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Department of Immunology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan;
Kirin Brewery Pharmaceutical Research Laboratory, Gunma, Japan;
Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tsukuba, Japan;
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Department of Biological Structure and Department of Immunology, University of Washington, Seattle, WA 98195;
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Precursory Research of Embryonic Science and Technology (PRESTO), Japan Science and Technology Corporation, Tokushima, Japan; and
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Division of Experimental Immunology, Institute for Genome Research, University of Tokushima, Tokushima, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With the aim of identifying new regulators of thymocyte development, we have found that Pref-1 is highly expressed by thymic stromal cells during early ontogeny. Pref-1 (4), also known as dlk (delta-like) (5), FA1 (fetal Ag 1) (6), or SCP-1 (stromal cell-derived protein 1) (7), is a Delta-like cell-surface transmembrane protein containing six tandem epidermal growth factor-like repeats, which are highly homologous to Delta/Notch-family proteins including Delta and Notch, that are involved in Drosophila neural development (8). Unlike other Delta-family molecules, Pref-1 lacks the DSL motif, which is suggested to be crucial for the interaction with Notch-family molecules (9), suggesting that Pref-1 may exert its biological function(s) independent of Notch-family receptors or their downstream signals, including the expression of HES family of basic helix-loop-helix transcription factors (10). Although the role of Pref-1 in thymocyte development was unknown, stromal cell expression of Pref-1 has been shown to play an important role in regulating the development and growth of adjacent cells; i.e., inhibiting adipocyte differentiation (4), promoting the proliferation of hematopoietic stem cells (11), and modulating IL-7 dependency of pre-B cell proliferation (12).
The present study shows that Pref-1 signals in the thymus regulate the cellularity of developing thymocytes. Despite previous assumptions based on the lack of the DSL motif, Pref-1 markedly increased immature thymocyte expression of the HES-1 transcription factor. Our data lend support to the notion that Pref-1 plays a role in thymocyte development by regulating thymocyte levels of HES-1 expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HES-1-deficient mice (13) were provided by Dr. Ryoichiro Kageyama (Institute for Virus Research, Kyoto University, Kyoto, Japan). Because HES-1-/- mice were embryonic lethal, HES-1-/- fetuses were obtained by crossing HES-1+/- mice. Day 14 embryos were screened for HES-1 genotype as previously described (13). C57BL/6 (B6) mice were purchased from SLC (Shizuoka, Japan).
Recombinant Pref-1 proteins
For monomeric recombinant soluble protein containing the extracellular domain of mouse Pref-1, the Pref-1-HA-His6 protein attached at C terminus with amino acid sequences corresponding to influenza hemagglutinin (HA)3 epitope and hexahistidine was expressed. For dimeric Pref-1, the Pref-1-Fc fusion protein attached with the Fc portion of human IgG1 was expressed. cDNA encoding extracellular domain of mouse Pref-1 was cloned from NIH-3T3 cells by RT-PCR using the following primers (sense, 5'-CGG AAT TCG AGA TGA TCG CGA CCG GAG CCC TC-3'; and antisense, 5'-TAA CTA GTG GTG AGG AGA GGG GTA CTC TT-3'). The PCR product was inserted into either an EcoRI site of the pSMT-201-HA-His6 vector (14) or a SpeI site of the pSMT-Fc vector (M. Ohashi, unpublished data). Pref-1-HA-His6 or Pref-1-Fc inserts were subcloned into the pcDNA3.1 mammalian expression vector (Invitrogen, Carlsbad, CA). CHO-ras clone I cells (provided by Dr. T. Shirahata, Kyusyu University, Kyusyu, Japan) were transfected with the expression vector and selected for high expression clones in the presence of 300 µg/ml hygromycin. Recombinant Pref-1-HA-His6 protein and Pref-1-Fc protein were purified from CHO cell culture supernatants by affinity chromatography over Hi-Trap Chelating-Sepharose and Hi-Trap Protein G-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden), respectively.
