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*
Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan;
CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation, Tokyo, Japan;
Department of Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan; and
§
Department of Joint Disease and Rheumatism, Nippon Medical School, Tokyo, 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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We previously demonstrated that CD27, which is a member of the TNF receptor superfamily and is constitutively expressed on most T cells, can transmit a CD28-independent costimulatory signal upon interaction with its ligand CD70 expressed on a mouse B lymphoma cell line (9). In addition, we and others have shown that OX40 ligand (OX40L),3 which is another member of the TNF superfamily, can provide a costimulatory signal to T cells, resulting in increased proliferation and cytokine production (10, 11, 12). OX40 was originally identified as a cell surface Ag on activated CD4+ T cells in rats (13). cDNA cloning of rat, mouse, and human OX40 showed that it belongs to the TNF receptor superfamily (14, 15, 16). It has been reported that expression of OX40 is restricted to activated T cells in rats, humans, and mice (13, 15, 17). On the other hand, expression of OX40L has been found on mouse activated B cells (15, 18), human dendritic cells (DC) (19), human vascular endothelial cells (20), and human and rat HTLV-1-transformed T cells (10, 21). Recent studies demonstrated that ligation of OX40L on human DC enhanced their maturation and production of cytokines (19) and that blockade of OX40L during naive T-DC interaction suppressed the development of IL-4-producing T cells (22), suggesting that OX40-OX40L may play an important role in T-DC interaction. In addition, OX40L expressed on activated B cells has been shown to transmit a signal that enhances the proliferation of and Ig secretion by B cells (18). This suggested that the cross-linking of OX40L on B cells by OX40 on T cells might deliver a B cell differentiation signal, and thus, the OX40-OX40L system might play a unique role in T-B interaction. To support this idea, it has been reported that administration of an anti-OX40 Ab inhibited the T cell-dependent humoral immune response in mice, possibly by interrupting the OX40-OX40L interaction (23).
To further characterize the role of OX40/OX40L in T-B cell interaction, we established a functional blocking mAb against mouse OX40L, which will be useful for interrupting the OX40-OX40L interaction in vivo. Our present study formally defined the expression of OX40 and OX40L on activated T and B cells. We found that the expression of OX40 on activated T cells is CD28 independent, and the CD28-independent costimulatory activity of surface Ig- and CD40-activated B cells is coordinately mediated by OX40L and CD70. The physiological relevance of these findings is discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to seven-week-old male DBA/2 and C57BL/6 mice and 6-wk-old female SD rats were purchased from Charles River Japan (Atsugi, Japan). Six- to seven-week-old C57BL/6 CD28-deficient (-/-) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Murine mastcytoma P815, murine T lymphoma L5178Y, murine B lymphomas A20.2J and 2PK-3, and murine myeloma P3U1 (P3 x 63Ag8U.1) were purchased from American Type Culture Collection (Manassas, VA). The hybridomas producing mAbs against MHC class II (M5/114), heat stable Ag (J11d), B220 (RA3-3A1), CD8 (3.155), and Thy1.2 (J1j) were also obtained from American Type Culture Collection. The hybridoma OX86 producing anti-mouse OX40 mAb (24) was obtained from European Collection of Cell Cultures (Wiltshire, U.K.). The hybridoma producing mAb against CD4 (RL172) was provided by Dr. T. Tanaka (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). A murine B lymphoma cell line BCL1-B20 (25) was provided by Dr. K. Takatsu (Institute of Medical Science, University of Tokyo, Tokyo, Japan). A murine T lymphoma cell line MBL2 (26) was provided by Dr. T. Nishimura (Tokai University, Kanagawa, Japan). These cells were cultured in RPMI 1640 medium containing 10% FCS, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mg/ml penicillin and streptomycin, and 50 µM 2-ME. A normal rat kidney cell line, NRK-52E (27), was provided by Dr. T. Otsuka (Institute of Cytosignal Research, Tokyo, Japan) and was cultured in DMEM containing 10% FCS, 10 mM HEPES, 4 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mg/ml penicillin and streptomycin, and 50 µM 2-ME.
