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*
Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, and
Genome Information Research Center, Osaka University, Suita, Osaka, 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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500 aa residues) in their
extracellular regions (1, 2). Many members of the
semaphorin family are known to play important roles in axon guidance,
acting as chemorepulsion factors during neuronal development
(2, 3, 4, 5, 6). Among the semaphorin family, CD100 is the first
semaphorin member, for which physiological expression has been
identified in the immune system (7). CD100 mRNA
has been detected in large quantities in lymphoid organs, including
spleen, thymus, and lymph nodes (8, 9). Although the
expression of CD100 is already abundant in resting T cells and is weak
in resting B cells, it is significantly up-regulated in both T and B
cells following activation of the respective cells
(10, 11, 12). Ab cross-linking of CD100 has been shown to provide proliferative signals to human peripheral blood T lymphocytes in the presence of submitogenic doses of CD3 or CD2 Abs (7, 10). In addition, tyrosine phosphatases, including CD45, have been shown to be associated with CD100 in either T or B cells (12, 13). Furthermore, a serine/threonine kinase activity has been reported to be associated with the cytoplasmic portion of CD100 (14, 15). These findings suggest that CD100 may function as a receptor.
Although CD100 is a semaphorin member of the transmembrane type, cumulative evidence has shown that, as is the case for other semaphorin members of the secreted type, CD100 can function as a ligand. Hall et al. (8) reported that human CD100-expressing transfectants promote aggregation and survival of B cells in vitro. Using a recombinant soluble mouse CD100 protein, constructed by fusing the extracellular region of CD100 with the Fc portion of human IgG1, or by using transfectants expressing mouse CD100, we have also demonstrated that CD100 enhances in vivo Ab production as well as in vitro B cell responses (11). Delaire et al. (16) also recently demonstrated that human soluble CD100 (sCD100)4 inhibits immune cell migration. Furthermore, we recently reported that CD100 is proteolytically cleaved into a 120-kDa sCD100 that has a costimulatory activity with CD40 mAb on B cells (17). High levels of sCD100 were observed in sera of mice immunized with T cell-dependent (TD) Ags or in sera of mice with autoimmune disease, suggesting that sCD100 plays a role in physiological and pathological immune responses. Interestingly, a recent report has shown that generation of human sCD100 is dependent on a metalloprotease process, which is regulated by a serine kinase activity associated with the cytoplasmic tail of CD100 (15).
It has been recently shown that CD100 uses two types of receptors in different tissues: plexin-B1, a receptor in nonlymphoid tissues (18, 19), widely expressed at prominent levels in the fetal brain and kidney (20), and CD72, a receptor found in lymphoid tissues (11). Interestingly, CD100-deficient mice have functional defects in lymphoid tissues where CD72 is expressed, but not in other tissues where plexin-B1 is abundantly expressed (21). Therefore, the interaction of CD100 with CD72 appears to play a nonredundant role in the immune system. As for the functions of CD72, CD72 mAbs have been reported to 1) block B cell Ag receptor (BCR)-mediated cell death, 2) promote B cell survival and proliferation, and 3) enhance the production and release of CD23 in B cells (22, 23, 24, 25, 26). Many of the effects due to CD72 mAbs, which appear to be very similar to those due to CD100 stimulation, imply that CD72 transmits positive signals for B cell activation (8, 11). On the other hand, recent findings suggest a potential role of CD72 as a negative regulator of B cell responses. Indeed, the cytoplasmic domain of CD72 has two immunoreceptor tyrosine-based inhibitory motifs. Adachi et al. (27) have shown that cross-linking of BCR enhances the phosphorylation of tyrosines on CD72 and association of CD72 with the protein tyrosine phosphatase Src homology 2 domain containing tyrosine phosphatase-1 (SHP-1), suggesting that CD72 might negatively regulate B cell activation. Consistent with this finding, B cells from CD72-deficient mice become hyperproliferative in response to various stimuli (28). These contradicting findings raise the question of how the positive outputs are generated from the binding of CD100 to CD72. Recently, we have demonstrated that CD100 binding induces the dephosphorylation of tyrosines on CD72 and the dissociation of SHP-1 from the latter. Therefore, CD100 appears to turn off the negative signaling effects of CD72, resulting in an enhancement of B cell activation (11). Indeed, CD100-deficient mice displayed hyporesponsiveness of B cells and impaired B-1 cell development, which are almost opposite the phenotypes of CD72-deficient mice (21, 28).