Anti-Pref-1 mAb
A hybridoma clone, 6C, producing a mAb against extracellular region of mouse Pref-1, was established by fusing mouse myeloma cells with spleen cells from Armenian hamsters immunized with the Pref-1-Fc protein. Hybridoma cells were initially screened for binding activity to Pref-1 recombinant proteins and then for staining activity to a stable line of CHO cells that had been transfected with full-length mouse Pref-1 cDNA. mAb 6C purified from culture supernatants over protein G affinity chromatography specifically bound to Pref-1 recombinant proteins but not to other Fc fusion proteins or HA-tagged proteins, and specifically stained Pref-1-transfected CHO cells but not untransfected CHO cells. To analyze the reactivity to membrane-bound form of mouse Pref-1, Pref-1-transfected or untransfected CHO cells were incubated with mAb, followed by staining with FITC-conjugated anti-hamster IgG Ab (PharMingen, San Diego, CA).
Thymic epithelial (TE) cell lines
Cortical TE cell lines used were TEP1-1 (15), TEC (16), and 1308.1 (17). Medullary TE cell lines used were TE71 (18) and Z210R (19).
Fetal thymus organ culture (FTOC)
Day 14 fetal thymus lobes from B6 mice were cultured in organ in the presence or absence of indicated reagents, as described previously (20).
Multicolor flow cytometry and cell sorting
Single cell suspensions were stained using mAbs (PharMingen) and analyzed by FACSCalibur flow cytometry (Becton Dickinson, San Jose, CA) as described previously (21). Adult thymocytes stained for CD4 (FITC) and CD8 (PE) were sorted for each CD4/CD8 fraction using a FACS-Vantage cell sorter (Becton Dickinson). The purity of CD4-CD8-, CD4+CD8+, CD4+CD8-, and CD4-CD8+ fractions was 97, 99, 98, and 98%, respectively.
RT-PCR analysis
Total RNA from indicated cells were reverse-transcribed using
the ThermoScript RT-PCR system (Life Technologies, Gaithersburg, MD)
and oligo(dT) primer. cDNAs were adjusted for their quantity by the
quantitative PCR using TaqMan Rodent GAPDH Control Reagent
(Perkin-Elmer Applied Biosystems, Foster City, CA), using the primers
for GAPDH (5'-TGC ACC ACC AAC TGC TTA G-3' and 5'-GGA TGC AGG GAT GAT
GTT C-3'). Relative amounts of GAPDH cDNA from real-time PCRs were
measured using the ABI PRISM 7700 analytical thermal cycler
(Perkin-Elmer Applied Biosystems). Semiquantitative PCRs were conducted
for 35 cycles using quantitatively normalized cDNAs with recombinant
Taq DNA polymerase (Takara, Shiga, Japan). The following PCR
primers were used: Pref-1 (GenBank accession number U15980; 5'-AAC ACA
TCC TGA AGG TGT CC-3' and 5'-TGG TCA TGT CAA TCT TCT CG-3'), DLL-1
(X80903; 5'-AAG ATG GAA GCG ATG TGG-3' and 5'-TCT TCA AAG CAA CTG
TCC-3'), DLL-3 (Y11895; 5'-TTG TGG TGT CCA ATC TCT AC-3' and
5'-TGG ATC TCT GTG AGT TAG AG-3'), Jagged-1 (cloned from AA182246
partial sequence; 5'-TGT GTG AAG TTG GAA GCA TCC-3' and 5'-ACC TTG AGC
TTG GTA ATA GCA C-3'), Jagged-2 (cloned from W13561 partial sequence;
5'-AAG GAC ATA CTC TAC CAG TGC-3' and 5'-ACG TCC TTG GTA CTT CTG
ACG-3'), Notch-1 (Z11886; 5'-TGC CTG TGC ACA CCA TTC TGC-3' and 5'-CAA
TCA GAG ATG TTG GAA TGC-3'), Notch-2 (D32210; 5'-ATG CAC CAT GAC ATC
GTT CG-3' and 5'-GAT AGA GTC ACT GAG CTC TCG-3'), Notch-3 (X74760;
5'-TTG GTC TGC TCA ATC CTG TAG C-3' and 5'-TGG CAT TGG TAG CAG TTG
CTG-3'), Notch-4 (M80456; 5'-AAG CGA CAC GTA CGA GTC TGG-3' and 5'-ATA
GTT GCC AGC TAC TTG TGG-3'), HES-1 (D16464; 5'-TCT ACA CCA GCA ACA GTG
G-3' and 5'-TCA AAC ATC TTT GGC ATC AC-3'), HES-3 (D32200; 5'-AGA ACT
CAC TGC AAG GAC TCT GG-3' and 5'-CTG GCA GTT TGA TGC AGG TTG-3'), HES-5
(D32132; 5'-AAG TGA CTT CTG CGA AGT TCC-3' and 5'-AAG GCC ATG TGG ACC
TTG AGG-3'), and hypoxanthine phosphoribosyltransferase (HPRT) (J00423;
5'-CAC AGG ACT AGA ACA CCT GC-3' and 5'- GCT GGT GAA AAG GAC CTC T-3'),
and ß2-microglobulin (20). PCR
primers for LEF-1, TCF-1, PEBP2
A, PEBP2B, and GATA-3 were
previously described (22). PCR products were
electrophoresed on a 2% agarose gel and visualized with ethidium
bromide staining.
