T cell fate determination is initiated independently of T cell receptor signaling. J. Exp. Med. 193: 689-698. [Abstract/Free Full Text]
22. Jochum, W., E. Passegue, E. F. Wagner. 2001. AP-1 in mouse development and tumorigenesis. Oncogene 20: 2401-2412. [Medline]
23. Angel, P., M. Karin. 1991. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochem. Biophys. Acta 1072: 129-157. [Medline]
24. Mechta-Grigoriou, F., D. Gerald, M. Yaniv. 2001. The mammalian Jun proteins: redundancy and specificity. Oncogene 20: 2378-2389. [Medline]
25. Davis, R. J.. 2000. Signal transduction by the JNK group of MAP kinases. Cell 103: 239-252. [Medline]
26. Angel, P., K. Hattori, T. Smeal, M. Karin. 1988. The jun proto-oncogene is positively autoregulated by its product, Jun/AP1. Cell 55: 875-885. [Medline]
27. Sabapathy, K., Y. Hu, T. Kallunki, M. Schreiber, J. P. David, W. Jochum, E. F. Wagner, M. Karin. 1999. JNK2 is required for efficient T-cell activation and apoptosis but not for normal lymphocyte development. Curr. Biol. 9: 116-125. [Medline]
28. Sabapathy, K., T. Kallunki, J. P. David, I. Graef, M. Karin, E. F. Wagner. 2001. c-Jun NH₂-terminal kinase (JNK)1 and JNK2 have similar and stage-dependent roles in regulating T cell apoptosis and proliferation. J. Exp. Med. 193: 317-328. [Abstract/Free Full Text]
29. Dong, C., D. D. Yang, C. Tournier, A. J. Whitmarsh, J. Xu, R. J. Davis, R. A. Flavell. 2000. JNK is required for effector T-cell function but not for T-cell activation. Nature 405: 91-94. [Medline]
30. Dong, C., D. D. Yang, M. Wysk, A. J. Whitmarsh, R. J. Davis, R. A. Flavell. 1998. Defective T cell differentiation in the absence of Jnk1. Science 282: 2092-2095. [Abstract/Free Full Text]
31. Yang, D. D., D. Conze, A. J. Whitmarsh, T. Barrett, R. J. Davis, M. Rincon, R. A. Flavell. 1998. Differentiation of CD4⁺ T cells to Th1 cells requires MAP kinase JNK2. Immunity 9: 575-585. [Medline]
32. Hilberg, F., A. Aguzzi, N. Howells, E. F. Wagner. 1993. c-jun is essential for normal mouse development and hepatogenesis. Nature 365: 179-181. [Medline]
33. Johnson, R. S., B. van Lingen, V. E. Papaioannou, B. M. Spiegelmann. 1993. A null mutation at the c-jun locus causes embryonic lethality and retarded cell growth in culture. Genes Dev. 7: 1309-1317. [Abstract/Free Full Text]
34. Chen, J., V. Stewart, G. Spyrou, F. Hilberg, E. F. Wagner, F. W. Alt. 1994. Generation of normal T and B lymphocytes by c-jun deficient embryonic stem cells. Immunity 1: 65-72. [Medline]
35. Williams, K. L., A. J. Zullo, M. H. Kaplan, R. R. Brutkiewicz, C. D. Deppmann, C. Vinson, E. J. Taparowsky. 2003. BATF transgenic mice reveal a role for activator protein-1 in NKT cell development. J. Immunol. 170: 2417-2426. [Abstract/Free Full Text]
36. Thornton, T. M., A. J. Zullo, K. L. Williams, E. J. Taparowsky. 2006. Direct manipulation of activator protein-1 controls thymocyte proliferation in vitro. Eur. J. Immunol. 36: 160-169.