The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsukumo, S.-i.
Right arrow Articles by Yasutomo, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsukumo, S.-i.
Right arrow Articles by Yasutomo, K.
The Journal of Immunology, 2006, 177: 8365-8371.
Copyright © 2006 by The American Association of Immunologists, Inc.

Lunatic Fringe Controls T Cell Differentiation through Modulating Notch Signaling1

Shin-ichi Tsukumo, Kayo Hirose, Yoichi Maekawa, Kenji Kishihara and Koji Yasutomo2

Department of Immunology and Parasitology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cells differentiate from bone marrow-derived stem cells by expressing developmental stage-specific genes. We here searched arrays of genes that are highly expressed in mature CD4–CD8+ (CD8 single-positive (SP)) T cells but little in CD4+CD8+ (double-positive (DP)) cells by cDNA subtraction. Lunatic fringe (Lfng), a modulator of Notch signaling, was identified to be little expressed in DP cells and highly expressed in CD8SP T cell as well as in CD4–CD8– (double-negative (DN)) and mature CD4+CD8– (CD4SP) T cells. Thus, we examined whether such change of expression of Lfng plays a role in T cell development. We found that overexpression of Lfng in Jurkat T cells strengthened Notch signaling by reporter gene assay, indicating that Lfng is a positive regulator for Notch signaling in T cells. The enforced expression of Lfng in thymocytes enhanced the development of immature CD8SP cells but decreased mature CD4SP and CD8SP cells. In contrast, the down-regulation of Lfng in thymocytes suppressed DP cells development due to the defective transition from CD44+CD25– stage to subsequent stage in DN cells. The overexpression of Lfng in fetal liver-derived hemopoietic stem cells enhanced T cell development, whereas its down-regulation suppressed it. These results suggested that the physiological high expression of Lfng in DN cells contributes to enhance T cell differentiation through strengthening Notch signaling. Shutting down the expression of Lfng in DP cells may have a physiological role in promoting DP cells differentiation toward mature SP cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Hemopoietic stem cells (HSC)3 differentiate to erythroid, myeloid, or lymphoid progenitor cells in bone marrow (1, 2). The lymphoid progenitors further differentiate to T cells in the thymus and B cells in bone marrow (1, 2). T cells in the thymus can be divided into four populations by their surface expression of CD4 and CD8 (3). The cells that lack expression of both CD4 and CD8 (double-negative (DN) cells) are the most immature population in thymocytes and they are further divided into four subpopulations by CD25 and CD44 expression (3). CD44+CD25– (DN1) cells are the most immature population in DN cells and they contain multipotent lymphoid progenitor cells. Subsequent CD44+CD25+ (DN2) cells still have the potential to differentiate to NK or dendritic cells (4, 5). The CD44–CD25+ (DN3) cells undergo beta-selection and CD44–CD25– (DN4) cells, after successful TCRbeta gene rearrangement, differentiate to CD4+CD8+ (double-positive (DP) cells) cells through transient TCRbetalowD4–CD8+ (immature CD8 single-positive (CD8SP)) cells (3). DP cells further differentiate to mature (TCRbetahigh) CD4+CD8– (CD4SP) or CD4–CD8+ (CD8SP) T cells (6). Although the interplays of intracellular and extracellular molecules have been reported to regulate each differentiation step of thymocytes (1, 2, 7, 8), key players, as well as regulatory mechanisms that control each differentiation, have not been fully understood.

Notch is a single-pass transmembrane receptor protein that regulates a broad range of cell fate decisions, such as embryogenesis, neurogenesis, and lymphopoiesis (9, 10, 11, 12). In mammals, four Notch genes (Notch1–4) and five ligands (Jagged 1 and 2; Delta-like ligands 1, 3, and 4) have been identified (13). When Notch receptors are stimulated by these ligands, the intracellular domain of Notch (Notch-IC) is cleaved by {gamma}-secretase and Notch-IC translocates to the nucleus (14). Notch-IC binds to RBP-J{kappa} in the nucleus and converts it from a transcriptional repressor to an activator together with mastermind-like family proteins (14).

Previous studies demonstrated that Notch signaling is involved in the differentiation from hemopoietic stem cells to mature T cells (10). During early T cell development, the gene disruption of Notch1 and RBP-J{kappa} leads to complete blockage of T cell development at early stages and ectopic differentiation of B cells in the thymus (15, 16). Notch signaling is also required after T cell lineage commitment, as reported that the disruption of Notch1 or RBP-J{kappa} at the DN2/DN3 boundary resulted in an arrest of the differentiation at DN3 (17, 18). We and others demonstrated that Notch signaling is also involved in the acquisition of the effector function of mature CD4SP T cells (19, 20, 21).

Notch signaling is modulated by several proteins (22). One of those modulators, fringe has glycosyltransferase activity, adding N-acetylglucosamine to O-fucose on the extracellular domain of Notch (23). In fruit flies, the glycosylation by fringe enhances Delta-stimulated Notch signaling, while it inhibits the serrate -stimulated one (24). In vertebrates, three fringe family genes have been identified and designated as lunatic, manic, and radical fringe (25). The effects of these mammalian fringes on Notch have been controversial, because each previous report claimed different results using different cell types and/or Notch receptors (26, 27, 28, 29).

