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Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, and
Core Research for Evolutional Science and Technology Program of the Japan Science and Technology Corporation, Sendai, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-2, and
IL-6 production in vitro. Adoptive transfer experiments however
revealed an efficient transfer of disease to OX40L-/-
mice using wild-type donor T cells, indicating an intact capacity of
OX40L-/- mice to initiate effector responses. On the
other hand, OX40L-/- donor T cells failed to transfer
disease to wild-type recipient mice. Furthermore, OX40L-Tg mice
developed a greater severity of EAE despite a delayed onset, while both
OX40L-Tg/CD28-/- and OX40L-Tg/CD40-/- mice
failed to develop EAE demonstrating a requisite for these molecules.
These findings indicate a pivotal role played by OX40L in the
pathogenesis of EAE. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, OX40L and OX40 have been detected in the tissues of several inflammatory disorders such as EAE, semiallogeneic graft-versus-host disease, and proliferative lupus nephritis (16, 17, 18, 19, 20). The multitude of functions attributable to OX40L is expanding. In an effort to reveal the biological function of OX40-OX40L interactions, we proceeded further by generating mice deficient in OX40L. These mice revealed substantial new information demonstrating a marked failure in APC function among dendritic cells when antigenically challenged (10). In addition, we and others have shown that the absence of OX40L signals failed to support potent CD4+ T cell responses, resulting in a reduced Th1 and Th2 cytokine profile in mice (10, 12, 21, 22). Beside providing potent costimulation, OX40-OX40L is suggested to be involved in redirecting a Th2-biased response (23, 24, 25, 26), maintenance, and survival of memory T cell populations (27). Amid much debate, the involvement of OX40-OX40L in altering Ab responses (10, 12, 21, 22, 28) and T cell migration (16, 29, 30, 31) has been suggested. Recently, in concert with CD28, OX40 has been further proposed to synergistically potentiate immune responses and participate in T cell migration to B cell areas (17, 32). Furthermore, antitumor immune responses have been attributable in part to OX40-OX40L signals (33, 34). Collectively, these data provide a potential key role for OX40-OX40L interaction in the immune regulation of various autoimmune diseases.
Experimental autoimmune encephalomyelitis (EAE), reminiscent of human multiple sclerosis, can be induced in C57BL/6 mice with either myelin oligodendrocyte glycoprotein peptide (MOG), a highly immunogenic myelin component, or adoptive transfer of TCR+ cells, specific for encephalitogenic antigens. Although recent studies blocking this interaction using anti-OX40L mAbs or an OX40-fusion protein have been shown to ameliorate not only EAE, but graft-versus-host disease, inflammatory bowel disease, and asthma in murine models (16, 17, 23, 29, 35), the molecular mechanisms by which OX40L exerts its influence in autoimmune disorders have remained unclear.
Using OX40L-deficient mice and mice constitutively expressing OX40L, we attempted to follow the immunological events during EAE induced by MOG and to provide an understanding of the underlying contributory mechanisms of the OX40L-OX40 interaction in autoimmune pathogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The C57BL/6 mice, homozygous for OX40L mutation previously reported (10), had been backcrossed between 9 and 11 generations and typed by PCR using ear biopsy-derived DNA. The OX40L-transgenic mice were constructed on the C57BL/6 background by using an lck promoter and constitutively expressed OX40L on T cells (K. Murata, N. Ishii, M. Nose, M. Yamada, L. C. Ndhlovu, and K. Sugamura, unpublished data). CD28-/- mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and CD40-/- mice were a kind gift from Dr. Kikutani (Osaka University, Osaka, Japan). All mice were 6–8 wk old at the start of experiments and wild-type littermates were used as controls.
Peptide and Abs
MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) peptide was provided by Sawady Technology (Tokyo, Japan) and had been synthesized by F-moc solid-phase methods to a purity of 95–100% through HPLC. Anti-mouse OX40L mAb, which blocks the OX40-OX40L interaction (10) and control rat IgG (Cappel, Durham, NC) were used during the experiments.