Antibodies
PE-conjugated goat anti-human and anti-rat IgG Abs were purchased from Caltag (South San Francisco, CA). Purified anti-CD16/32 (2.4G2), CD3 (145-2C11) mAbs, biotinylated anti-CD80 (16-10A1), CD86 (GL1), CD69 (H1.2F3) mAbs, FITC-conjugated anti-CD3 (145-2C11), CD4 (RM4-4), CD8 (53-6.7), B220 (RA3-6B2), CD11b (M1/70) mAbs, PE-conjugated anti-CD25 (3C7), control rat IgG2b, and PE-labeled streptavidin were purchased from PharMingen (San Diego, CA). Anti-CD40 (HM40–3), anti-CD80 (RM80), anti-CD86 (PO3), and anti-CD70 (FR70) mAbs were prepared as described previously (9, 28, 29). Anti-CD28 mAb (PV-1) (30) was provided by Dr. R. Abe (Science University of Tokyo, Chiba, Japan). Goat anti-mouse IgM F(ab')2 Ab was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). A fusion protein consisting of the extracellular portion of murine OX40 and the Fc portion of human IgG1 (OX40-Ig) was prepared as described previously (10).
Preparation of mouse OX40L transfectants
A cDNA fragment encoding the entire open reading frame of mouse OX40L was prepared by RT-PCR from BCL1-B20 mRNA. 5'-GACCTCGAGATGGAAGGGGAAGGGGTTCAA-3' corresponding to nucleotides 123–142 of murine OX40L cDNA (11) tagged with an XhoI site and 5'-GACGCGGCCGCGTCCACCCTCAGAGTGGAA-3' corresponding to nucleotides 721–739 tagged with a NotI site were used as 5' and 3' primers, respectively. The PCR product was cloned into pMKITneo vector, provided by Dr. K. Maruyama (Tokyo Medical and Dental University, Tokyo, Japan), and transfected into L5178Y, NRK-52E, and P815 cells by electroporation. Mock-transfected cells were also produced by electroporation of the pMKITneo vector without an insert. After selection by 1 mg/ml of geneticin, transfectants stably expressing OX40L were identified by staining with OX40-Ig.
Generation of anti-mouse OX40L mAb
An SD rat was immunized with OX40L-transfected NRK cells three
times at 7-day intervals. Three days after final immunization, the
splenocytes were fused with P3U1 cells as previously described (31).
After hypoxanthine-aminopterin-thymidine selection, one hybridoma
producing mAb RM134L (rat IgG2b, 
) was identified by its strong
reactivity with OX40L-transfected L5178Y cells, but not with
mock-transfected L5178Y cells, and was cloned by limiting dilution.
RM134L was purified from ascites by standard procedures with caprylic
acid, and purity was verified by SDS-PAGE analysis.
Preparation and culture of splenic T cells and B cells
Splenic T and B cells were purified as previously described (9). Briefly, splenic T cells were purified by passage through nylon wool column (Wako, Osaka, Japan) and treatment with a mixture of hybridoma supernatants (anti-MHC class II, anti-heat stable Ag, and anti-B220) and low-tox rabbit complement (Cedarlane, Hornby, Canada). For preparing small resting B cells, splenocytes were treated with a mixture of hybridoma supernatants (anti-Thy-1.2, anti-CD4, and anti-CD8) and low-tox rabbit complement. After Percoll (Pharmacia, Piscataway, NJ) gradient centrifugation, small B cells were collected from the 60/70% interface. The purity of the T and B cell population was >95% CD3+ and >95% B220+, respectively, as determined by flow cytometry. In some experiments purified T cells (3 x 106/ml) were stimulated with immobilized anti-CD3 mAb (10 µg/ml) with or without soluble anti-CD28 mAb (5 µg/ml) for 24–120 h. Purified B cells (3 x 106/ml) were stimulated with anti-IgM Ab (10 µg/ml) and/or anti-CD40 mAb (5 µg/ml) for 24–96 h.