We here examined the genes that specifically change their expression during differentiation from DP cells to mature CD8SP cells, and found that lunatic fringe (Lfng) was highly expressed in DN, CD4SP, and CD8SP T cells, but was little expressed in DP cells. Thus, we investigated whether Lfng was critical for on/off regulation of Notch signaling during T cell development. We revealed that the enforced expression of Lfng in thymocytes leads to the increased differentiation of immature CD8SP cells, whereas it suppressed the development of mature CD4SP and CD8SP T cells. In contrast, the down-regulation of Lfng in DN cells inhibited DP cell development. Furthermore, overexpression of Lfng in fetal liver cells-derived HSCs (FL-HSC) enhanced T cell development, while its down-regulation suppressed them. These results indicated that Lfng promotes T cell development from HSC/DN cells to DP cells. Furthermore, little expression of Lfng in DP cells may have a physiological role in enhancing the production of mature T cells by shutting down Notch signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female nonpregnant (7-wk-old) and pregnant (gestational day 15) C57BL/6 mice were purchased from Japan SLC. The mice were maintained in a specific pathogen-free animal facility and treated in accordance with the institutional guidelines for animal care of the University of Tokushima.

Cell preparation for total RNA

For the subtractive cloning, CD4 and CD8 SP cells were isolated from splenocytes of C57BL/6 adult mice. Single-cell suspensions were stained by anti-CD4 or anti-CD8 MACS beads (Miltenyi Biotec). Cells were washed with MACS buffer and positively selected by MACS columns. DN cells were isolated from fetal thymocytes (gestational day 15), which did not contain CD4/CD8 SP or DP cells. DP thymocytes were purified from adult mice by panning on peanut agglutinin-coated plates (Sigma-Aldrich) (20 µg/ml) in PBS containing 5% FCS for 75 min at 4°C. After three washes, bound cells were eluted with 0.2 M D-galactose in PBS/FCS for 10 min at room temperature, and washed twice in medium. For RT-PCR experiments, single-cell suspensions of C57BL/6 thymocytes (7-wk old) were stained by CD4-PE and CD8-FITC. Then, 2 x 106 cells of subpopulations were sorted by the JSAN cell sorter (Bay Bioscience).

RNA extraction and RT-PCR

Total RNA was extracted from cell suspensions by using TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer’s instruction. Reverse transcription from 1 µg of total RNA was conducted with the Omniscript RT kit (Qiagen) and hexanucleotide mix (Roche Molecular Biochemicals). Two microliters of the diluted or undiluted first strand cDNA was used in 20-µl PCRs containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.1 µM of each primer, and 2 U of rTaq DNA polymerase (Toyobo). The PCR consisted of 95°C for 5 min, followed by 24 or 30 cycles of 94°C for 1 min, 58°C for 1.5 min, and 72°C for 1 min, with a final extension for 10 min at 72°C. The primers for PCR were listed in Table I.


View this table:
[in this window]
[in a new window]

  		Table I. Summary of primers used for PCR

 
Representational difference analysis (RDA)

Total RNA was prepared from DP thymocytes and CD8SP splenocytes of adult mice. RDA was preformed essentially as described previously (30). The second-round product was cloned into the pUC19 vector. The samples were sequenced and compared with GenBank database by using the basic local alignment search tool (BLAST) program.

Plasmid construction

The full-length Lfng cDNA was amplified by PCR from splenocytes of mice and cloned into pKE004, which is a bicistronic retrovirus vector encoding internal ribosomal entry site-GFP as a marker (provided from Dr. R. Germain, National Institute of Allergy and Infectious Diseases, Bethesda, MD) (19). The partial cDNA of Lfng, including the target sequence for small hairpin RNA (shRNA), was amplified by PCR and cloned into the 3'-UTR region of pFB-DsRed2. The retrovirus vector for shRNA was constructed by replacing the H1 promoter and hCD4 of pRVH1 (provided by Dr. R. Medzhitov, Yale University, New Haven, CT) by the U6 promoter and CMV promoter-driven GFP of pLL3.7 (31, 32). The oligonucleotide for Lfng knockdown, 5'-TGGAGATGACGTTCATCTTCTTCAAGAGAGAAGATGAACGTCATCTCCTTTTTT-3', was cloned into the HpaI and XhoI site in pRU6G.

Retrovirus preparation and transduction

The retroviral vector constructs were transfected into a retrovirus-packaging cell line Plat-E (provided by Dr. T. Kitamura, University of Tokyo, Tokyo, Japan) (33). Briefly, 1 x 106 cells/well were seeded into a 6-well plate in 1.5 ml DMEM supplemented with 10% FCS, 0.05 mM 2-ME, 100 µg/ml penicillin/streptomycin. Then, the subconfluent cells were transfected with 1 µg of plasmid DNA and 5 µl of FuGene 6 (Roche Molecular Biochemicals). The virus-containing sup was recovered 2 days after transfection. For retrovirus transduction, the retroviral supernatants were filtrated through a 0.45-µm filter, and added to cells with 10 µg/ml polybrene (Chemicon International). The retroviral infections were facilitated by centrifugation at 2600 rpm for 1 h.

Fetal thymus organ cultures

Fetal thymic lobes were obtained from fetal day 15 C57BL/6 mice embryos and placed on Nucleopore Track-Etch membranes (Whatman) in 24-well culture plates. The lobes were cultured in RPMI 1640 medium supplemented with 10% FCS, 100 µg/ml penicillin, 100 µg/ml streptomycin, 0.05 mM 2-ME, and 1.35 mM 2-deoxyguanosine (dGuo) for 7 days. The GFP-positive cells from the virus-transduced fetal thymocytes or FL-HSC were sorted by the JSAN cell sorter (Bay Bioscience) and then the sorted cells were aliquoted at 2000 cells/well in Terasaki plates, and one dGuo-treated lobe per well was added. The cells and lobes were incubated for 24 h as hanging drop cultures and then placed on the membranes in 24-well plates for 9 days.