In the present study, to elucidate in vivo functions of CD100 as a ligand and to clarify a role of the cytoplasmic tail in the expression and functions of CD100, we generated transgenic mice expressing a truncated form of CD100 (CD100-Tg). The results indicate that CD100 is critically involved in the regulation of B-1 cell development and immune responses and suggest the importance of sCD100 in regulating immune reactions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A truncated form of CD100 cDNA was prepared by PCR
from a full-length mouse CD100 cDNA derived from
CD40-stimulated WEHI-231 cells (11). A pair of
oligonucleotide primers made of a sense sequence including a
SalI site (5'-GCTGTCGACTGTGTGCCCGTTGCTGAAGGCCT-3') and an
antisense sequence including a XhoI site and a Flag sequence
(DYKDDDDK)
(5'-CACTCGAGTCACTTGTCATCGTCGTCCTTGTAGTCGCCCTTGTAGCAGTTGTAGGAA-3')
were used. The resulting mutant CD100 cDNA contained the
complete extracellular and transmembrane regions, but not the
intracellular region. The SalI-XhoI cDNA fragment
was inserted into the unique XhoI site of the rabbit
-globin gene in the pBluescript II KS+
cassette, which carries the enhancer µ, the promoter of Ig heavy
chain VH4C8, and 3'-untranslated rabbit -globin gene. The vector was
linearized by the SalI and NotI restriction
enzymes and microinjected into the pronuclei of fertilized C57BL/6
mouse eggs. Offspring from the microinjected eggs were selected by PCR
and Southern blot analysis. The following oligonucleotide primers were
used to identify endogenous CD100 expression or
transgenic CD100 expression. Primer 1
(5'-TGGTAGACAGGACCCAG-3') was made of a sense sequence, and primer
2 (5'-TCAGTCCCCGTCAGCATCCGAATC-3') was made of an antisense sequence
complementary to cDNA of CD100. Primer 3
(5'-CTTGTCATCGTCGTCCTTGTAGTC-3') was made of an antisense sequence
complementary to a Flag sequence. Primer 1 was used in
conjugation with primer 2 to identify the endogenous CD100
expression and was used in conjugation with primer 3 to identify the
transgene. Two lines (lines 6 and 22) were established. The copy number
of the transgenes was determined by Southern blot analysis of tail DNA.
To introduce the transgene into a CD100-null background, the
transgenic mice were backcrossed with CD100-deficient mice
(21). All these mice were maintained in a pathogen-free
environment.
Real-time monitoring quantitative PCR (QPCR)
The relative abundance of CD100 and GAPDH mRNAs was assessed by real-time monitoring QPCR. Briefly, B220+ B cells or Thy1+ T cells were purified from the spleen by MACS (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity of the cells was >95%. Total RNA was isolated from B220+ B cells or Thy1+ T cells using Isogen (Nippon Gene, Toyama, Japan). Total RNA was treated with DNase I to remove any contaminating genomic DNA before RT. Then, cDNA were synthesized using SuperScript II reverse transcriptase (Life Technologies). Primer 643-F (5'-AGTCCCTTGAGGACGGAGTATG-3') was used in conjugation with primer 719-R (5'-CACACTTGGCCTGTATCTGCA-3') to amplify a 76-bp fragment in the transmembrane region of CD100. A TaqMan probe, 5'-Fam (6-carboxy fluorescein)-CCCGTGGCTGAACGAGCCTAGCTT-3' TAMRA (6-carboxy-tetramethylrhodamine), was included during QPCR. PCR primers and TaqMan probe for GAPDH were obtained from PerkinElmer (Norwalk, CT). QPCR amplification was conducted in triplicate in 50-µl reaction volumes consisting of TaqMan Universal PCR Master Mix, 900 nM of each primer, and 250 nM TaqMan probe. cDNA (500 ng) was amplified according to the thermal profile of 50°C for 2 min and 95°C for 10 min (95°C for 15 s, 60°C for 1 min) for 40 cycles and incubated at 25°C using an ABI 7700 (Applied Biosystems, Foster City, CA). Quantification was performed using software from ABI 7700 sequence detection systems.