Flow cytometric analysis
Cells (5 x 105) were first preincubated with
anti-CD16/32 mAb to avoid nonspecific binding of Abs to Fc
R and
then incubated with a saturating amount of OX40-Ig, FITC- or PE-labeled
mAb, or biotinylated mAb. After washing with PBS twice, the cells were
incubated with PE-conjugated goat anti-human IgG Ab for OX40-Ig or
PE-labeled streptavidin for biotinylated mAb. After washing with PBS
twice, the stained cells (live-gated on the basis of forward and side
scatter profiles and propidium iodide exclusion) were analyzed on a
FACScan (Becton Dickinson, San Jose, CA), and data were processed using
the CellQuest program (Becton Dickinson).
T cell proliferation and IL-2 assay
Purified splenic T cells (1 x 105/well) were cocultured with irradiated (100 Gy) transfectants (2 x 104/well) or irradiated (20 Gy) preactivated B cells (5 x 104/well) in the presence or the absence of anti-CD3 mAb (2C11, 1 µg/ml) in 96-well round-bottomed culture plates at 37°C for 48 h. For estimating proliferative responses, the cultures were pulsed with 0.5 µCi/well of [3H]thymidine (DuPont-New England Nuclear, Boston, MA) for the last 9 h and then harvested using a Micro 96 Harvester (Skatron, Lier, Norway). Incorporated radioactivity was measured in a micro beta counter (Micro ß Plus, Wallac, Turku, Finland). To determine IL-2 production, cell-free supernatants were collected at 48 h and subjected to ELISA using OptEIA Mouse IL-2 Set (PharMingen) according to the manufacturer’s instruction.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To isolate murine OX40L cDNA, we first tested the reactivities of
various mouse cell lines with OX40-Ig. A B lymphoma cell line,
BCL1-B20, exhibited high reactivity with OX40-Ig (data not shown).
Then, OX40L cDNA was cloned into a mammalian expression vector from RNA
of BCL1-B20 cells by RT-PCR and transfected into NRK-52E, L5178Y, and
P815 cells. Stable expression of OX40L on the cDNA transfectants
(OX40L/NRK, OX40L/L5178Y, and OX40L/P815), but not on mock-transfected
cells (mock/NRK, mock/L5178Y, and mock/P815), was verified by binding
of OX40-Ig (data not shown). We then immunized an SD rat with OX40L/NRK
cells and fused the splenocytes with P3U1 myeloma cells. The hybridomas
producing anti-OX40L mAb were screened by specific reactivity to
OX40L/L5178Y cells. As represented in Fig. 1
A, one mAb, designated
RM134L, was obtained, which bound to OX40L/L5178Y and OX40L/NRK cells
but not to mock/L5178Y or mock/NRK cells. A number of mouse cell lines
were also tested for OX40L expression by staining with RM134L and
Northern blot analysis. BCL1-B20 and T lymphoma cell line MBL2, which
expressed OX40L mRNA as estimated by Northern blot analysis (not
shown), were reactive with RM134L (Fig. 1A), but the other T
(WR19L, YAC-1, and BW5147) or B (A20.2J, 2PK-3, CH1, and WEHI231)
lymphoma cell lines, which did not express OX40L mRNA, were not
reactive with RM134L (Fig. 1A and data not shown).
Macrophage cell lines (J774.1 and P388D1) also did not express OX40L
mRNA or react with RM134L (not shown). These results indicated a strict
coincidence of OX40L mRNA expression and RM134L reactivity. Moreover,
preincubation with RM134L blocked OX40-Ig binding to OX40L/L5178Y cells
(Fig. 1B), further substantiating that RM134L is specific
for mouse OX40L.
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It has been shown that OX40L provided a costimulatory signal for T
cell proliferation in mice, humans, and rats (10, 11, 12). To examine
whether RM134L can block the costimulatory activity of mouse OX40L,
splenic T cells were cocultured with OX40L/P815 or mock/P815 cells in
the presence of anti-CD3 mAb and RM134L, and the proliferative
responses were assessed. As shown in Fig. 2A, while OX40L/P815 cells
expressed a high level of mouse OX40L, mock/P815 cells also expressed
OX40L at a low level, which was verified by Northern blot analysis (not
shown). As shown in Fig. 2B, OX40L/P815 cells costimulated
the proliferation of anti-CD3-stimulated T cells far more
efficiently than mock/P815 cells. The peak proliferative response on
day 2 was blocked by RM134L, but not by control IgG, in a
dose-dependent manner (Fig. 2C). In addition, the
proliferative response costimulated by mock/P815 cells was abrogated by
RM134L (data not shown), indicating a substantial contribution of
endogenously expressed OX40L to the costimulatory activity of mock/P815
cells. These results indicated that mouse OX40L exhibits a potent
costimulatory activity for anti-CD3-stimulated T cell
proliferation, which can be blocked by RM134L.