Flow cytometric analysis

Four-color flow cytometry was performed on a dual-laser FACSCalibur flow cytometer and analyzed by CellQuest software (Nippon/BD Biosciences). Cells were stained with mAbs as follows: PE-conjugated anti-CD4 mAb (GK1.5; Biolegend), allophycocyanin-conjugated anti-Thy1.2 mAb (53-2.1; eBiosciences), CyChrome-conjugated anti-CD8 mAb (53-6.7; eBiosciences), CyChrome-conjugated anti-CD19 mAb (MB19-1; eBiosciences), and allophycocyanin-conjugated anti-TCRbeta mAb (H57-597; Biolegend).

Luciferase assay

Jurkat cells were transiently transfected with the RBP-J{kappa}-responsible luciferase reporter gene with pRL-CMV Renilla luciferase vector (Promega) by DMRIE-C reagent (Invitrogen Life Technologies), according to the manufacturer’s instruction. The cells were transferred to Delta1-Fc- or Jagged1-Fc- (19) coated plates 24 h after the transfection. Twenty-four hours after starting the stimulation, the luciferase activities were determined by the dual-luciferase assay system (Promega).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PCR-based cDNA subtraction between DP thymocytes and CD8SP peripheral T cells

To identify genes critical for T cell differentiation, we screened genes differentially expressed in DP cells and mature CD8SP T cells by PCR-based cDNA subtraction. We succeeded in identifying nine genes that are up-regulated during the maturation from DP cells to CD8SP cells (Table II). They included genes that had been already reported to be up-regulated during T cell maturation, such as class I MHC, RANTES, CCR7, and IAN4 (34, 35, 36, 37). In addition, we newly identified IGFBP4, Edg1, Arl7, Amigo2, and Lfng as candidate genes differentially expressed between the two T cell subsets. Then, we confirmed whether these genes were differentially expressed in DP and CD8SP cells by RT-PCR. The results revealed that all of the genes were highly expressed in CD8SP and CD4SP T cells and little in DP cells (Figs. 1 and 2).


View this table:
[in this window]
[in a new window]

  		Table II. Genes identified by RDA

 

Figure 1
View larger version (63K):
[in this window]
[in a new window]

  		FIGURE 1. The expression of the genes identified by the subtraction in T cells. The expressions of each gene were assessed by RT-PCR. Four serial dilutions (2-fold) of template cDNA were used. The samples without reverse transcription were indicated by –. G3PDH was used as standard.

 

Figure 2
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 2. The expression pattern of the Lfng gene in T cells and fetal liver cells. RT-PCR was performed to detect the Lfng transcript in the indicated cells. Four serial dilutions (2-fold) of template cDNA were used. The samples without reverse transcription were indicated by –. G3PDH was used as standard.

 
We further examined the expression pattern of Lfng in other T cell subsets, because Lfng was reported to be a modulator of Notch signaling, which has critical roles in the various steps during T cell differentiation (10, 11, 12, 14, 38, 39). We found that the mRNA of Lfng was highly expressed not only in mature SP cells but also in DN cells and fetal liver cells (Fig. 2), in which the Notch activation was critical for T cell differentiation (15, 17, 18, 40, 41, 42). These findings allowed us to investigate whether Lfng had any roles in the regulation of Notch signaling that controls T cell development.

Overexpression of Lfng in T cells enhances Delta1-stimulated Notch signaling

The experiments using fruit flies have indicated that the modification of Notch by fringe enhances the Delta-stimulated signal but suppresses the serrate (Jagged)-stimulated signal (23, 24, 43), although the effects of mammalian fringes are controversial (27, 28, 29, 44, 45). To examine the effect of Lfng on Notch signals in T cells, Jurkat T cells transduced with Lfng cDNA and a Notch signal reporter gene were stimulated by Delta1-Fc or Jagged1-Fc. The increase of luciferase activity by Lfng was observed when Jurkat T cells were stimulated with 2 and 10 µg/ml Delta1-Fc, but not with Jagged1-Fc (Fig. 3). The results indicated that the glycosylation of Notch by Lfng positively regulated the Delta1-Notch signal in T cells.


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 3. Lfng enhances Delta-stimulated Notch signaling. Jurkat T cells were transduced with retrovirus vector encoding Lfng ({blacksquare}) and control vector ({square}). The transduced cells were further transfected with RBP-J{kappa} luciferase reporter plasmid. Then the cells were stimulated by plate-bound Delta1-Fc or Jagged1-Fc. After 24 h of stimulation, the luciferase activities were analyzed and normalized to Renilla luciferase activity. The results are shown as mean ± SD from three samples. *, p < 0.05. The results are from one representative experiment of three independent experiments.

 
Overexpression of Lfng in thymocytes enhanced the development of the immature CD8SP cell, but suppresses mature CD4SP and CD8SP T cell development