Flow cytometry and Abs
Single-cell suspensions were prepared from mouse spleen (8–12 wk old). Bone marrow cells were obtained from one tibia and one femur. Peritoneal cavity cells were collected by flushing the peritoneal cavity with PBS containing 2% FCS and 10 U/ml heparin. One million cells of the various tissues tested were stained with the following Abs: anti-IgD, anti-CD43 (S7), anti-CD8 (Ly-2), and anti-B220 (RA3-6B2), Thy1.2 (30H12), anti-CD5 (53-7.3), anti-Mac 1 (M1/70), anti-IgM (R6-60.2), and anti-CD4 (GK1.5), and anti-CD100 (BMA-12) conjugated with FITC, PE, or biotin. Streptavidin-FITC, -PE, and -allophycocyanin were used as second-step reagents for biotinylated Abs. These Abs and reagents, except for anti-IgD and anti-CD100, were purchased from BD PharMingen (San Diego, CA). Goat anti-IgD was purchased from Nordic Immunological Laboratories (Tilburg, The Netherlands), and anti-CD100 (BMA-12) was established at our laboratory (11). Data analysis was performed using FlowJo software (Treestar, San Carlos, CA).
Serum sCD100 assays
The levels of serum sCD100 were determined by sandwich ELISA as previously described (17). Ninety-six-well microplates (MaxiSorp Nunc-Immunoplate; Nunc, Roskilde, Denmark) were coated with BMA-12 (100 µl of 5 µg/ml in 0.1 mM NaHCO3 pH 9.0) at 4°C overnight. The wells were incubated in a blocking solution (200 µl of 50 mM Tris-HCl (pH 8.1), 1 mM MgCl2, 0.15 M NaCl, 1% BSA, and 0.05% Tween 20) for 1 h at room temperature. Samples and controls (recombinant sCD100 protein consisting of the extracellular region of the mouse CD100 fused with a Flag peptide) (11) diluted with the blocking solution were added to the wells (50 µl/well) and were incubated to determine the concentration of sCD100. Samples and controls diluted with the blocking solution were added to the wells (50 µl/well) and incubated for 2 h at room temperature. The wells were washed three times with PBS containing 0.05% Tween 20 and incubated with biotinylated BMA-8 (2 µg/ml) for 2 h at room temperature (11). The incubation continued with alkaline phosphatase-conjugated streptavidin and was subsequently developed by phosphatase substrate (Sigma-104; Sigma, St. Louis, MO). Absorbance values were obtained at 405 nm (correction wavelength set at 620 nm).
Assays for proliferation and Ig production
Nonadherent splenic B cells were isolated with a combination of anti-Thy1.2 (F7D5; Serotec, Oxford, U.K.) and rabbit complement (Wako Pure Chemical Industries, Osaka, Japan) to remove T cells. The remaining B cells were fractionated with a Percoll gradient of 50, 60, 66, and 70%, and cells at the interface between 66 and 70% were collected. The purity of the resting B cell preparations was >90%. Purified B cells (1 x 105 cells/well) were cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% FCS (Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 2-ME (5 x 10-5 M) in the presence or the absence of IL-4 (Genzyme, Cambridge, MA), CD40 mAb (HM40–3; BD PharMingen), or 0.2 µg/ml LPS (Sigma) in 96-well microtiter plates. For B cell proliferation assays, cultures were pulsed with 2 µCi of [3H]thymidine for the last 16 h of culture period, and the incorporation of [3H]thymidine was measured. For Ig production assays, culture supernatants stimulated with 10 U/ml IL-4 and 0.5 µg/ml CD40 mAb were harvested from 7-day cultures, and the levels of Igs were measured by ELISA (29).