Induction and evaluation protocol for EAE
Active induction. A single s.c. injection of 100 µg of MOG35–55 peptide dissolved in PBS, emulsified in an equal volume of CFA (Difco, Detroit, MI) containing 4 mg/ml Mycobacterium tuberculosis H37Ra (Difco), was administered on both flanks followed by an i.v. injection of 500 ng pertussis toxin (List Biological Laboratories, Campbell, CA) dissolved in 200 µl sterile PBS administered on the day of immunization and 48 h later.
Passive induction. Wild-type mice were immunized with 100 µg MOG35–55 in CFA (Difco). Fifteen to 20 days later, draining lymph nodes were harvested, and MOG-specific lymphocytes were cultured with 10 µg/ml MOG35–55 in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 1x nonessential amino acids, 50 µM 2-ME, and heat-inactivated 10% FCS. Human rIL-2 (15 pg/ml; Ajinomoto, Kanagawa, Japan) was added on day 2. After 4 days of culture, cells were washed and resuspended in PBS and adjusted to the required concentration for transfer. Wild-type or OX40L-/- recipients received 3 x 107 cells i.v. in a 0.2-ml suspension via the tail vein, as well as 200 ng of pertussis toxin (List Biological Laboratories) immediately after cell transfer and 2 days later.
Clinical evaluation. In both groups, mice were examined daily for signs of EAE and scored as follows (36): 0, no disease; 1, tail paralysis; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limb plus forelimb paralysis; or 5, moribund and dead.
Proliferative response of T cells to MOG Ags
Mice were immunized s.c. with 100 µg of MOG35–55 emulsified 1:1 in CFA (Difco). Draining lymph nodes were harvested at the indicated days, and single-cell suspensions were prepared. Whole draining (axillary and inguinal regions) lymph node cells were cultured in 96-well plates at 5 x 105 cells/well with a range of concentrations of MOG35–55 peptide in RPMI 1640 medium supplemented with 10% FCS. Plates were pulsed with [3H]thymidine (New England Nuclear, Boston, MA) at 1 µCi/well on day 3 of culture for the final 18 h. Mean incorporation of thymidine in DNA was measured in triplicate wells by a 1450 Microbeta Plus liquid scintillation counter (Wallac, Turku, Finland). Thymidine incorporation was used as a measure of cell proliferation and DNA synthesis.
Preparation and evaluation of CNS cells
Mononuclear cells were isolated from murine brains as
previously described (37). Briefly, brains were removed
from donor animals, and a single-cell suspension was prepared by
passage through a wire mesh. Cells were washed in RPMI 1640 medium and
mononuclear cells were isolated using a discontinuous Percoll gradient
(Pharmacia, Piscataway, NJ). The cells were washed three times and the
CD11b+ and CD4+/CD8+ cell fractions
were isolated from the CNS by magnetic separation. Initially, the cells
were magnetically labeled with either MACS CD11b+ or MACS
CD4+ and CD8+ Microbeads (Miltenyi Biotec,
Gladbach, Germany) and incubated at 4°C for 20 min, washed, and
passed through a nylon mesh. The different cell subsets were then
purified by using an autoMACS (Miltenyi Biotec). Magnetic separation
with positive selection columns was performed twice. Analysis using a
FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA)
determined the cells to be >90% pure for microglia/macropages and T
cells using CD11b+-FITC/CD45+-PE and
TCR
+-FITC mAbs (BD PharMingen, San Diego, CA),
respectively. After blocking the cells with the relevant ascitic fluid,
cells were incubated directly with the following conjugated mAb in FACS
buffer (1% BSA, 01% sodium azide in PBS); CD11b+ FITC (BD
PharMingen), Bio-MGP34 (10), streptavidin-allophycocyanin
and control human CD8+-FITC (both from BD Biosciences); and
Bio-CD80+, Bio-CD86+, and CD45+-PE
(all obtained from Beckman Coulter (Fullerton, CA)).