Loss-of-function experiments indicated that Notch1 and RBP-J{kappa} is required for differentiation from DN to DP cells (17, 18). In addition, Delta-like ligand 1-stimulation of DN cells can promote the differentiation toward DP cells in vitro (41, 46). To examine the role of Lfng in these differentiation steps, Lfng cDNA was transduced into DN cells by a retroviral vector and such cells were cultured in fetal thymus lobes. The results revealed that the relative percentage of CD8SP cells was increased by Lfng overexpression, while that of the other populations was decreased (Fig. 4A). Because CD8SP cells contain immature and mature cells, the expression level of TCRbeta on these cells was examined. We found that the percentage of the TCRbetahigh population in CD8SP cells (i.e., mature CD8SP cells) became low when Lfng was overexpressed (Fig. 4B). The absolute cell numbers of TCRbetahighCD8SP as well as TCRbetahighCD4SP were decreased whereas the number of TCRbetalowCD8SP cells was increased when Lfng was overexpressed (Fig. 4C). The numbers of DN and DP cells were not significantly affected (Fig. 4C). These results indicated that the overexpression of Lfng in thymocytes suppressed the differentiation from DP to mature CD4SP and CD8SP T cells. In contrast, the increased development of immature CD8SP cells in Lfng overexpressed DN cells suggested the enhanced differentiation from DN to DP cells or the selective proliferation of immature CD8SP cells. The same phenotypic changes of T cells were observed in the culture of active Notch1-overexpressed DN cells (47) (S. Tsukumo and K. Yasutomo, unpublished observation), suggesting that the overexpression of Lfng in thymocytes enhanced Delta-Notch activation.


Figure 4
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 4. The overexpression of Lfng affects thymocyte differentiation. To examine whether the expression of Lfng affects thymocyte differentiation, fetal thymocytes (gestational day 15) were transduced with the bicistronic retrovirus vector encoding Lfng cDNA and GFP. Twenty-four hours later after the transduction, GFP-positive cells were sorted and transferred into dGuo-treated fetal thymus lobes, and then further cultured on filters for 9 days. The empty vector was used as a control. A, The result of flow cytometric analysis for CD4 and CD8 expression when Lfng was overexpressed. The small panels indicates GFP expression of each sample. The GFP+ cells were gated and further analyzed. The percentages of each population are indicated. B, The flow cytometric analysis of TCRbeta expression of each population in A are shown. The percentage of TCRbetahigh cells are indicated in each panel. C, Statistical analyses of the cell numbers of the indicated subpopulations are shown. The results are shown as mean ± SD from four samples. *, p < 0.05. The results are from one representative experiment of two independent experiments.

 
Down-regulation of Lfng in thymocytes suppresses T cell differentiation of DN cells toward DP cells

To further elucidate the role of Lfng in thymocyte differentiation, we used shRNA-encoding retroviral vector to inhibit Lfng expression. The efficiency of shRNA was estimated in DO11.10 T cell hybridoma cells (Fig. 5A). Retroviral vector encoding DsRed2 with Lfng in its 3'-UTR region (DsRed2-Lfng) was transduced into DO11.10. A cell expressing DsRed2; Lfng at relatively high levels was cloned and further transduced with the shRNA-expressing retroviral vector. The expression level of DsRed2 was ~3-fold down-regulated in GFP-positive cells (i.e., shRNA-expressing cells) (Fig. 5A).


Figure 5
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 5. The down-regulation of Lfng affects thymocyte differentiation. A, The efficiency of Lfng shRNA. T cell hybridoma DO11.10 cells were transduced with the retrovirus vector encoding DsRed2 with Lfng cDNA fragment in 3'-UTR. High DsRed2-expressing cells were further transduced with the Lfng shRNA expression retrovirus vector encoding GFP as a marker. The panel shows flow cytometric analysis of the cells. Mean fluorescence intensities are indicated in each region. B, The result of flow cytometer analysis for the CD4 and CD8 profile when Lfng was down-regulated in thymocytes. The retrovirus vectors were introduced in thymocytes as described in Fig. 4. The percentages of each population are indicated in the quadrants. The empty vector was used as a control. C, Statistical analyses of the cell numbers of DN and DP cells in B are shown. The results are shown as mean ± SD from four samples. *, p < 0.05. The results are from one representative experiment of three independent experiments. The results are shown as mean ± SD from four samples. *, p < 0.05. The results are from one representative experiment of three independent experiments.

 
The shRNA retroviral vector was transduced into fetal thymocytes and such cells were cultured in fetal thymus lobes. The down-regulation of Lfng suppressed the relative and the absolute cell number of DP cells, whereas DN cell number was not significantly affected (Fig. 5, B and C). The result suggested that Lfng was required for the differentiation from DN cells to DP cells.

Lfng affects the differentiation from FL-HSC to T cells

Notch signaling has been suggested to be involved in the T cell lineage commitment stage (38), because disruptions of Notch1 or RBP-J{kappa} genes result in lack of T cells and enhanced differentiation of B cells in mice (15, 16). Furthermore, Delta-like ligand 1-stimulation of FL-HSC supports T cell differentiation and suppresses B cell differentiation (40). Koch et al. (45) reported that the transgene of Lfng driven by the lck-proximal promoter suppressed T cell differentiation but enhanced B cell differentiation. Because our results did not show the increase of B cells when Lfng was overexpressed in thymocytes, we investigated whether Lfng had a suppressive effect on T cell differentiation at the step of T/B lineage commitment by using FL-HSC.

We examined the roles of Lfng in this Notch-dependent T cell lineage commitment of FL-HSC by introducing Lfng-targeted shRNA or cDNA. The FL-HSC transduced with the shRNA/cDNA for Lfng was cultured in fetal thymus lobes. The overexpression of Lfng in FL-HSC increased the percentage and the absolute number of T cells but did not increase B cell development (Fig. 6, A and B). The down-regulation of Lfng in FL-HSC decreased the relative and the absolute number of T cells (Fig. 6, C and D). These results indicated that Lfng enhanced T cell differentiation through promoting Delta-Notch signaling in the early stage of T cell lineage progression but did not affect B cell development in our system.