Immunization and serum Ab assays
To induce Ab responses to TD Ag, 8- to 12-wk-old mice were
immunized i.p. with 100 µg of 4-hydroxy-3-nitrophenylacetyl-chiken
-globulin conjugates (NP-CGG) as an alum-precipitated complex
on day 0, boosted on day 28, and bled on the indicated days after the
first immunization. NP-specific Abs were detected with
NP12-BSA-coated plates as previously described
(30). To induce Ab responses to T cell-independent (TI)
Ags, 8- to 12-wk-old mice were immunized i.p. with 50 µg of
2,4,6-trinitrophenyl (TNP)-LPS (Sigma) or 25 µg of NP-Ficoll in PBS
and were bled before and 7 days after the immunization. The
anti-TNP and anti-NP specific Abs were detected using
DNP-conjugated-BSA or NP12-BSA-coated ELISA
plates, respectively, and were quantified by isotype-specific
ELISA.
T cell priming
Mice were immunized by injecting 1, 10, or 100 µg of keyhole
limpet hemocyanin (KLH) in CFA into the hind footpads. Nine days after
priming, CD4+ T cells were purified from the
draining lymph nodes using MACS, and 1 x
105 cells were stimulated for 72 h with
various concentrations of KLH in the presence of irradiated (3000 rad)
splenocytes (5 x 105) of wild-type
littermates in 96-well microtiter plates. For proliferation assays,
cells were pulsed with 2 µCi of [3H]thymidine
for the last 12 h. Levels of IL-4 and IFN- in the culture
supernatants were measured using ELISA kits (R&D Systems,
Minneapolis, MN).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of CD100 is relatively weak in resting B cells, but
is up-regulated upon activation (10, 11, 12). To investigate
the effect of increased CD100 expression on immune responses, we
generated transgenic mice expressing increased levels of CD100. As
shown in Fig. 1
A, the
transgene contains the cDNA sequence encoding the extracellular and
transmembrane regions of CD100, but not the cytoplasmic region, next to
the µ enhancer and the promoter of Ig heavy chain gene. Offspring
from eggs in which the fragment was microinjected were selected by PCR.
Among several lines of mice carrying the transgene, two lines (lines 6
and 22) with higher expression of the transgene were selected by
RT-PCR. Since these two lines showed essentially the same phenotypes,
line 6 was subjected to further detailed analysis as described below.
From Southern blot analysis, 20 copies of the transgenes were found
to be present in this transgenic line. The real-time monitoring QPCR
revealed that B cells and T cells from CD100-Tg mice expressed 8.8-
and 2.7-fold more CD100 mRNA than those from wild-type mice,
respectively (Fig. 1B). However, only a slight increase in
CD100 expression was observed on the cell surface of CD100-Tg B cells
(Fig. 1C). We could not detect any increase in CD100
expression on the cell surface of T cells. Interestingly, a large
amount of sCD100 was detected in sera of CD100-Tg mice, but not of
wild-type littermates (Fig. 1D). We have previously shown
that the extracellular region of CD100 on the surface of lymphocytes
was cleaved and released in the form of sCD100 upon activation
(17). It thus appears that a truncated form of CD100 is
unstable on the surface of lymphocytes and is easily cleaved and
released from the surface of lymphocytes without stimulation, resulting
in the accumulation of sCD100 in sera of CD100-Tg mice.