RT-PCR protocol
CD11b+ brain cells were extracted from OX40L+/+ and OX40L-/- at the onset and peak of disease as described above and total RNA was extracted using TRizol reagent (LTI; Life Technologies). First-strand cDNA was prepared using the Superscript RT-PCR system (Life Technologies) from 5 µg of total RNA. The cDNA amount was normalized by measuring the CD86 expression, and OX40L transcripts were analyzed by RT-PCR using specific primer sequences for mouse (m)OX40L and mCD86. Reactions were performed in a programmable thermal controller (PerkinElmer/Cetus, Norwalk, CT) and after amplification, 20 µl of PCR product was separated by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining.
Cytokines and ELISA
Lymph node cells were prepared and cultured as described
above with medium alone or in the presence of 50 µg/ml
MOG35–55 peptide in RPMI 1640 medium supplemented
with 10% FCS. Supernatants were harvested at 48 and 72 h of
culture. The concentrations of IL-2, IL-4, IL-6, IL-10, and IFN-
were measured by quantitative capture ELISA according to the guidelines
of the manufacturers (R&D Systems, Minneapolis, MN).
Histopathological examination
Brains were removed and fixed in 10% Formalin. Six-µm-thick paraffin-embedded sections were stained with Luxol fast blue or H&E for light microscopic examination. Evidence of meningeal and parenchymal inflammatory foci and features of demyelination were examined by an unbiased observer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the role of OX40L-OX40 interactions in the
regulation of MOG-induced EAE, we first compared the severity of
actively induced EAE between OX40L-deficient (OX40L-/-)
mice and wild-type (OX40L+/+) littermates. The
susceptibility in the development of clinical signs of EAE was
significantly reduced in the OX40L-/- mice when compared
to the OX40L+/+ littermates (Fig.
1A and Table I
). Consistently the wild-type
littermates developed more severe EAE signs and the chronic pattern of
disease characteristic of MOG-induced EAE was observed. We also
observed a similar reduction in disability of MOG-induced EAE in mice
treated with our anti-OX40L mAb, MGP-34 (data not shown), an
inhibitory mAb blocking the interaction between OX40L and OX40
(10).
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Altered recall T cell priming responses to MOG in OX40L-deficient mice
We previously demonstrated a defective generation of
Ag-specific T cell responses in OX40L-/- mice
(11). To test whether this defect participated in the
reduced progression of EAE in the OX40L-/- mice, we
restimulated draining lymph node cells from both the
OX40L-/- and OX40L+/+ mice extracted at
several time points after immunization with MOG. A series of
proliferative assays consistently presented a picture of profound
impairment in the T cell responses in the OX40L-/- mice,
with a 3-fold reduction in proliferative response as compared to the
OX40L+/+ mice, both early (day 13; Fig.
2A) and late (day 29; data not
shown) during the disease. Supernatants extracted from the cultures
were analyzed for cytokine release of IFN-, IL-2, IL-4, IL-6, and
IL-10. The cells derived from OX40L-/- mice produced
lower levels of IL-2 and IFN- at day 13 (Fig. 2B) and day
29 (Fig. 2C). Although IL-6 levels were lower at day 13,
comparable levels of IL-6 by day 29 were seen when compared with those
derived from the OX40L+/+ mice (Fig. 2, B and
C). Levels of IL-10 were not significantly different and
IL-4 production was undetectable in the two groups (data not shown),
suggesting a redirection to a Th2 bias unlikely. In summary, the
compiled analysis of the T cell responses in the OX40L-/-
mice suggests impairment in proliferative responses and Th1 cytokine
release.
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In an effort to determine the localization of OX40 and OX40L
expression in the brains of mice during EAE, we initially carried out
flow cytometric analysis of the inflammatory cells in the brains
extracted on day 35 after immunization. OX40L was undetectable
throughout the disease in the Mac1+ fraction of both
OX40L+/+ and OX40L-/- mice (Fig.
3A). RT-PCR was next performed
using RNA extracted from the Mac1+ CNS population to help
determine whether OX40L is indeed expressed in the CNS during EAE. Two
distinct sets of primers each for murine OX40L and CD86 were used. The
amplification products were visible for OX40L mRNA at the onset of
disease in OX40L+/+ mice and not in OX40L-/-
mice (Fig. 3C). Expression of CD86 (Fig. 3, A and
C) and CD80 (data not shown) were comparable between the two
groups at the peak of disease both by flow cytometry and RT-PCR. On
examination of the infiltrating T cells in the brain, only OX40 was
clearly expressed on the T cell population (Fig. 3B) at the
peak of disease in both OX40L+/+ and
OX40L-/- mice.
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We generated a transgenic mouse line, overexpressing OX40L
specifically in T cells under the control of the lck
promoter (Fig. 4A). The mice
developed normally but exhibited a markedly enhanced proliferative and
cytokine responses to protein Ags (K. Murata, N. Ishii, M. Nose, M.
Yamada, L. C. Ndhlovu, and K. Sugamura, unpublished data). We
here induced EAE in the OX40L-transgenic (OX40L-Tg) and their
counterpart wild-type littermates by immunization with MOG. As
predicted, a more severe progression of EAE and greater mortality as
compared to the wild-type mice developed, and, interestingly, the
OX40L-Tg mice developed a delayed onset of symptoms (Fig. 4B
and Table I
). Comparable perivascular infiltrations of mononuclear
cells (data not shown) and extensive demyelination (Fig. 4C)
between the OX40L-Tg mice and the wild-type mice was observed in
histological brain sections. Next, draining lymph node cells derived
from the OX40L-Tg and wild-type mice immunized with MOG in vivo were
then restimulated with MOG in vitro and assayed for proliferation and
cytokine production. The OX40L-Tg lymph node cells hyperproliferated in
response to MOG as compared to control wild-type lymph nodes (Fig.
5A). Significantly elevated
levels of IL-2, IL-6, and IFN- were present in the culture
supernatants of lymph node cells derived from the OX40L-Tg mice as
compared with the wild-type mice (Fig. 5B). No significant
difference in the levels of IL-10 was seen between the two groups, and
as expected IL-4 production was undetectable (data not shown). These
results appeared to be a reversal of the patterns observed in the
OX40L-deficient mice.
Passive transfer of encephalitogenic donor T cells
Given the diminished clinical disease upon active EAE
induction in the OX40L-/- mice, and the observation that
OX40L-Tg mice developed a more severe disease, we raised the question
of how the alteration in OX40L expression influenced Ag-specific
reactivity of the primed encephalitogenic T cells. Hence, we carried
out a combination of adoptive transfer experiments. First,
OX40L+/+ MOG-specific donor T cells were transferred into
OX40L-/- and OX40L+/+ recipient mice. The
incidence of disease was 100% in both groups of mice with an earlier
onset of EAE in the OX40L-/- mice (day 15.4) as compared
to the OX40L+/+ mice (day 18.6; Fig. 6 and Table II). The disease severity in the
OX40L-/- mice (maximum disability score (DAS), 2.8) was
remarkably similar to the wild-type mice (maximum DAS, 2.9;
Table II). However subsequent recovery of disability was noted in the
OX40L-/- mice but not in the OX40L+/+ mice
after adoptive transfer (Fig. 6).
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). On the
other hand, when we transferred OX40L-Tg donor T cells to wild-type
recipient mice, despite a delayed onset of disability, a severe disease
pattern with a higher mortality among the mice was observed (Table II).
These data reinforce the data demonstrating the requirement for OX40L
signals in providing adequate costimulation for Th cell differentiation
and thus enabling them to provide an environment conducive for EAE
progression. Costimulatory molecules, CD28 and CD40, are required for OX40L function during EAE
CD28 and OX40 have been reported to synergistically provide
additional T cell costimulation in vitro (2, 17).
Furthermore, it has been documented that both molecules may also be
involved in the migration of T cells into B cell follicles through the
up-regulation of CXCR5 (11, 32). To further dissect
whether OX40L could compensate for CD28 signals, OX40L-Tg mice were
crossed with CD28-/- mice to eliminate the CD28
costimulation component. These mice were immunized with MOG/CFA and
observed for signs of EAE. When compared to the wild-type littermates
both OX40L-Tg/CD28-/- (incidence, 0/3) and
CD28-/- (incidence, 0/4) mice failed to develop EAE (Fig. 7). CD28-/- mice have been
reported to have a marked failure to develop EAE (36).
Thus, although CD28 is dispensable in some immune responses (38, 39), OX40L function requires initial CD28 costimulation.
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). OX40L-Tg mice consistently presented
with severe disease (mean peak DAS, 4.0 ± 0.0) (Fig. 8). The
findings here further emphasize that effective OX40L function requires
both intact CD28 and CD40 signals during EAE pathogenesis, possibly
through their ability to up-regulate OX40.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effective transmission of EAE to OX40L-/- recipient mice using wild-type encephalitogenic T-cells indicated that these effector T cells were able to induce disease in OX40L-/- mice and function normally. However, early recovery from paralysis in the OX40L-/- recipient mice suggests that these effector T cells are unable to sustain the chronic nature of disease in the absence of continued OX40L signals. This is in line with reports suggesting that OX40-OX40L interactions may function effectively in sustaining the survival of memory T cells (27). On the other hand, the failure to transduce disease to wild-type recipient mice, using encephalitogenic T cells lacking OX40L, suggests that the priming of these T cells was adversely affected and hence their ability to induce disease diminished. Adoptive transfer of OX40L-Tg T cells further reinforced our findings using OX40L-/- mice. The delayed onset pattern seen in the OX40L-Tg mice is still an unresolved phenomenon. The kinetics of OX40 and OX40L expression on T cells and APCs, respectively, may not be responsible as both are expressed within a few days after antigenic challenge (10).
OX40L appears to promote Th1 cytokine production stimulated by
MOG in vitro. A significant reduction in both IFN- and IL-2 of in
vitro-stimulated OX40L-/- T cells was observed. OX40L-Tg
mice, as expected, had higher proliferative and cytokine responses as
compared to the wild-type T cells and appears to correlate with
responses we revealed using keyhole limpet hemocyanin peptide (our
unpublished data). EAE is usually referred to as being a Th1-mediated
disease (42, 43); however, the role of Th1 cytokines
remains highly contentious. Both IFN-- and TNF-
-deficient
mice have been shown to be highly susceptible to EAE
(43, 44, 45). Thus, the altered Th1 cytokine responses we
observed could be due to an overall inability of the T cells to
differentiate into both Th1/Th2 effectors. Noteworthy however was the
finding that a reduction of IL-6 production by OX40L-/-
draining lymph node cells and a concomitant increase in IL-6 production
in the OX40L-Tg lymph node cells may render this cytokine paramount in
the pathogenesis of EAE. Indeed, elevated IL-6 levels in the culture
medium just prior to adoptive transfer further highlights the ability
of OX40L to enhance IL-6 secretion, at least in the early stages of the
disease (data not shown). In fact, IL-6-/- mice have
complete suppression of EAE, making IL-6 an essential inflammatory
cytokine involved in EAE pathogenesis (46).
The nonlymphoid cells such as CNS resident cells and infiltrating macrophages were examined in light of the limited demyelination seen in the brain tissues of OX40L-/- mice. Myelin lacks MHC expression, suggesting that effector T cells cannot directly recognize and damage myelin, and thus microglia/macrophages in addition may participate directly in myelin destruction (47). OX40L was not detected among the CD11b+ microglia/macrophage population by flow cytometry throughout the disease. This result is in direct conflict with a report showing strong OX40L expression in SJL/J mice after proteolipid protein-induced EAE in vivo (17). We however were able to detect mRNA expression in the same population in OX40L+/+ mice but not in the OX40L-/- mice. The continued presence of OX40 may be responsible for the down-regulation/internalization/shedding of OX40L expression in vivo. Indeed, activated OX40-/- T cells in vitro allow for the easy detection of OX40L on T cells which are not readily detectable in wild-type mice (12).
Although there is no direct evidence, OX40-OX40L has been suggested to participate both directly and indirectly in the migration of immune cells (11, 16, 32). This has further been highlighted by reports observing OX40 and OX40L expression in vascular endothelial cells (16, 30, 48). Interrupting OX40L signals however did not alter the expression of several integrin family members (16). Furthermore, normal migration of OX40L-/- dendritic cells has been described after sensitization (21). Most significant however is a recent study demonstrating that migratory encephalitogenic T cells did not express OX40 in the periphery (spleen), but this molecule was then re-expressed in the brain, thus making OX40 an unlikely candidate involved in cell migration (31). We observed a large number of inflammatory cells in the brain sections of OX40L-/- diseased mice. In addition, adoptive transfer of encephalitogenic T cells derived from wild-type mice to recipient OX40L-/- mice were able to migrate to the CNS in sufficient numbers to induce disease. From our findings using OX40L-Tg, it is clear that CD28 signals are essential for OX40-OX40L interactions and may affect expression of the T cell activation Ag OX40 both in vivo and in vitro. However, CD28 is dispensable in some immune responses (38, 39), and it has been documented that they may also synergistically coordinate with OX40 in providing significant T cell costimulation (17). Recent suggestions go further, theorizing that OX40L costimulation alone is sufficient to drive significant costimulation in a CD28-independent manner (49). Our result demonstrates that blockade of CD28 signals interrupts the sequential costimulatory pathway necessary for OX40L function, rendering additional interference of OX40L unnecessary. Similarly, the essential requirement for CD40 signals has been further analyzed.
We initially described the presence of OX40L on human T cell lymphotropic virus type 1-infected T cells and here determine that OX40L expressed on T cells is capable of inducing significant T cell costimulation. Thus OX40-OX40L interaction during EAE is a complicated interplay between T-APC and T-T interactions in the CNS. In conclusion, this interaction appears to alter the generation of MOG-responsive T cells and production of proinflammatory cytokines due to dysfunctional CNS APC signals, detrimental in the priming of pathogenic T cells. Furthermore, as a consequence, OX40L function is both strongly CD40 and CD28 dependent and thus clearly participates in both the priming and maintenance of autoreactive T cells. The multiple roles of OX40L interactions make it an important candidate for controlling autoimmune disorders.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Professor Kazuo Sugamura, Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan. E-mail address: sugamura{at}mail.cc.tohoku.ac.jp
3 Abbreviations used in this paper: OX40L, OX40 ligand; EAE, experimental autoimmune encephalomyelitis; Tg, transgenic; MOG, myelin oligodendrocyte; PLP, proteolipid protein; m, mouse; DAS, disability score.
Received for publication April 27, 2001. Accepted for publication June 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis. J. Exp. Med. 192:123.This article has been cited by other articles:
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C. S. Schmidt, J. Zhao, J. Chain, D. Hepburn, B. Gitter, G. Sandusky, S. Chintalacharuvu, A. Glasebrook, and S. Na Resistance to Myelin Oligodendrocyte Glycoprotein-Induced Experimental Autoimmune Encephalomyelitis by Death Receptor 6-Deficient Mice J. Immunol., August 15, 2005; 175(4): 2286 - 2292. [Abstract] [Full Text] [PDF]
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B. Valzasina, C. Guiducci, H. Dislich, N. Killeen, A. D. Weinberg, and M. P. Colombo Triggering of OX40 (CD134) on CD4+CD25+ T cells blocks their inhibitory activity: a novel regulatory role for OX40 and its comparison with GITR Blood, April 1, 2005; 105(7): 2845 - 2851. [Abstract] [Full Text] [PDF]
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 S. Andarini, T. Kikuchi, M. Nukiwa, P. Pradono, T. Suzuki, S. Ohkouchi, A. Inoue, M. Maemondo, N. Ishii, Y. Saijo, et al. Adenovirus Vector-Mediated in Vivo Gene Transfer of OX40 Ligand to Tumor Cells Enhances Antitumor Immunity of Tumor-Bearing Hosts Cancer Res., May 1, 2004; 64(9): 3281 - 3287. [Abstract] [Full Text] [PDF]
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I. Takeda, S. Ine, N. Killeen, L. C. Ndhlovu, K. Murata, S. Satomi, K. Sugamura, and N. Ishii Distinct Roles for the OX40-OX40 Ligand Interaction in Regulatory and Nonregulatory T Cells J. Immunol., March 15, 2004; 172(6): 3580 - 3589. [Abstract] [Full Text] [PDF]
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G. Demirci, F. Amanullah, R. Kewalaramani, H. Yagita, T. B. Strom, M. H. Sayegh, and X. C. Li Critical Role of OX40 in CD28 and CD154-Independent Rejection J. Immunol., February 1, 2004; 172(3): 1691 - 1698. [Abstract] [Full Text] [PDF]
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N. Martin-Orozco, Z. Chen, L. Poirot, E. Hyatt, A. Chen, O. Kanagawa, A. Sharpe, D. Mathis, and C. Benoist Paradoxical Dampening of Anti-Islet Self-Reactivity but Promotion of Diabetes by OX40 Ligand J. Immunol., December 15, 2003; 171(12): 6954 - 6960. [Abstract] [Full Text] [PDF]
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B. R. Blazar, A. H. Sharpe, A. I. Chen, A. Panoskaltsis-Mortari, C. Lees, H. Akiba, H. Yagita, N. Killeen, and P. A. Taylor Ligation of OX40 (CD134) regulates graft-versus-host disease (GVHD) and graft rejection in allogeneic bone marrow transplant recipients Blood, May 1, 2003; 101(9): 3741 - 3748. [Abstract] [Full Text] [PDF]
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G. Gri, E. Gallo, E. Di Carlo, P. Musiani, and M. P. Colombo OX40 Ligand-Transduced Tumor Cell Vaccine Synergizes with GM-CSF and Requires CD40-Apc Signaling to Boost the Host T Cell Antitumor Response J. Immunol., January 1, 2003; 170(1): 99 - 106. [Abstract] [Full Text] [PDF]
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K. Murata, M. Nose, L. C. Ndhlovu, T. Sato, K. Sugamura, and N. Ishii Constitutive OX40/OX40 Ligand Interaction Induces Autoimmune-Like Diseases J. Immunol., October 15, 2002; 169(8): 4628 - 4636. [Abstract] [Full Text] [PDF]
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 A. Yamada, A. D. Salama, and M. H. Sayegh The Role of Novel T Cell Costimulatory Pathways in Autoimmunity and Transplantation J. Am. Soc. Nephrol., February 1, 2002; 13(2): 559 - 575. [Full Text] [PDF]
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) mice
(n = 5/group). Animals were immunized with MOG/CFA in
both flanks and received pertussis toxin on days 0 and 2. Mice were
monitored for symptoms daily. One representative experiment of three
separate experiments is shown. B, Histopathological profiles
of brains of affected OX40L+/+ and OX40L-/-
mice on day 35 after immunization. Brains were harvested, fixed in
Telly’s fixture solution, and embedded in paraffin. Six-micrometer
sections were stained with H&E (upper panel) and
Kluver-Barrera (lower panel) stain; Blue staining is
indicative of myelin. On average, 15 sections were made per mouse and
results are based on sections from 5 mice in each group with DAS
ranging from 2 to 3. Original magnification, x100. WT, Wild type; KO,
knockout.