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We next examined the expression of OX40L and OX40 on splenic T
cells by flow cytometric analysis using RM134L and an anti-mouse
OX40 mAb (MRC OX86) (24). An anti-CD25 mAb was also included as an
activation marker. Purified splenic T cells were stimulated for 24–120
h with immobilized anti-CD3 mAb in the presence or the absence of
soluble anti-CD28 mAb. As shown in Fig. 3A, OX40 expression was
detected on T cells after stimulation with anti-CD3 mAb alone,
which appeared at 24 h and reached a peak at 72 h. The
addition of anti-CD28 mAb significantly increased and sustained the
expression of OX40 on anti-CD3-stimulated T cells (Fig. 3B). In contrast, OX40L was not found on T cells even when
stimulated with both anti-CD3 and anti-CD28 mAbs (Fig. 3B). In the mouse system, it has been reported that OX40 was
expressed on both activated CD4+ and CD8+ T
cells (24), while OX40 was expressed predominantly on CD4+
T cells in the rat system (13). As shown in Fig. 3C, we
found mouse OX40 expression on both CD4+ and
CD8+ T cells after stimulation with anti-CD3 mAb.
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D, a comparable level of OX40 expression was found on
CD28- T cells to that on wild-type T cells. Collectively,
these results indicated that mouse OX40 is expressed on
TCR/CD3-stimulated CD4+ and CD8+ T cells in a
CD28-independent manner and that OX40L is not expressed on normal mouse
T cells. Expression of OX40L and OX40 on activated B cells
We next examined the expression of OX40L and OX40 on splenic B
cells. Purified splenic B cells were stimulated with anti-IgM Ab,
anti-CD40 mAb, or both for 24–96 h and stained with RM134L or
anti-OX40 mAb. An anti-CD69 mAb was included as an activation
marker. As shown in Fig. 4C,
OX40L expression was detected on B cells after stimulation with the
combination of anti-IgM Ab and anti-CD40 mAb, which appeared at
24 h and reached a peak at 72 h. In contrast, stimulation
with either anti-IgM Ab or anti-CD40 mAb alone did not induce
OX40L expression (Fig. 4, A and B). Unexpectedly,
marginal expression of OX40 was reproducibly found on
anti-IgM-stimulated B cells, which was apparent at 48–72 h (Fig. 4A). The combination with anti-CD40 mAb markedly
enhanced OX40 expression (Fig. 4C), whereas stimulation with
anti-CD40 mAb alone did not induce OX40 expression (Fig. 4B). These results indicated that OX40L is specifically
expressed on surface Ig- and CD40-stimulated B cell and that either
stimulation alone was not effective. Moreover, a novel expression of
OX40 on B cells was revealed.
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The above observations of a potent T cell costimulatory activity
of OX40L and the expression of OX40L on anti-IgM- plus
anti-CD40-stimulated B cells suggested that OX40L might play a
substantial role in T cell costimulation by activated B cells. It has
been shown that surface Ig- and/or CD40-stimulated B cells expressed
the CD28 ligands CD80 and CD86 (32, 33, 34, 35). We also previously
demonstrated that anti-IgM- plus anti-CD40-stimulated B cells
expressed CD70, which is another member of the TNF superfamily and
provides a costimulatory signal via CD27 constitutively expressed on
most T cells (9). Consistent with these observations, the anti-IgM
plus anti-CD40-stimulated B cells expressed CD80, CD86, CD70, and
OX40L at high levels (Fig. 5A). We then examined the
relative contributions of CD80/86, CD70, and OX40L to T cell
costimulation by anti-IgM- plus anti-CD40-stimulated B cells.