Figure 6
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 6. The expression of Lfng affects the differentiation of fetal liver cells toward T cells. Fetal liver cells were transduced with the retrovirus vector encoding Lfng cDNA or shRNA with GFP. The GFP-positive cells were sorted and transferred into dGuo-treated fetal thymus lobes, and then cultured on filters for 9 days. The empty vector was used as a control. A and C, The results of flow cytometric analysis for Thy1.2 and CD19 profiles of the Lfng-overexpressed cells (A) or Lfng-down-regulated cells (C). The percentages of each population are indicated in the quadrants. B and D, Statistical analyses of the percentages and the cell numbers of the Thy1.2+ cells when Lfng was overexpressed (B) or down-regulated (D). The results are shown as mean ± SD from four samples. *, p < 0.05. The results are from one representative experiment of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
To elucidate the molecular mechanisms of T cell differentiation, we identified genes which were highly expressed in mature CD8SP cells but not in DP cells (Table I). Among them, CCR7 and Edg1 have been reported to be involved in T cell migration (48, 49, 50). However, the other genes have not been fully investigated for their roles during T cell differentiation, although some genes were reported to be up-regulated during T cell differentiation (34, 35, 36, 37). In this research, we focused on investigating the role of Lfng because Notch signaling is required for several steps during T cell development (10, 11) and the interaction between Notch and Notch ligands is generally regulated by fringe that glycosylates the extracellular domain of Notch (22). The modification of Notch by fringe controls the growth, patterning, and compartmentalization of the wings in fruit flies (24), although the fringe modulation is not required for all actions of Notch signaling (22). Despite of the importance of Notch signaling in T cell differentiation, it has been unclear whether fringe is physiologically involved in T cell differentiation although Lfng-transgenic mice shows reduced T cell development (45). Thus, we investigated whether Lfng regulates T cell development through modulating Notch signaling by overexpressing or down-regulating Lfng expression in immature T cells.

The overexpression of Lfng in fetal thymocytes increased the percentage and the number of TCRbetalowCD8SP cells that are intermediates from DN to DP differentiation. In contrast, the overexpression of Lfng decreased the mature TCRbetahighCD4SP and TCRbeta highCD8SP cells. On the other hand, DP and DN cell numbers were not affected. These phenotypes were quite similar to the ones observed in constitutively active Notch1-overexpressing fetal thymocytes (47) (S. Tsukumo and K. Yasutomo, unpublished observation), which suggested that the effects of Lfng in our studies were due to the enhancement of Notch signaling. This notion was supported by the luciferase assay in Jurkat cells, which indicated that the overexpression of Lfng promoted Delta-stimulated Notch signaling. Thus, the physiological low level of Lfng expression in DP cells is supposed to be important for shutting down Notch signaling. In contrast, the increase of immature CD8 SP cells might be due to the enhancement of the differentiation/proliferation during the stage between DN and DP cells, as reported that Delta-Notch stimulation promoted this stage of differentiation (4, 40).

To evaluate the physiological roles of endogenous Lfng in T cell development, we examined whether the down-regulation of Lfng in thymocytes affected their differentiation. The results indicated that the down-regulation of Lfng decreased the DP cell number and the differentiation was inhibited during the transition during the DN1 to subsequent stage. The inhibitor of {gamma}-secretase, which suppresses Notch activation, arrested the differentiation at the DN1 stage in vitro (51). In addition, conditional Notch1 inactivation showed that the Notch1 deficiency resulted in the arrest of differentiation at the DN1 stage (15), and the differentiation assays in vitro indicated that recurrent Delta stimulation is required for differentiation throughout DN1 to DN3 (4). Therefore, these results suggest that the expression of Lfng in DN cells is necessary for the activation of Notch1 signaling during the differentiation of DN cells.

In addition to DN cell differentiation, Notch signaling is critical for T cell commitment from lymphoid progenitor cells (11). For instance, Notch1 conditional-deficient mice lacked T cells but have enhanced development of B cells in the thymus (15). To evaluate the contribution of Lfng during the T cell commitment stage, we modified the expression of Lfng in FL-HSC. We demonstrated that the overexpression of Lfng in FL-HSC enhanced T cell differentiation, whereas its down-regulation suppressed it. In both cases, B cell development was not increased. We used retrovirus-based shRNA system to down-regulate Lfng and started culture of retrovirus infected FL-HSC soon after infection. Thus, it was possible that the efficacy of down-regulation of Lfng itself was not enough to inhibit Notch signaling that suppressed B cell development. It would be important to use Lfng-deficient mice to address this question. In addition, Guidos and colleagues (45) demonstrated that Lfng-transgenic mice driven by the lck-proximal promoter showed reduced T cell and increased B cell development in the thymus, although our results indicated that overexpression of Lfng in DN cells promoted Notch-mediated T cell development and did not enhance B cell development. Regarding this discrepancy, very high expression of Lfng in their transgenic mice might strongly enhance the interaction between Delta and Notch, which decreases the availability of Delta for neighboring cells (42). In contrast, relatively low expression of Lfng in our retroviral gene transduction system might not be able to induce such strong interaction. As another possibility, because they used Lfng tagged by FLAG C-terminal in their transgenic mouse system (45), the fusion with FLAG might influence the localization or the enzyme specificity of Lfng.

In conclusion, the present results demonstrated that the expression of Lfng regulates T cell differentiation through on/off of Delta-Notch signaling (Fig. 7). Lfng is highly expressed in FL-HSC and DN cells, which enhances Delta-Notch signaling to promote T cell differentiation from those stages. The expression of Lfng is faint in DP cells, so that Notch signaling is not effectively transduced. This inactivation of Notch signaling during DP cells may be required for the optimal production of mature CD4SP and CD8SP cells. The Lfng is highly expressed in mature SP cells, but Notch signaling in SP cells does not seem to be activated in thymus (52). As we reported that Delta-Notch signaling was required for Th1 differentiation of peripheral T cells (19), the high expression of Lfng in mature SP T cells might be important for the acquisition of effector function of mature T cell. The mammals have other two fringes, radical and manic fringes, and those two fringes are also expressed in DN and mature T cells (S. Tsukumo and K. Yasutomo, unpublished observation). It would be important to reveal how those three fringes control T cell development by modulating Notch signaling.