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We first performed flow cytometric analyses on cells from the bone
marrow, spleen, and peritoneal cavity of CD100-Tg mice to examine the
development of lymphocytes. The mutant mice had relatively increased
numbers of CD5+B220+ B-1
cells (wild type, 13.7 ± 2.0%; CD100-Tg, 17.2 ± 1.0%;
p < 0.05; Fig. 2F). This is in good agreement
with our previous observations in CD100-deficient mice, where the
number of B-1 cells was reduced (21), suggesting a role of
CD100 in the development of B-1 cells. We also analyzed the
subpopulations of B-1, B-1a
(CD5+B220+
Mac-1+), and B-1b
(CD5-B220+Mac-1+)
cells by three-color flow cytometric analysis. B-1a cells were found to
increase in CD100-Tg (wild type, 9.4 ± 2.9%; CD100-Tg, 14.7
± 1.9%; p < 0.05). However, we could not detect
significant changes in B-1b cells in CD100-Tg mice (wild type, 4.2
± 1.7%; CD100-Tg, 2.5 ± 0.87%). Except for these phenotypic
changes, we did not observe any other differences in the surface
phenotypes (Fig. 2, A–E), numbers, and ratios of T and B
cells between CD100-Tg mice and wild-type mice (data not shown). T
cells and conventional B cells appeared to develop normally in the
transgenic mice.
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CD100 stimulation synergistically enhances CD40-induced B cell
proliferation and Ig production (8, 11). CD100-deficient B
cells show hyporesponsiveness to various stimuli (21). To
examine the influence of increased CD100 expression on B cell
responses, small resting spleen B cells were purified from CD100-Tg or
wild-type mice and stimulated with various stimuli. As shown in Fig. 3A, B cells from CD100-Tg mice
showed enhanced proliferative responses to CD40 mAb compared with those
from wild-type mice, although the differences were obscured when cells
were stimulated with a high dose of IL-4. CD100-Tg B cells also showed
enhanced responses to LPS stimulation (Fig. 3B). In
addition, in vitro IgG1 production of B cells induced by the
combination of CD40 mAb and IL-4 was elevated in CD100-Tg mice (Fig. 3C). These results are inconsistent with our previous
observations that either sCD100- or CD100-expressing transfectants
enhance in vitro B cell responses (11, 17).
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To explore the influence of the excessive CD100 on in vivo Ab
responses, CD100-Tg mice were immunized with a TD Ag, NP-CGG. Although
the expression of the transgene did not have SO much effect
on the primary Ab responses, Ab titers were still sustained at the
later phase of primary responses (day 28). The effects of the transgene
were much more obvious in the secondary immune responses (Fig. 4). Particularly, Ab responses persisted
for a long period in CD100-Tg mice. We next examined the Ab responses
against TI Ags: TNP-LPS as a type I TI-Ag (TI-1) and NP-Ficoll as a
type II TI-Ag (TI-2). We could not observe clear differences in
comparable Ab responses against TNP-LPS between CD100-Tg and wild-type
mice (Fig. 5A), although Ab
titers fluctuated among individual mice. On the other hand, CD100-Tg
mice mounted relatively reduced responses against NP-Ficoll,
particularly in the IgG1 Ab response (Fig. 5B), suggesting
that CD100 may modulate BCR-mediated signaling.
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We examined the generation of Ag-specific T cells in CD100-Tg mice
because CD100-deficient mice exhibited severe impairments of T cell
priming (21). We immunized wild-type or CD100-Tg mice with
a low dose (1 µg/mouse; Fig. 6A), an intermediate dose (10
µg/mouse; Fig. 6B), or a high dose (100 µg/mouse; Fig. 6C) of KLH in CFA s.c. in the hind foot pad. Nine days after
immunization, CD4+ T cells were prepared from the
draining lymph nodes and stimulated with various concentrations of KLH
in the presence of APC from normal mice. Enhancement of Ag-induced
proliferation and cytokine production of CD4+ T
cells was observed in CD100-Tg mice when immunized with a low dose of
KLH. Enhanced IFN- production was still observed in CD100-Tg mice
immunized with an intermediate dose of KLH, although there was no
difference when mice were immunized with a high dose of Ags. Taken
together, these findings suggest that excessive amounts of sCD100
result in enhancement of T cell priming in vivo when the mice are
immunized with suboptimal doses of Ags.