Purified splenic T cells were cocultured with preactivated B cells in
the presence of anti-CD3 mAb and combinations of anti-CD80/86,
anti-CD70, and/or anti-OX40L mAb for 48 h, and then the
proliferative response and IL-2 production were assessed. As shown in
Fig. 5, B and C, the anti-IgM- plus
anti-CD40-stimulated B cells exhibited a potent costimulatory
activity for anti-CD3-stimulated T cell proliferation and IL-2
production. Anti-CD80/86 mAbs only partially inhibited T cell
proliferation, and further inhibition was obtained by their combination
with either anti-OX40L or anti-CD70 mAb
(p < 0.05; Fig. 5B). The strongest
inhibition was obtained by the combination of all these mAbs. While
anti-OX40L or anti-CD70 mAb alone only modestly inhibited T
cell proliferation, the combination of these two mAbs exhibited a
significant inhibitory effect (p < 0.01)
comparable to that of anti-CD80/86 mAbs (Fig. 5B). On
the other hand, IL-2 production was strongly inhibited by
anti-CD80/86 mAbs, and further inhibition was observed by their
combination with anti-OX40L and/or anti-CD70 mAb (Fig. 5C). Anti-OX40L or anti-CD70 mAb alone significantly
inhibited IL-2 production (p < 0.01), and
further inhibition was observed by the mixture of these two mAbs (Fig. 5C). These results indicated the substantial contributions
of OX40L and CD70 to the costimulatory activity of anti-IgM- plus
anti-CD40-stimulated B cells, which can mostly account for the
CD80/86-independent part.
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The above experiment implied that the OX40L and CD70 expressed on
activated B cells could costimulate T cells independently of the
CD80/86 and CD28 interaction. To further address this idea, we examined
the contributions of OX40L and CD70 to costimulation of
CD28- T cells from CD28-deficient mice by preactivated B
cells. As shown in Fig. 6A,
the anti-IgM- plus anti-CD40-stimulated B cells from
CD28-deficient mice expressed CD80, CD86, CD70, and OX40L at levels
comparable to those in B cells from wild-type C57BL/6 mice (Fig. 5A). As shown in Fig. 6, B and C, when
costimulated by these preactivated B cells, the CD28- T
cells exhibited 50–60% reduced proliferation and 60–70% reduced
IL-2 production compared with wild-type T cells, which were almost
comparable to those of wild-type T cells in the presence of
anti-CD80/86 mAbs (Fig. 5, B and C). As
expected, the addition of anti-CD80/86 mAbs showed no inhibitory
effect. In contrast, both T cell proliferation (Fig. 6B) and
IL-2 production (Fig. 6C) were significantly inhibited by
either anti-OX40L or anti-CD70 mAb alone. Notably, T cell
proliferation and IL-2 production were mostly inhibited by the mixture
of these two mAbs (75.8 and 70.6% inhibition, respectively). These
results indicated that the CD28-independent T cell costimulatory
activity of surface Ig- and CD40-stimulated B cells are mostly mediated
by OX40L and CD70, although a minor contribution of some other molecule
was also suggested.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OX40 has been originally identified as an activation marker
preferentially expressed on CD4+ T cells in the rat system
(13). In contrast, a recent study demonstrated that OX40 was expressed
on both CD4+ and CD8+ T cells after Con A
stimulation of mouse splenocytes, suggesting a differential expression
of OX40 on activated rat and mouse T cells (24). We confirmed this in
the present study and observed that both CD4+ and
CD8+ T cells expressed OX40 after anti-CD3 stimulation
in the mouse system (Fig. 3C). Although the expression of
OX40 on human T cells has not been well characterized, our preliminary
results showed that OX40 is expressed on both CD4+ and
CD8+ T cells after anti-CD3 stimulation of human
peripheral blood lymphocytes (unpublished observations). Therefore, the
rather restricted expression of OX40 on CD4+ T cells
appears to be a unique feature of the rat system. We also examined the
stimulatory requirement for OX40 expression on T cells, which has not
been determined in previous studies. We found that OX40 can be
expressed on anti-CD3-stimulated T cells without costimulation with
anti-CD28 mAb (Fig. 3A) and also on CD28- T
cells from CD28-deficient mice (Fig. 3D). This independence
of OX40 expression from CD28 costimulation underlies the
CD28-independent costimulatory activity of OX40L as represented in Fig. 6.
A previous report (11) showed that OX40L was expressed on a murine T
lymphoma cell line and anti-TCR-stimulated murine splenic T cells
as estimated by OX40-Ig binding, whereas staining of the latter was
marginal. We found a high expression of OX40L in a Moloney
virus-induced T lymphoma cell line MBL2 (Fig. 1A), but not
in the other T cell lines tested or in anti-CD3- or
anti-CD3/CD28-stimulated splenic T cells as estimated by staining
with anti-OX40L mAb. Consistently, we could detect OX40L mRNA in
MBL2, but not in anti-CD3- or anti-CD3/CD28-stimulated splenic
T cells, by Northern blot analysis (data not shown). Therefore, OX40L
appears not to be expressed on splenic T cells under these conditions,
although it remains to be determined whether OX40L can be expressed on
T cells under a particular condition. In this respect, it is noteworthy
that OX40L is frequently expressed on HTLV-1-transformed human and rat
T cell lines, possibly due to trans-activation by Tax
protein encoded by the pX region of HTLV-1 (10, 21, 36).
HTLV-1 is not only the etiological agent of adult T cell leukemia but
also causes HTLV-1-associated diseases, such as myelopathy, uveitis,
arthropathy, and Sjögren’s syndrome (37). It has been shown that
the HTLV-1 pX transgenic mice developed inflammatory
arthropathy, neurofibronatosis, and exocinopathy (38, 39, 40, 41). The
anti-mouse OX40L mAb we established in the present study will be
useful to address the pathological function of OX40L in these mouse
models.
It has been reported that OX40L, as estimated by OX40-Ig binding, was
expressed on mouse splenic B cells upon stimulation with anti-IgD
dextran or CD40 ligand (CD40L)-expressing L cells, and the highest
expression levels were obtained when stimulated by both (18). In
contrast, we could not detect significant levels of OX40L expression on
anti-IgM Ab- or anti-CD40 mAb-stimulated B cells (Fig. 4, A and B). This discrepancy may be due to higher
cross-linking of surface Ig by anti-IgD dextran than by
anti-IgM Ab or a signaling difference between surface IgD and IgM
and higher cross-linking of CD40 by CD40L-expressing L cells than
anti-CD40 mAb. In our present results, however, a high expression
of OX40L was obtained by the stimulation with anti-IgM Ab plus
anti-CD40 mAb, consistent with the previous observation that the
highest OX40L expression was obtained after stimulation by anti-IgD
dextran and CD40L. Therefore, it is likely that OX40L is preferentially
expressed on surface Ig-stimulated, Ag-specific B cells upon
interaction with CD40L-expressing Th cells, especially when surface Ig
cross-linking by Ags and CD40 cross-linking by CD40L are limiting.
Stüber et al. also demonstrated that cross-linking of OX40L on
activated B cells by OX40-Ig resulted in an enhanced proliferation and
an increase in Ig secretion in vitro (18), and that administration of
anti-OX40 Ab profoundly inhibited the anti-hapten IgG response
in vivo and the development of periarteriolar lymphoid
sheath-associated B cell foci probably by interrupting the interaction
of OX40 on activated T cells with OX40L on activated B cells (23),
suggesting a critical role for the OX40-OX40L interaction in
differentiation of activated B cells into Ig-producing plasma cells. In
this respect, our novel finding of concomitant expression of OX40 and
OX40L on activated B cells is intriguing, since it suggests that the
OX40/OX40L-mediated proliferation and differentiation could be driven
by B-B cell interactions in the B cell foci. Further studies are now
under way to characterize the function of OX40 expressed on activated B
cells.