Figure 7
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 7. The current model regarding the roles of Lfng in T cell development. FL-HSC/DN cells, Lfnghigh DN cells and FL-HSC receive strong Notch signaling that promotes T cell development (physiological condition). Lfngvery high cells by transducing Lfng receive stronger Notch signaling, which further enhances T cell development. Lfnglow cells by transducing Lfng-targeted shRNA receive weaker Notch signaling, which inhibits T cell development. DP cells, Lfnglow DP cells do not have sufficient interaction between Notch and Delta-like ligand (physiological condition). Lfnghigh DP cells that receive strong Notch signaling, which antagonizes positive selection signaling. SP cells, Lfng is highly expressed in SP cells, but the role of Lfng in SP cells has not been evaluated.

 
Note added in proof.

During submission of this manuscript, a study investigating the role of lunatic fringe in T cell development, which reached the similar conclusions of this study, was published (53).


   Acknowledgments
 
We thank Dr. S. Chiba for providing fusion proteins, Dr. C. Guidos for communicating unpublished results, Dr. Y. Takahama for technical help about hanging drop culture, Maki Fukuhara and Mariko Suga for assistance with experiments, and Kiyoka Yamakawa for secretarial assistance.


   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 This work was supported by a grant from the Mitsubishi Foundation and Grants-In-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (17047027, 17046013 (to K.Y.) and 18015035, 1806003), and Princess Takamatsu Cancer Research Fund. Back

2 Address correspondence and reprint requests to Dr. Koji Yasutomo, Department of Immunology and Parasitology, Institute of Health Biosciences, University of Tokushima Graduate School, 3-18-15 Kuramoto, Tokushima 770-8503, Japan. E-mail address: yasutomo{at}basic.med.tokushima-u.ac.jp Back

3 Abbreviations used in this paper: HSC, hemopoietic stem cell; DN, double negative; DP, double positive; SP, single positive; Lfng, lunatic fringe; FL-HSC, fetal liver-derived HSC; shRNA, small hairpin RNA; dGuo, 2-deoxyguanosine. Back