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To test whether the transgenic expression of CD100 can restore
the impaired in vitro and in vivo B cell responses in
CD100-deficient mice, CD100-Tg mice were backcrossed with
CD100-deficient mice. As expected, the transgenic expression of CD100
could restore in vitro responses of CD100-deficient B cells to various
stimuli (Fig. 7A). When
CD100-Tg mice in the CD100-deficient background were immunized with a
TD Ag, NP-CGG, they produced Ag-specific Abs comparable to those of
wild-type mice (Fig. 7B). This result confirms the
involvement of CD100 in immune responses and suggests the importance of
elevated amounts of sCD100 in immune reactions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies suggest that CD100 may exert its functions as both a ligand and a receptor (7, 8, 10, 11, 14, 15, 16, 17). To study the functions of CD100 as a ligand and the role of its cytoplasmic tail in functions and expression of CD100, we generated transgenic mice expressing the truncated CD100 lacking its cytoplasmic region. A large amount of sCD100 was observed in sera of CD100-Tg mice, although the expression of transgenic CD100 on B cells was only slightly elevated. These findings suggest that a truncated form of CD100 may be rapidly cleaved and shed from the cell surface. A serine kinase activity has previously been shown to be associated with the cytoplasmic tail of CD100 (14). In addition, it has recently been reported that the release of sCD100 is significantly enhanced by a serine kinase inhibitor (15). Taken together, all these findings suggest that the cytoplasmic tail of CD100 plays a role in the regulation of proteolytic cleavage of its extracellular domain.
CD100-Tg mice displayed an increased number of B-1 cells, enhanced in vitro B cell responses, elevated levels of Ab production against TD Ags, and accelerated in vivo T cell priming, which are phenotypes almost opposite to those of CD100-deficient mice (21). Furthermore, introduction of the transgene could restore in vitro and in vivo B cell responses of CD100-deficient mice. Such effects of transgene expression can be largely attributed to increased sCD100 rather than to the truncated membrane-bound CD100 on B cells because of only a small increase in CD100 on the surface of B cells. We have recently shown that there is a good correlation between the levels of sCD100 and the titers of specific Abs against TD Ag in sera of immunized mice or the titers of autoantibodies in sera of MRL/lpr mice (17). Furthermore, in vivo treatment with recombinant sCD100 protein has been shown to enhance humoral immune responses of normal mice and to restore Ag-specific T cell priming of CD100-deficient mice (11, 21). Thus, our present and previous observations strongly indicate that sCD100 plays a critical role in the regulation of physiological and pathological immune responses.
Although the semaphorin family consists of secreted-type and transmembrane-type proteins, their representative biological functions, such as repulsive axon guidance, have been reported on secreted-type members, acting as soluble factors (2). In contrast, the biological functions of transmembrane-type members have not been fully elucidated. CD100 has been reported to exist not only as a membrane-bound form, but also as a soluble form (10, 11, 15, 16, 17). We have directly demonstrated that sCD100 generated from the cell surface of lymphocytes by proteolytic cleavage had potent costimulatory activity as well as recombinant sCD100 protein (17). Many receptors, including IL-6R and CD62 ligand, are known to be cleaved by proteases such as metalloproteases and released as soluble forms (31, 32, 33). Although it is not clear whether the transmembrane-type CD100 needs to be converted into a soluble form to exert its functions, sCD100 seems to work as the active form in immune responses. From this point of view, the transmembrane-type of CD100 may exist as a reservoir for sCD100 to allow for a broader spectrum of immune reactions. If this is the case, proteolytic cleavage of CD100 would be one of the critical steps in regulating the function of CD100 in immune response (10). The phenotypes of CD100-Tg mice can be mostly explained by biological activities of sCD100 as a ligand. However, human CD100 has been shown to mediate signals as a receptor (7, 10, 14), suggesting that some phenotypes may be due to a dominant negative effect of sCD100 on signals via CD100. At present we cannot rule out this possibility. Further studies will be necessary to elucidate the functions of mouse CD100 as a receptor.