It has been shown that ligation of OX40 by anti-OX40 mAbs or OX40L
provided a costimulatory signal for T cell proliferation (10, 11, 12). We
also observed a potent costimulatory activity of mouse OX40L for
proliferation of anti-CD3-stimulated naive T cells (Fig. 2). We
then examined the costimulatory function of OX40L expressed on
activated B cells. The anti-IgM plus anti-CD40-stimulated B
cells expressed multiple costimulatory molecules, including CD80, CD86,
CD70, and OX40L (Fig. 5A). Anti-CD80/86 mAbs only partially
inhibited the costimulatory activity of activated B cells, and the
CD80/86-independent part was mostly inhibited by the combination of
anti-OX40L and anti-CD70 mAbs (Fig. 5B), indicating
substantial contributions of OX40L and CD70. The residual part may be
mediated by other costimulatory molecules, such as CD44H (42) and CD54
(43), which have also been implicated in the costimulatory activity of
CD40L-activated B cells. Although it is possible that the
anti-OX40L and anti-CD70 mAbs might act in an inhibitory manner
by negatively signaling T cells, this is unlikely, since neither mAb
inhibited anti-CD3- and anti-CD28-stimulated T cell
proliferation (data not shown). The additional experiment with
CD28- T cells from CD28-deficient mice (Fig. 6) clearly
indicated that OX40L and CD70 expressed on activated B cells can
costimulate T cells in a CD28-independent manner. It has been reported
that CD4+ T cells from CD28-deficient mice can express
CD40L upon anti-CD3 stimulation (44), which is a prerequisite for
the expression of OX40L and CD70 on B cells. We also showed that OX40
can be expressed on CD28- T cells upon anti-CD3
stimulation (Fig. 3). Taken together, these results suggest that OX40L
and CD70 may mostly account for the CD28-independent pathway of T cell
costimulation during Ag-specific T-B cognate interactions. It has been
shown that CD28-deficient mice can be primed with soluble Ags in vivo
so as to generate Ag-specific T cell proliferative responses in vitro
(6, 7). CD40/CD40L have been implicated as the predominant mediator of
the CD28-independent pathway of T cell costimulation (45, 46, 47). Although
some reports suggested transmission of a costimulatory signal via CD40L
into T cells (48, 49), it is generally accepted that CD40L acts
indirectly by up-regulating the expression of costimulatory molecules
on APC. Our present results suggest that OX40L and CD70 can at least
partly account for the CD40/CD40L-dependent pathway of T cell
costimulation. We are now addressing this possibility by administrating
anti-OX40L and/or anti-CD70 mAb into CD28-deficient or
CD40L-deficient mice immunized with various Ags.
Our present study revealed substantial contributions of OX40L and CD70 to CD28-independent T cell costimulation. CD28- T cells are not unique to CD28-deficient mice. CD28 is expressed on most CD4+ T cells, but on only half of CD8+ T cells (50, 51). It has been suggested that CD8+ CD28- T cells might represent suppressor T cells in humans (52, 53). It has also been reported that CD4+ CD28- T cells accumulated in PBL from SLE patients, which might be relevant to autoantibody production in these patients (51). At present, costimulatory molecules mediating activation of these CD28- T cells have not been well characterized. Our present results suggest that OX40L and CD70 may be responsible for the costimulation of these CD28- T cells.
In the present study we demonstrated the T cell costimulatory function of OX40L expressed on activated B cells. It has been reported that OX40L was also expressed on human DC and endothelial cells. We observed that murine DC and endothelial cell lines express OX40L, which costimulated CD28- T cells (unpublished data). OX40/OX40L have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis in rats and in multiple sclerosis, rheumatoid arthritis, and acute graft-vs-host disease in humans (54, 55). The anti-OX40L mAb we established in the present study will be useful to further explore the physiological and pathological functions of OX40L in the mouse model systems.
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Acknowledgments |
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2 Address correspondence and reprint requests to Dr. Ko Okumura, Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail address:
3 Abbreviations used in this paper: OX40L, OX40 ligand; DC, dendritic cells; HTLV-1, human T cell leukemia virus type I; CD40L, CD40 ligand.
Received for publication February 9, 1999. Accepted for publication March 31, 1999.
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production. Eur. J. Immunol. 26:1621.[Medline]
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