Received for publication May 9, 2006. Accepted for publication October 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Zuniga-Pflucker, J. C., T. M. Schmitt. 2005. Unraveling the origin of lymphocyte progenitors. Eur. J. Immunol. 35: 2016-2018. [Medline]
  2. Baba, Y., R. Pelayo, P. W. Kincade. 2004. Relationships between hematopoietic stem cells and lymphocyte progenitors. Trends Immunol. 25: 645-649. [Medline]
  3. Ceredig, R., T. Rolink. 2002. A positive look at double-negative thymocytes. Nat. Rev. Immunol. 2: 888-897. [Medline]
  4. Schmitt, T. M., M. Ciofani, H. T. Petrie, J. C. Zuniga-Pflucker. 2004. Maintenance of T cell specification and differentiation requires recurrent notch receptor-ligand interactions. J. Exp. Med. 200: 469-479. [Abstract/Free Full Text]
  5. Shen, H. Q., M. Lu, T. Ikawa, K. Masuda, K. Ohmura, N. Minato, Y. Katsura, H. Kawamoto. 2003. T/NK bipotent progenitors in the thymus retain the potential to generate dendritic cells. J. Immunol. 171: 3401-3406. [Abstract/Free Full Text]
  6. Laky, K., B. J. Fowlkes. 2005. Receptor signals and nuclear events in CD4 and CD8 T cell lineage commitment. Curr. Opin. Immunol. 17: 116-121. [Medline]
  7. Ladi, E., X. Yin, T. Chtanova, E. A. Robey. 2006. Thymic microenvironments for T cell differentiation and selection. Nat. Immunol. 7: 338-343. [Medline]
  8. Berg, L. J., M. Nussenzweig. 2004. Signals that regulate lymphocyte development and differentiation. Curr. Opin. Immunol. 16: 163-166. [Medline]
  9. Yoon, K., N. Gaiano. 2005. Notch signaling in the mammalian central nervous system: insights from mouse mutants. Nat. Neurosci. 8: 709-715. [Medline]
  10. Radtke, F., A. Wilson, S. J. Mancini, H. R. MacDonald. 2004. Notch regulation of lymphocyte development and function. Nat. Immunol. 5: 247-253. [Medline]
  11. Maillard, I., T. Fang, W. S. Pear. 2005. Regulation of lymphoid development, differentiation, and function by the Notch pathway. Annu. Rev. Immunol. 23: 945-974. [Medline]
  12. Pear, W. S., F. Radtke. 2003. Notch signaling in lymphopoiesis. Semin. Immunol. 15: 69-79. [Medline]
  13. Artavanis-Tsakonas, S., M. D. Rand, R. J. Lake. 1999. Notch signaling: cell fate control and signal integration in development. Science 284: 770-776. [Abstract/Free Full Text]
  14. Robey, E. A., J. A. Bluestone. 2004. Notch signaling in lymphocyte development and function. Curr. Opin. Immunol. 16: 360-366. [Medline]
  15. Radtke, F., A. Wilson, G. Stark, M. Bauer, J. van Meerwijk, H. R. MacDonald, M. Aguet. 1999. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10: 547-558. [Medline]
  16. Han, H., K. Tanigaki, N. Yamamoto, K. Kuroda, M. Yoshimoto, T. Nakahata, K. Ikuta, T. Honjo. 2002. Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision. Int. Immunol. 14: 637-645. [Abstract/Free Full Text]
  17. Wolfer, A., A. Wilson, M. Nemir, H. R. MacDonald, F. Radtke. 2002. Inactivation of Notch1 impairs VDJbeta rearrangement and allows pre-TCR-independent survival of early {alpha}beta lineage thymocytes. Immunity 16: 869-879. [Medline]
  18. Tanigaki, K., M. Tsuji, N. Yamamoto, H. Han, J. Tsukada, H. Inoue, M. Kubo, T. Honjo. 2004. Regulation of {alpha}beta/{gamma}{delta} T cell lineage commitment and peripheral T cell responses by Notch/RBP-J signaling. Immunity 20: 611-622. [Medline]
  19. Maekawa, Y., S. Tsukumo, S. Chiba, H. Hirai, Y. Hayashi, H. Okada, K. Kishihara, K. Yasutomo. 2003. Delta1-Notch3 interactions bias the functional differentiation of activated CD4+ T cells. Immunity 19: 549-559. [Medline]
  20. Amsen, D., J. M. Blander, G. R. Lee, K. Tanigaki, T. Honjo, R. A. Flavell. 2004. Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells. Cell 117: 515-526. [Medline]
  21. Minter, L. M., D. M. Turley, P. Das, H. M. Shin, I. Joshi, R. G. Lawlor, O. H. Cho, T. Palaga, S. Gottipati, J. C. Telfer, et al 2005. Inhibitors of {gamma}-secretase block in vivo and in vitro T helper type 1 polarization by preventing Notch upregulation of Tbx21. Nat. Immunol. 6: 680-688. [Medline]
  22. Haines, N., K. D. Irvine. 2003. Glycosylation regulates Notch signalling. Nat. Rev. Mol. Cell Biol. 4: 786-797. [Medline]
  23. Haltiwanger, R. S.. 2002. Regulation of signal transduction pathways in development by glycosylation. Curr. Opin. Struct. Biol. 12: 593-598. [Medline]
  24. Panin, V. M., V. Papayannopoulos, R. Wilson, K. D. Irvine. 1997. Fringe modulates Notch-ligand interactions. Nature 387: 908-912. [Medline]
  25. Johnston, S. H., C. Rauskolb, R. Wilson, B. Prabhakaran, K. D. Irvine, T. F. Vogt. 1997. A family of mammalian Fringe genes implicated in boundary determination and the Notch pathway. Development 124: 2245-2254. [Abstract]
  26. Dale, J. K., M. Maroto, M. L. Dequeant, P. Malapert, M. McGrew, O. Pourquie. 2003. Periodic notch inhibition by lunatic fringe underlies the chick segmentation clock. Nature 421: 275-278. [Medline]
  27. Hicks, C., S. H. Johnston, G. diSibio, A. Collazo, T. F. Vogt, G. Weinmaster. 2000. Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2. Nat. Cell. Biol. 2: 515-520. [Medline]
  28. Shimizu, K., S. Chiba, T. Saito, K. Kumano, T. Takahashi, H. Hirai. 2001. Manic fringe and lunatic fringe modify different sites of the Notch2 extracellular region, resulting in different signaling modulation. J. Biol. Chem. 276: 25753-25758. [Abstract/Free Full Text]
  29. Yang, L. T., J. T. Nichols, C. Yao, J. O. Manilay, E. A. Robey, G. Weinmaster. 2005. Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1. Mol. Biol. Cell 16: 927-942. [Abstract/Free Full Text]
  30. Hubank, M., D. G. Schatz. 1999. cDNA representational difference analysis: a sensitive and flexible method for identification of differentially expressed genes. Methods Enzymol. 303: 325-349. [Medline]
  31. Barton, G. M., R. Medzhitov. 2002. Retroviral delivery of small interfering RNA into primary cells. Proc. Natl. Acad. Sci. USA 99: 14943-14945. [Abstract/Free Full Text]