Role of CD100 in B cell development
The CD5+ B-1 cell population in the adult mouse is thought to be a remnant of a distinct fetal B cell differentiation pathway that is maintained without the help of T cells (34, 35). The number of CD5+ B-1 cells was significantly reduced in the peritoneal cavity of CD100-deficient mice (21). In contrast, the number of B-1 cells is reported to be elevated in CD72-deficient mice (28). In CD100-Tg mice the number of B-1 cells was elevated as well. Other mutant mice carrying defects in SHP-1 signals, such as SHP-1- and CD22-deficient mice, also show an expansion of B-1 cells (36, 37, 38). Collectively, these observations indicate that SHP-1 participates in the negative regulation of development or maintenance of B-1 cells. Our findings, therefore, suggest that CD100 may contribute to autonomous expansion of B-1 cells by inactivating SHP-1 signals through CD72.
The transgenic expression of the truncated form of CD100 did not show any effect on the development of conventional B cells at present. This is in good agreement with our previous observation that conventional B cells develop normally in CD100-deficient mice (21). It thus appears that CD100 is not involved in the regulation of conventional B cell development. However, CD72-deficient mice have been reported to display a subtle defect in early B cell development in the bone marrow (28). Although we currently do not have any clue to explain this discrepancy, it may be possible that an unknown ligand other than CD100 plays a role in regulating CD72 signals in bone marrow.
Role of CD100 in B cell responses
The fact that in vitro B cell responses and in vivo Ab responses against TD Ags were enhanced in CD100-Tg mice substantiates an important role of CD100 in B cell functions, which has been proposed from our previous observations of recombinant sCD100 or CD100-deficient mice (11, 21). Furthermore, the phenotypes of CD100-Tg mice are similar to those of CD72-deficient mice (28). Thus, the present findings confirm our assumption that CD100 turns off negative signals of CD72 (11). However, Ab responses against TI-2 Ags in CD100-Tg mice were weaker than those in wild-type control mice. Conversely, we previously showed that TI-2 Ab responses of certain isotypes, such as IgG1, were enhanced in CD100-deficient mice (21). TI-2 Ags, which have highly repetitive structures and can efficiently cross-link surface Igs, are known to have biphasic effects on B cells: heavy cross-linking of BCR renders B cell unresponsive, while moderate cross-linking of BCR stimulates B cells (35). Therefore, it is possible that CD100 may attenuate signals from heavily cross-linking of BCR and rescue B cells from apoptosis or anergy, resulting in the modulation of Ab responses against TI-2 Ag. Although it is not statistically as significant, some CD100-Tg mice showed greater IgG3 responses to TI-1 Ag. It may reflect enhancing effects of CD100 on LPS-induced B cell responses.
Role of CD100 in activation of Ag-specific T cells
Ag-specific T cell responses in CD100-deficient mice have been previously shown to be impaired, which was shown to be rescued by in vivo administration of recombinant sCD100 protein (21). In the present study, we demonstrated that CD100-Tg mice showed enhanced Ag-specific T cell responses. Although Ab cross-linking of CD100 has been shown to provide a proliferative signal to human T cells in the presence of submitogenic doses of anti-CD3 or anti-CD2 Abs (7, 10), our results instead suggest that CD100 plays the role of a ligand in T cell activation as well as in B cell activation. Then, the question is whether CD100 acts directly on T cells or on APCs. We have previously shown that defects in CD100 do not show any effect on anti-CD3- or mitogen-stimulated T cells, suggesting an indirect involvement of CD100 in T cell activation (21). CD72, the lymphocyte receptor for CD100, is known to be expressed on some fractions of APCs (26). We also observed that CD40 stimulation significantly induced CD72 expression on APCs, including dendritic cells and macrophages (A. Kumanogoh, C. Watanabe, and H. Kikutani, unpublished observations). In addition, sCD100 significantly enhanced the costimulatory activity of dendritic cells (A. Kumanogoh, C. Watanabe, and H. Kikutani, unpublished observations), suggesting the involvement of CD100 in the priming of Ag-specific T cells through modulating the functions of APCs.
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Acknowledgments |
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Footnotes |
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2 C.W. and A.K. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Hitoshi Kikutani, Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail address: kikutani{at}ragtime.biken.osaka-u.ac.jp
4 Abbreviations used in this paper: sCD100, soluble form of CD100; BCR, B cell Ag receptor; KLH, keyhole limpet hemocyanin; NP-CGG, 4-hydroxy-3-nitrophenylacetyl-chiken -globulin conjugates; QPCR, quantitative PCR; TD, T cell dependent; TI, T cell independent; TNP, 2,4,6-trinitrophenyl; SHP-1, Src homology 2 domain-containing tyrosine phosphatase-1.
Received for publication March 12, 2001. Accepted for publication August 17, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, interferons or anti-CD19 antibody is overridden on engaging CD40. Eur. J. Immunol. 21:1917.[Medline]
converting activaty. Eur. J. Immunol. 27:1275.[Medline]
and notch. Cell 90:589.[Medline]
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 Y. Rao, K. Wong, M. Ward, C. Jurgensen, and J. Y. Wu Neuronal migration and molecular conservation with leukocyte chemotaxis Genes & Dev., December 1, 2002; 16(23): 2973 - 2984. [Full Text] [PDF]
|
|
|
|
|
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A. Kumanogoh, K. Suzuki, E. Ch'ng, C. Watanabe, S. Marukawa, N. Takegahara, I. Ishida, T. Sato, S. Habu, K. Yoshida, et al. Requirement for the Lymphocyte Semaphorin, CD100, in the Induction of Antigen-Specific T Cells and the Maturation of Dendritic Cells J. Immunol., August 1, 2002; 169(3): 1175 - 1181. [Abstract] [Full Text] [PDF]
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 G. Bismuth and L. Boumsell Controlling the Immune System Through Semaphorins Sci. Signal., April 16, 2002; 2002(128): re4 - re4. [Abstract] [Full Text] [PDF]
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), the promoter of the Ig heavy chain VH4C8 (
), the coding
sequence of the truncated mouse CD100 cDNA including the
extracellular region (EC; 
), the transmembrane region (TM; 
), and
the rabbit 
). B,
Real-time monitoring QPCR analysis for expression of the transgene.
Total RNA was isolated from splenic T and B cells of wild-type (
) or CD100-Tg (•) mice and cultured
with or without CD40 mAb (0, 0.1, 0.4, and 1 µg/ml) and IL-4 (0, 2,
and 20 U/ml) for 24 or 72 h. Cells were pulsed with 2 µCi of
[3H]thymidine for the last 16 h. B,
Enhanced proliferative B cell responses to LPS in CD100-Tg mice. Small
resting B cells were purified from wild-type (
; n = 5) mice were
immunized i.p. with 100 µg of NP-CGG as an alum-precipitated complex
on days 0 and 21 (arrows) and bled at the indicated times. Levels of
anti-NP Abs were determined using ELISA. For detection of
NP-specific IgG1, Abs were quantified by means of plates coated with
NP12-conjugated BSA. The results shown are representative
of two independent experiments. *, p < 0.05.
Statistical analysis was performed using two-way repeated measures
ANOVA.