Immunohistochemical staining
Frozen sections (5 µm) of day 14 fetal, day 0 neonatal, and 7-wk-old adult thymus lobes were incubated with anti-Pref-1 mAb, followed by biotinylated anti-hamster IgG Ab and HRP-conjugated streptavidin. The sections from organ-cultured fetal thymus lobes were incubated with anti-HES-1 Ab, followed by HRP-conjugated anti-rabbit IgG Ab. Slides were developed in diaminobenzidine and counterstained with methyl green.
Immunohistochemical detection of Pref-1 and HES-1 in frozen tissue sections also utilized indirect immuno-enzyme procedures (23). The anti-Pref-1 mAb was modified with N-hydroxysuccinimidyl-digoxygenin (NHS-DIG) (Boehringer Mannheim, Indianapolis, IN) according to the instructions of the manufacturer. Detection of NHS-DIG modified Abs was accomplished with HRP-conjugated Fab fragments of sheep anti-digoxygenin Abs (Boehringer Mannheim). Unconjugated polyclonal rabbit anti-HES-1 Abs were detected by sequential application of NHS-DIG-modified goat anti-rabbit IgG Abs followed by the same anti-digoxygenin reagent. Peroxidae activity was revealed with 3,3'-diaminobenzidine (Sigma, St. Louis, MO) in the presence of hydrogen peroxide.
Western blotting
Fetal thymus lobes cultured in organ for 3 days were lysed in a buffer containing 1% Nonidet P-40. The lysates of equal numbers of the cells were electrophoresed in a SDS-polyacrylamide gel, transferred to a nylon membrane, and detected for HES-1 using anti-HES-1 Ab (provided by Dr. T. Sudo, Toray, Japan) followed by HRP-conjugated anti-rabbit IgG Ab and ECL detection reagents (Amersham Pharmacia). Anti-Flag mAb (Eastman Kodak, Tokyo, Japan) was used for the detection of Flag-tagged HES-1 proteins. Densitometric analysis was done by electronic scanning of the film followed by the measurement using NIH Image software (version 1.61, http://rsb.info.nih.gov/nih-image/).
Retrovirus transfer of HES-1 in developing thymocytes
The pSK plasmid (Stratagene, La Jolla, CA) was modified to
contain synthetic oligonucleotide corresponding to the Flag peptide
(MDYKDDDDKI) with a BglII restriction site at the 3' end. To
generate the BglII site at the 5' end of rat HES-1 cDNA,
pCMV-2-HES-1 plasmid (24) was PCR amplified with the
following primers (sense, 5'-GGA AGA TCT CCA GCT GAT ATA ATG GAG AAA
AAT-3'; and antisense, 5'-AGC GGC GGT CAT CTG CGC CCG CTG CAG GTT-3').
BglII-PstI double digest of the PCR product was
cloned into BglII-BamHI sites of the pSK-Flag
plasmid. The Flag-HES-1 insert was subcloned into the EcoRI
site of the pMSCV-IRES-GFP plasmid (25). Retrovirus vector
was transfected into GP+E-86 packaging cells, and clones producing
106 CFU/ml were selected (25).
Retrovirus infection into immature thymocytes and the detection of
their development in FTOC have been described (25, 26).
Briefly, single cell suspensions from day 14 fetal thymocytes were
cultured for 1–2 days with virus-producing packaging cells in the
presence of recombinant mouse IL-7 in 96-well flat-bottom culture
plates. Virus-infected thymocytes identified as
GFP+CD45.2+ were purified
using a FACS-Vantage cell sorter equipped with Clone-Cyt hardware and
software (Becton Dickinson). Equal numbers (1000–2500/lobe) of sorted
GFP+ cells (CD45.1-
CD45.2+) were transferred into
2-deoxyguanosine-treated B6-Ly5.1 (CD45.1+
CD45.2-) fetal thymus lobes in a hanging-drop in
an inverted Terasaki well, and were organ-cultured at the interface
between a collagen sponge-supported filter and 5%
CO2-humidified air. All experiments using
retroviruses were conducted in accordance with the guidelines of
University of Tsukuba.
Measurement of HES-1 activity by luciferase reporter assay
293 T cells were transfected with 5 µg of the plasmid containing luciferase reporter gene under the control of the HES-1 promoter (27). Luciferase activity in cell lysates on day 2 was measured as described (27).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To begin analyzing biological function of Pref-1, we first
prepared recombinant soluble proteins containing the extracellular
region of murine Pref-1, a Pref-1 monomer, and a Pref-1 homodimer fused
to Fc region of human IgG1 (Fig. 1
A). We also immunized
hamsters with recombinant Pref-1 to generate a mAb that specifically
reacted with Pref-1 recombinant proteins as well as Pref-1-transfected
CHO cells but not with untransfected CHO cells (Fig. 1B).
Immunohistochemical analyses of thymus tissues with this Ab revealed a
developmentally regulated pattern of reactivity, with widespread
staining of stromal cells in the fetal thymus, and progressive loss of
reactivity with increasing age, such that scattered
Pref-1+ cells were detected in the adult thymus
(Fig. 1, C and D).
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). cDNAs from
various thymocyte samples were normalized using the real-time
quantitative PCR assay for a house-keeping enzyme GAPDH (Fig. 2A), and an equivalent amount of cDNA was employed for
further semiquantitative analysis (Fig. 2B). Jagged-1,
Jagged-2, Notch-1, Notch-2, Notch-3, Notch-4, HES-1, and HES-5, in
addition to another house-keeping enzyme HPRT, were consistently
expressed during thymus ontogeny and among adult thymocyte
subpopulations (Fig. 2B). The expression of HES-3 was barely
detected in the thymus samples (Fig. 2B). In contrast,
however, variable expression of Pref-1, DLL-1, and DLL-3 was noticed
among thymocyte subpopulations. Pref-1, DLL-1, and DLL-3 were expressed
higher in stromal cells than in lymphoid cells within adult thymus
(Fig. 2B). Unlike DLL-1 and DLL-3, Pref-1 in the thymus was
expressed higher in early ontogeny, consistent with immunohistochemical
results (Fig. 1
C). More interestingly, within TE cell lines,
Pref-1 was expressed only by a group of cortical TE cell lines such as
TEP1-1 and TEC but not by other cortical TE cell line such as 1308.1 or
by medullary TE cell lines such as TE71 and Z210R (Fig. 2B).
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Effects of recombinant Pref-1 and anti-Pref-1 Ab in FTOC
To examine the function of Pref-1 in T lymphocyte development,
thymocyte development was analyzed in FTOC in the presence of
recombinant Pref-1 proteins. As shown in Fig. 3A, the Pref-1-Fc dimer
increased the numbers of developing thymocytes in a dose-dependent
manner, whereas the Pref-1 monomer decreased the cell numbers. Other
than the cellularity, however, we detected no alterations in T cell
development including CD4/CD8 phenotype and TCR-ß/TCR-
ratio
(Fig. 3B). Time course analysis showed that the Pref-1-Fc
dimer and the Pref-1 monomer affected thymocyte cellularity within 2–3
days in the culture (Fig. 3C). The effects by Pref-1
recombinant proteins appeared to be mediated by biological functions of
Pref-1 proteins, because other Fc-containing proteins such as normal
human IgG1 and CTLA-4-Ig fusion protein did not affect the cellularity
in parallel cultures (data not shown). The cellularity was not affected
by the addition of the Pref-1 dimer in single cell suspension cultures
of fetal thymocytes in the absence or presence of IL-7 (Fig. 3D), suggesting that Pref-1 may be capable of supporting
thymocytes only in the presence of other factors that are supplied in
organ cultures but are not present in suspension cultures.
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A, anti-Pref-1 Ab
decreased the cellularity of thymocytes in the culture without
affecting the CD4/CD8 phenotype or TCR-ß/TCR- ratio (Fig. 4B).
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Pref-1 affects the expression of HES-1 in developing thymocytes
To explore molecular mechanism by which Pref-1 regulates thymocyte
cellularity, the expression levels of various molecules including
transcription factors were examined by semiquantitative RT-PCR for
Pref-1-treated thymocytes recovered from FTOC. Consequently, we found
that mRNA expression of a basic helix-loop-helix transcription factor
HES-1 was up-regulated by Pref-1-treated thymocytes within 3 days in
the culture (Fig. 5A). The
expression levels of other transcription factors including HES-5,
GATA-3, LEF-1, TCF-1, PEBP2A, and PEBP2B as well as Notch-family
molecules including Notch-1, Notch-2, Notch-3, and Notch-4 were not
markedly affected (Fig. 5A and data not shown). Pref-1
treatment indeed up-regulated protein levels of HES-1 in fetal
thymocytes (Fig. 5, B and C). Though consistent,
the Pref-1-mediated increase in HES-1 protein levels was always modest
by the immunoblot analysis (Fig. 5B). We think that this
modest increase in total thymocyte lysates reflected the basal
expression of HES-1 proteins by many subcapsular thymocytes even in
control FTOC without Pref-1 treatment (Fig. 5C, left
panel). Although it is still unclear how to reconcile the clear
increase in total RNA levels (Fig. 5A) and the modest
increase in total protein levels (Fig. 5B), it is important
to note that majority of thymocytes in Pref-1-treated FTOC now
expressed markedly higher levels of HES-1 proteins (Fig. 5C). Thus, the addition of Pref-1 in FTOC increases HES-1
expression by developing thymocytes, the increase coincided with the
increase in thymocyte cellularity.
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To directly test whether HES-1 is involved in the effects of
Pref-1 onto thymocytes, FTOC was conducted using thymus lobes from
HES-1-deficient mice. As has been recently reported, HES-1-deficient
mice are embryonic lethal during late stage of fetal development
(13). As summarized in Table I, some, but not all, HES-1-deficient
fetuses were athymic. Organ culture of HES-1-deficient fetal thymus
lobes revealed a markedly reduced cellularity compared with normal
littermate controls (Table I and Fig. 6A). Importantly, the addition
of a dimeric Pref-1-Fc fusion protein did not significantly increase
thymocyte cellularity in HES-1-deficient cultures (Fig. 6A).
It should be noted that, other than cellularity, the CD4/CD8 phenotype
and the TCR-ß/TCR- ratio in HES-1-deficient FTOC was
virtually normal in the absence or presence of Pref-1 (Table I, Fig. 6B, and data not shown).
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Retroviral overexpression of HES-1 increases thymocyte cellularity
To further examine the possibility that HES-1 in thymocytes is
involved in supporting the cellularity of thymocytes, we examined the
consequence of HES-1 overexpression in developing thymocytes using the
retrovirus-mediated gene transfer technique (25, 26).
Retroviral coexpression of GFP along with HES-1 allowed the detection
and purification of gene-transferred cells (25). Fig. 7, A and B, shows
that HES-1 expression levels markedly increased in the cells that had
been infected with this HES-1-expressing retrovirus. Introduced HES-1
indeed exhibited the regulatory activity to the transcription at the
HES-1 promoter (Fig. 7C).
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D). CD4/CD8 phenotype and
TCR-ß/TCR- ratio of thymocytes were not affected by HES-1
gene transfer (Fig. 7, E and F). These results indicate that HES-1 overexpression in developing thymocytes increased their cell numbers, supporting the possibility that HES-1 expression level is involved in supporting the cellularity of developing thymocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The selective Pref-1-mediated up-regulation of HES-1 expression among the transcription factors assayed suggests that elevated levels of HES-1 might be involved in the mechanism supporting increased thymocyte cellularity in FTOC. We have described two sets of experimental data that are consistent with this possibility. First, thymocyte cellularity is consistently elevated in FTOC following retrovirus-mediated overexpression in immature thymocytes. The lineage pattern of thymocytes under these conditions was identical to that observed following administration of exogenous dimeric Pref-1 to FTOC. The second line of supportive evidence comes from analyses of thymuses from HES-1-deficient mice, where thymus cellularity was dramatically reduced and accompanied by qualitatively normal thymocyte development. The hypocellularity of HES-1-deficient FTOC and the inability of dimeric Pref-1-fusion protein to augment thymocyte cellularity in these cultures is significant in several respects. First, these data provide additional support for the notion that levels of HES-1 expression may represent an important regulator supporting thymocyte cellularity. In addition, the inability of Pref-1 to enhance cellularity in HES-1-deficient FTOC indicates that Pref-1 exerts its growth-promoting effects through a HES-1-dependent mechanism. While we favor the interpretation that these results reflect a mechanism whereby Pref-1-mediated interactions directly affect levels of HES-1 expression, we cannot presently exclude the possibility that the action of Pref-1 is indirect, involving other molecules but requiring HES-1 expression.
The present study shows that Pref-1 and HES-1 are both involved in
supporting a number of developing thymocytes. It has been suggested
that the DSL motif in the extracellular region of Delta-family
molecules is crucial for the interaction with Notch-family receptor
molecules (9). Because Pref-1 lacks the DSL motif, it has
been speculated that Pref-1 might function through a receptor
independent of Notch-family molecules, and thus independent of
HES-family-mediated downstream signals. However, our results show that
Pref-1 markedly up-regulate the expression of HES-1 in developing
thymocytes, and that HES-1-deficient thymocytes do not respond to
Pref-1, suggesting that Pref-1 affects immature thymocytes via signals
through the HES-1 transcription factor. Still, it is not clear yet how
Pref-1 up-regulates HES-1 expression in developing thymocytes. It is
possible that immature thymocytes express the Pref-1 receptor, which
directly transmits the Pref-1 binding signal to enhance HES-1
expression. However, Pref-1 in suspension culture did not affect the
cellularity of immature thymocytes even in the presence of IL-7 (Fig. 3D). We have further found that, in such a suspension
culture condition, Pref-1 did not increase HES-1 expression by immature
thymocytes (M. Kaneta and Y. Takahama, unpublished observation). Thus,
Pref-1 expressed by TE cells may affect other cells, such as
mesenchymal stromal cells, which in turn produce other factors inducing
HES-1 expression by thymocytes. Alternatively, Pref-1 interaction with
its receptor on thymocytes may support HES-1 expression in thymocytes
only in the presence of other molecular interactions between epithelial
cells and thymocytes, interactions that are lost in suspension
cultures. To better understand the molecular mechanism for
Pref-1-induced nourishment of developing thymocytes, we aim to identify
the molecular nature of the Pref-1 receptor.
Immunohistochemical analysis (Fig. 1, C and D)
and RT-PCR analysis (Fig. 2B) showed that Pref-1 in the
thymus is expressed at higher levels during fetal development than in
adult life. We think that the decrease in Pref-1 expression along the
ontogeny is well coordinated with the function of Pref-1 presented in
this study, thus supporting thymocyte numbers, because thymocyte
numbers rigorously increase during early ontogeny but not in adulthood.
Whether or not Pref-1 is also involved in T lymphocyte generation that
is maintained in adult thymus (28) and that is crucial for
adult T-lymphopoiesis after chemotherapy or radiation exposure would be
an issue of interest and of clinical importance.
Our results show that neither Pref-1 nor HES-1 is involved in
developmental choice between CD4 and CD8 T cell subsets and between
TCR-ß and TCR- T cell lineages. On the other hand, Robey and
colleagues (29, 30) have shown that Notch-1 is involved in
these lineage decisions rather than affecting the cellularity of
thymocytes. These results suggest a possibility that Notch-1 signal and
HES-1 signal are not always identical, at least in developing T cells.
This possibility is supported by the recent results showing that the
Notch signal is capable of initiating CD4 silencer function even in the
absence of a functional HES-1 binding site, although Notch signal and
HES-1 signal can both repress CD4 gene expression (31).
More compatible with our results are recently published results showing
that Notch-1 signals are capable of rendering thymomas resistant to
glucocorticoid-induced apoptosis (32), and that Notch-1
signals protect TCR-induced apoptosis (33), which suggest
that the Notch-HES signaling cascade is likely involved in supporting
survival of immature thymocytes.
It should be mentioned that lineage patterns of thymocytes including
CD4/CD8 phenotype and TCR-ß/TCR- ratio were essentially
normal either by blocking Pref-1 interactions with anti-Pref-1 Ab
or in the absence of HES-1 in HES-1-deficient FTOC, suggesting that the
signals through Pref-1 and HES-1 equally regulate cell numbers of every
thymocyte lineage, possibly by affecting cellular survival at an early
CD4-CD8- stage of T cell
development before the division of cell lineages. Although thymocyte
cellularity is also critically regulated by IL-7, the disruption of
IL-7 signals more severely reduces TCR- cells than TCR-ß
cells during thymocyte development (34, 35), suggesting
that the Pref-1/HES-1 signal might regulate the cellularity of
thymocyte lineages in a manner more distinct than the IL-7 signal.
Further analysis of the relationship between these two signal pathways
as well as Bcl-2-family-mediated anti-apoptotic signals, for
example antagonizing TCR signals or steroid signals, would be of great
interest toward better understanding of the mechanism for
growth/survival signals regulating thymocyte development.
It has been reported recently that the deficiency of either HES-1 or
Notch-1 in hematopoietic precursor cells results in the arrest of T
cell development at early
CD4-CD8- thymocytes
(36, 37), suggesting that a signaling cascade through
Notch-1 and HES-1 is essential for the specification of lymphoid
precursors into the T cell lineage. These results appear contradictory
with our results which show hypocellularity but normal lineage
distribution of HES-1-deficient thymocytes in FTOC (Table I and Fig. 6). However, the developmental arrest of thymocytes in these reports
(36, 37) was observed when examining in vivo transfer of
lymphopoietic precursor cells from either HES-1-deficient or
Notch-1-deficient mice, unlike our results obtained from organ cultures
of intact thymus lobes from HES-1-deficient mice. It is possible that
the deficiency in HES-1 or Notch-1 could cause low efficiency of
T-precursor cells in either migration into the thymus or early survival
in the thymus. Consistent with this possibility, ex vivo analysis of
Notch-1-deficient thymocytes showed hypocellularity but normal T cell
lineage distribution (37). In addition, normal lineage
distribution of thymocytes were generated in irradiated mice
transferred with higher numbers of HES-1-deficient fetal liver cells
(Dr. N. Minato, unpublished data). Consequently, we think that
HES-1-mediated and perhaps Notch-1-mediated signals are critical for
early survival and/or expansion of immature T-precursor cells, rather
than essential for lineage specification into T lymphocytes.
Finally, our results show that the addition of a Pref-1 monomer decreased the cellularity of thymocytes, unlike Pref-1-Fc dimer proteins that increased thymocytes. We think that this decrease is due to the competitive block of endogenous Pref-1 by the tailless exogenous monomer protein. Indeed, like the Pref-1 monomer, the anti-Pref-1 mAb exhibited the similar activity in FTOC, decreasing thymocyte cellularity, thus supporting the interpretation that competitive inhibition of Pref-1 in the thymus cancels the function of Pref-1 supporting developing thymocytes.
In conclusion, the present results identify a role for Pref-1 in
supporting developing T lymphocytes in the thymus. However, Pref-1
stimulation could not directly support the survival of immature
thymocytes even in the presence of IL-7 (Fig. 3D),
suggesting that Pref-1 and IL-7 signals are not sufficient for fully
nourishing the cellularity of developing thymocytes. Attempts to
identify further molecular signals supporting developing T cells are
currently under the investigation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yousuke Takahama, Division of Experimental Immunology, Institute for Genome Research, University of Tokushima, 3-18-15 Kuramoto, Tokushima, 770-8503, Japan. E-mail address:
3 Abbreviations used in this paper: HA, hemagglutinin; TE, thymic epithelial; FTOC, fetal thymus organ culture; HPRT, hypoxanthine phosphoribosyltransferase; GFP, green fluorescence protein.
Received for publication September 10, 1999. Accepted for publication October 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Cell. Immunol. 119:427.[Medline]
ß+ thymocytes. J. Immunol. 147:2883.[Abstract]
ß and T cell lineage decision. Cell 88:833.[Medline]
/ T cells. Proc. Natl. Acad. Sci. USA 93:7172. T cell development and early thymocyte maturation in IL-7-/- mice. J. Immunol. 157:2366.[Abstract]
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Y. Wang and H. S. Sul Ectodomain Shedding of Preadipocyte Factor 1 (Pref-1) by Tumor Necrosis Factor Alpha Converting Enzyme (TACE) and Inhibition of Adipocyte Differentiation. Mol. Cell. Biol., July 1, 2006; 26(14): 5421 - 5435. [Abstract] [Full Text] [PDF]
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A. Dumortier, R. Jeannet, P. Kirstetter, E. Kleinmann, M. Sellars, N. R. dos Santos, C. Thibault, J. Barths, J. Ghysdael, J. A. Punt, et al. Notch Activation Is an Early and Critical Event during T-Cell Leukemogenesis in Ikaros-Deficient Mice Mol. Cell. Biol., January 1, 2006; 26(1): 209 - 220. [Abstract] [Full Text] [PDF]
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K. Murata, M. Hattori, N. Hirai, Y. Shinozuka, H. Hirata, R. Kageyama, T. Sakai, and N. Minato Hes1 Directly Controls Cell Proliferation through the Transcriptional Repression of p27Kip1 Mol. Cell. Biol., May 15, 2005; 25(10): 4262 - 4271. [Abstract] [Full Text] [PDF]
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J. M. Lefebvre, M. C. Haks, M. O. Carleton, M. Rhodes, G. Sinnathamby, M. C. Simon, L. C. Eisenlohr, L. A. Garrett-Sinha, and D. L. Wiest Enforced Expression of Spi-B Reverses T Lineage Commitment and Blocks {beta}-Selection J. Immunol., May 15, 2005; 174(10): 6184 - 6194. [Abstract] [Full Text] [PDF]
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M. Miyazaki, H. Kawamoto, Y. Kato, M. Itoi, K. Miyazaki, K. Masuda, S. Tashiro, H. Ishihara, K. Igarashi, T. Amagai, et al. Polycomb Group Gene mel-18 Regulates Early T Progenitor Expansion by Maintaining the Expression of Hes-1, a Target of the Notch Pathway J. Immunol., March 1, 2005; 174(5): 2507 - 2516. [Abstract] [Full Text] [PDF]
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K. Masuda, M. Itoi, T. Amagai, N. Minato, Y. Katsura, and H. Kawamoto Thymic Anlage Is Colonized by Progenitors Restricted to T, NK, and Dendritic Cell Lineages J. Immunol., March 1, 2005; 174(5): 2525 - 2532. [Abstract] [Full Text] [PDF]
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12) are shown. Asterisks
indicate significant (*, p < 0.01; **,
p < 0.001 by Student’s t test)
differences in cell numbers from control cell numbers in the absence of
Ab. The addition of normal hamster IgG did not significantly (N.S.)
alter the cell numbers. B, Thymocytes were two-color
stained as indicated. Representative results from five
(A) and three (B) independent
measurements are shown.