  32. Rubinson, D. A., C. P. Dillon, A. V. Kwiatkowski, C. Sievers, L. Yang, J. Kopinja, D. L. Rooney, M. M. Ihrig, M. T. McManus, F. B. Gertler, et al 2003. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33: 401-406. [Medline]
  33. Morita, S., T. Kojima, T. Kitamura. 2000. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 7: 1063-1066. [Medline]
  34. Mick, V. E., T. K. Starr, T. M. McCaughtry, L. K. McNeil, K. A. Hogquist. 2004. The regulated expression of a diverse set of genes during thymocyte positive selection in vivo. J. Immunol. 173: 5434-5444. [Abstract/Free Full Text]
  35. Puthier, D., F. Joly, M. Irla, M. Saade, G. Victorero, B. Loriod, C. Nguyen. 2004. A general survey of thymocyte differentiation by transcriptional analysis of knockout mouse models. J. Immunol. 173: 6109-6118. [Abstract/Free Full Text]
  36. Campbell, J. J., J. Pan, E. C. Butcher. 1999. Cutting edge: developmental switches in chemokine responses during T cell maturation. J. Immunol. 163: 2353-2357. [Abstract/Free Full Text]
  37. Nitta, T., M. Nasreen, T. Seike, A. Goji, I. Ohigashi, T. Miyazaki, T. Ohta, M. Kanno, Y. Takahama. 2006. IAN family critically regulates survival and development of T lymphocytes. PLoS. Biol. 4: e103[Medline]
  38. Radtke, F., A. Wilson, H. R. MacDonald. 2004. Notch signaling in T- and B-cell development. Curr. Opin. Immunol. 16: 174-179. [Medline]
  39. Tsukumo, S., K. Yasutomo. 2004. Notch governing mature T cell differentiation. J. Immunol. 173: 7109-7113. [Abstract/Free Full Text]
  40. Schmitt, T. M., J. C. Zuniga-Pflucker. 2002. Induction of T cell development from hematopoietic progenitor cells by Delta-like-1 in vitro. Immunity 17: 749-756. [Medline]
  41. Ciofani, M., T. M. Schmitt, A. Ciofani, A. M. Michie, N. Cuburu, A. Aublin, J. L. Maryanski, J. C. Zuniga-Pflucker. 2004. Obligatory role for cooperative signaling by pre-TCR and Notch during thymocyte differentiation. J. Immunol. 172: 5230-5239. [Abstract/Free Full Text]
  42. Tan, J. B., I. Visan, J. S. Yuan, C. J. Guidos. 2005. Requirement for Notch1 signals at sequential early stages of intrathymic T cell development. Nat. Immunol. 6: 671-679. [Medline]
  43. Klein, T., A. M. Arias. 1998. Interactions among Delta, serrate and fringe modulate Notch activity during Drosophila wing development. Development 125: 2951-2962. [Abstract]
  44. Morales, A. V., Y. Yasuda, D. Ish-Horowicz. 2002. Periodic lunatic fringe expression is controlled during segmentation by a cyclic transcriptional enhancer responsive to notch signaling. Dev. Cell. 3: 63-74. [Medline]
  45. Koch, U., T. A. Lacombe, D. Holland, J. L. Bowman, B. L. Cohen, S. E. Egan, C. J. Guidos. 2001. Subversion of the T/B lineage decision in the thymus by lunatic fringe-mediated inhibition of Notch-1. Immunity 15: 225-236. [Medline]
  46. Lehar, S. M., J. Dooley, A. G. Farr, M. J. Bevan. 2005. Notch ligands Delta 1 and Jagged1 transmit distinct signals to T-cell precursors. Blood 105: 1440-1447. [Medline]
  47. Izon, D. J., J. A. Punt, L. Xu, F. G. Karnell, D. Allman, P. S. Myung, N. J. Boerth, J. C. Pui, G. A. Koretzky, W. S. Pear. 2001. Notch1 regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength. Immunity 14: 253-264. [Medline]
  48. Kurobe, H., C. Liu, T. Ueno, F. Saito, I. Ohigashi, N. Seach, R. Arakaki, Y. Hayashi, T. Kitagawa, M. Lipp, et al 2006. CCR7-dependent cortex-to-medulla migration of positively selected thymocytes is essential for establishing central tolerance. Immunity 24: 165-177. [Medline]
  49. Ueno, T., F. Saito, D. H. Gray, S. Kuse, K. Hieshima, H. Nakano, T. Kakiuchi, M. Lipp, R. L. Boyd, Y. Takahama. 2004. CCR7 signals are essential for cortex-medulla migration of developing thymocytes. J. Exp. Med. 200: 493-505. [Abstract/Free Full Text]
  50. Matloubian, M., C. G. Lo, G. Cinamon, M. J. Lesneski, Y. Xu, V. Brinkmann, M. L. Allende, R. L. Proia, J. G. Cyster. 2004. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 427: 355-360. [Medline]
  51. Hadland, B. K., N. R. Manley, D. Su, G. D. Longmore, C. L. Moore, M. S. Wolfe, E. H. Schroeter, R. Kopan. 2001. {gamma}-Secretase inhibitors repress thymocyte development. Proc. Natl. Acad. Sci. USA 98: 7487-7491. [Abstract/Free Full Text]
  52. Duncan, A. W., F. M. Rattis, L. N. DiMascio, K. L. Congdon, G. Pazianos, C. Zhao, K. Yoon, J. M. Cook, K. Willert, N. Gaiano, T. Reya. 2005. Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance. Nat. Immunol. 6: 314-322. [Medline]
  53. Visan, I., J. B. Tan, J. S. Yuan, J. A. Harper, U. Koch, C. J. Guidos. 2006. Regulation of T lymphopoiesis by Notch1 and lunatic fringe-mediated competition for intrathymic niches. Nat. Immunol. 7: 634-643. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. Kijima, A. Iwata, Y. Maekawa, H. Uehara, K. Izumi, A. Kitamura, H. Yagita, S. Chiba, H. Shiota, and K. Yasutomo
Jagged1 Suppresses Collagen-Induced Arthritis by Indirectly Providing a Negative Signal in CD8+ T Cells
J. Immunol., March 15, 2009; 182(6): 3566 - 3572.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Kijima, T. Yamaguchi, C. Ishifune, Y. Maekawa, A. Koyanagi, H. Yagita, S. Chiba, K. Kishihara, M. Shimada, and K. Yasutomo
Dendritic cell-mediated NK cell activation is controlled by Jagged2-Notch interaction
PNAS, May 13, 2008; 105(19): 7010 - 7015.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
N. A. Gude, G. Emmanuel, W. Wu, C. T. Cottage, K. Fischer, P. Quijada, J. A. Muraski, R. Alvarez, M. Rubio, E. Schaefer, et al.
Activation of Notch-Mediated Protective Signaling in the Myocardium
Circ. Res., May 9, 2008; 102(9): 1025 - 1035.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsukumo, S.-i.
Right arrow Articles by Yasutomo, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsukumo, S.-i.
Right arrow Articles by Yasutomo, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS