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*
Department of Paediatric Gastroenterology, St. Bartholomew’s and The Royal London School of Medicine and Dentistry, St. Bartholomew’s Hospital, London, United Kingdom; and
Cantab Pharmaceuticals Limited, Cambridge, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, IL-12, and
IFN-
; and a reduction in the T cell infiltrate. Targeting OX40
therefore shows considerable promise as a new strategy to inhibit
ongoing T cell reactions in the gut. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß (8). The cytoplasmic tail of
OX40 interacts with the TNFR-associated factor 2 and 3, which regulate
activation of nuclear factor-
B (11). Blocking OX40L with an OX40-IgG
fusion protein has been shown to inhibit these responses (8). Signaling
also occurs through OX40L and is important in T cell-dependent terminal
differentiation of activated B cells (12). In addition, the expression
of OX40L on vascular endothelium suggests the involvement of OX40 in T
cell migration into tissues (13). The therapeutic potential of
targeting OX40 lies in its limited cellular expression, predominantly
on activated CD4+ T cells, which are thought to be central
to the pathogenesis of many human diseases, including inflammatory
bowel disease (IBD), multiple sclerosis, rheumatoid arthritis, and
graft-versus-host disease (14, 15, 16, 17). The efficacy of anti-OX40 Abs
in the treatment of the rat experimental allergic encephalomyelitis
model of MS has been demonstrated, in which the use of a ricin-A
anti-OX40 immunotoxin ameliorated disease (18).
In this study, we have constructed OX40-IgG and mOX40L-IgG fusion
proteins and have examined their effect on the in vitro proliferation
and cytokine production of T cells to mitogenic and antigenic
stimulation. In addition, we have tested their efficacy at inhibiting
the ongoing Th1-type responses responsible for colonic tissue injury in
hapten-induced colitis and IBD in IL-2 knockout mice (19, 20). In both
models, disease is mediated by Th1 type T cells through massive
TNF-, IL-1, and IFN- release (19, 21).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice (8–10 wk old) were obtained from A. Tuck & Sons (Southend-on-Sea, U.K.). IL-2+/- C3H mice (20) were bred under standard conditions, and mice homozygous for the null mutation were identified by genotyping, as described previously (21). All mice were housed under standard conditions with free access to food and water.
Induction of colitis
BALB/c mice were weighed before procedure. Trinitrobenzene sulfonic acid (TNBS; Fluka, Gillingham, U.K.) was prepared in a 50% ethanol solution diluted to give a final concentration of 2 mg TNBS in 75 µl total volume. Mice were lightly anesthetized using 200 µl of a 1/10 aqueous dilution of Hypnorm (Janssen-Cilag, High Wycombe, U.K.). Colitis was induced by intrarectal administration of 75 µl of the TNBS solution using a plastic catheter. Control mice received 50% aqueous ethanol only. Mice were checked daily with respect to general condition and body weight.
Myeloperoxidase assay
Myeloperoxidase (MPO) was measured in snap-frozen samples of colonic tissue (22). Tissue (75–150 mg) was homogenized in 400 µl of cold 1% (w/v) hexadecyl trimethyl ammonium bromide (Sigma, Poole, U.K.) in phosphate buffer, pH 6. The homogenate was then sonicated for 15 s. After snap freezing (in liquid nitrogen) and thawing three times, the homogenate was centrifuged for 15 min at 12,000 x g at 4°C. The supernatant was then removed for MPO assay. To 10 µl of supernatant in a flat-bottom 96-well microtiter plate (Philip Harris, London, U.K.), 200 µl of 50 mM phosphate buffer, pH 6, containing 0.4 mg/ml of substrate o-phenylenediamine (Sigma) and 0.05% H2O2 (Sigma) was added. After 20 min, the reaction was stopped by the addition of 50 µl 0.4 M H2SO4, and absorbance at 490 nm was determined using a plate reader (Titertek Multiscan, Eflab, Finland). Sample enzyme activity was measured from a standard curve of horseradish peroxidase activity (Boehringer Mannheim, Lewes, U.K.). Assay sensitivity was 10-7 U/µl.
Generation of fusion proteins
For the hOX40-hIgG1 fusion protein, the construct was as described previously (8). This construct was used to transfect Chinese hamster ovary cells, and positive clones were selected using G418. Fusion protein secretion was detected by incubation of supernatants with OX40L-transfected Sp2/0 cells and detection of binding by flow-cytometric analysis. Cells secreting high levels of fusion protein were expanded, and fusion protein from the supernatant was purified on a protein G-Sepharose column. Eluted material was electrophoresed on SDS-PAGE (12%), and the gel was stained with Coomassie blue to confirm purity. The mOX40L-hIgG1 fusion protein was prepared by a similar method (all enzymes were purchased from New England BioLabs, Hitchin, U.K.). A fragment encoding the extracellular domain of mOX40L was PCR cloned with the introduction of PstI and HindIII sites at the 5' and 3' ends, respectively. To form the hIgG1-mOX40L fusion construct, this fragment was ligated into PstI-digested plasmid that encoded for the hinge CH2 and CH3 domains of hIgG1. This gene was then isolated as a HindIII fragment and transferred to the pCR3 expression vector (Invitrogen, Abingdon, U.K.) containing the hCMV promoter and neoR selectable marker. Clones were screened for inserts in the correct orientation, and then grown up for transfection and expression experiments, as above.
Treatment with fusion proteins
TNBS colitic mice and ethanol-treated controls were injected i.p. with hOX40-IgG (100 or 10 µg) or mOX40L-IgG (100 µg) on days 4, 5, and 6 after induction of colitis, or one single dose of hOX40-IgG (100 µg) on day 4. All mice were killed on day 7. IL-2-deficient mice, aged over 35 days, showing physical signs of deterioration and weight loss, indicative of colitis, were treated with three consecutive daily doses of hOX40-IgG (100 µg) and were killed one day later. Since IL-2 knockout mice developed disease unpredictably, the choice of treating a mouse with weight loss with either IgG or OX40-IgG was decided by tossing a coin. In all cases, hIgG (100 µg) (Sigma) was used as a control.
RNA extraction and quantitative RT-PCR
Constructs encoding standard RNAs (pMCQ1, pMCQ2, pMCQ3, and pMCQ4) kindly provided by Dr. M. F. Kagnoff (Department of Medicine, University of California, San Diego) (23) were used for quantitative competitive RT-PCR. To generate standard RNA, plasmids were linearized with SalI (pMCQ1) or NotI (pMCQ2, 3, 4) and transcribed in vitro using T7 RNA polymerase, under conditions recommended by the supplier (Promega, Southampton, U.K.).
Gut tissue and cell pellets were snap frozen in liquid nitrogen and stored at -70°C. Total cellular RNA was isolated by homogenizing tissue or cells in TRIzol (Life Technologies, Paisley, U.K.) and incubating at room temperature for 5 min. RNA was extracted using chloroform (Sigma), followed by centrifugation for 15 min at 12,000 x g at 4°C. The aqueous phase was precipitated with an equal volume of isopropanol (Sigma), followed by centrifugation for 15 min at 12,000 x g at 4°C. The pellet was washed with 70% ethanol and resuspended in 50 µl water. Total RNA was determined by spectrometric analysis.
RT-PCR amplification
Serial 10-fold dilutions of standard RNA (1 pg to 1 fg) were co-reverse transcribed with total cellular RNA (2 µg) at 42°C for 50 min in a 20 µl reaction volume containing 50 mM Tris, pH 8.3, 75 mM KCl, 3 mM MgCl2, 3 mM DTT, 10 mM dNTP mix, and 0.5 µg oligo(dT) (Pharmacia Biotech, Herts, U.K.), using 100 U of reverse transcriptase (Superscript II RNase H-; Life Technologies). The reaction was terminated by heat inactivation at 70°C for 10 min. PCR amplification was conducted routinely in 50 µl reaction volumes (10 mM Tris, pH 9, 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTPs, 10 pmol 5' and 3' primers, as described elsewhere (23), and 1 U Taq polymerase (Pharmacia Biotech, U.K.)). Forty amplification cycles of 45-s denaturation at 94°C, 45-s annealing at 58°C, and 75-s extension at 72°C were used.
After amplification, PCR products were analyzed on 1% agarose gels and bands were visualized by ethidium bromide staining. Band intensities were quantified by densitometry (Seescan, Cambridge, U.K.). The sensitivity of this technique enables the detection of >103 mRNA transcripts per µg of total RNA.
Immunohistochemistry
Three-step avidin peroxidase staining was performed on 5-µm frozen sections, as described previously (24), using mAbs 145-2C11 (anti-CD3), YTS 191 (anti-CD4), YTS 169 (anti-CD8), and OX86 (anti-mOX40). Biotin-conjugated rabbit anti-rat IgG (Dako, High Wycombe, U.K.) and goat anti-hamster IgG (Vector Laboratories, Peterborough, U.K.) were used at a 1/50 dilution in TBS, pH 7.6, containing 4% (v/v) normal mouse serum (Harlan Seralab, Oxon, U.K.). Avidin peroxidase (Sigma) was used at a dilution of 1/200 in TBS. Peroxidase activity was detected with 3,3'-diaminobenzidine-tetra-hydrochloride (Sigma) in 0.5 mg/ml Tris-HCl, pH 7.6, containing 0.01% H2O2. The density of positive cells in the lamina propria was determined by image analysis, as described previously (21).
Ag-specific T cell responses
Female BALB/c (2–4 mo old) mice were immunized (s.c.) with keyhole limpet hemocyanin (KLH; 100 µg; Sigma) in 200 µl CFA (Sigma). Draining lymph nodes and spleens were removed 14 days postimmunization.
Preparation of cells and proliferation assays
Single cell suspensions of mesenteric lymph node (MLN) and spleen cells were prepared by gently teasing apart in RPMI cell culture medium, supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin, using sterile forceps. Cell aggregates were removed by passing suspensions through sterile cell strainers (Falcon, London, U.K.). Single cell suspensions were washed three times with RPMI/FCS. A total of 200 µl cell suspensions (5 x 105 cells/ml) was incubated in a 96-well microtiter plate with or without Con A (5 µg/ml) or KLH (100 µg/ml). hOX40-IgG, mOX40L-IgG, or hIgG was added at 5, 25, or 50 µg/ml to cell cultures. Con A-stimulated cells were incubated for 3 days, and KLH-stimulated cells for 5 days at 37°C, and were pulsed with 1 µCi per well of [3H]thymidine during the last 15 h before being harvested onto filters.
Isolation of lamina propria cells and flow cytometry
Lamina propria lymphocytes were prepared by enzymic digestion. Briefly, colon was washed out with HBSS (Sigma) and was cut into 0.5-cm pieces. Epithelial cells were removed by incubating gut segments in 25 ml HBSS, without Ca/Mg, and supplemented with 1 mM EDTA (Sigma), for 20 min at 37°C. After removing supernatant, gut segments were washed with HBSS resuspended in RPMI/FCS containing collagenase (90 U/ml; Sigma) and dispase (2.5 U/ml; Sigma), and tissue was left to digest for 1 h at 37°C, with stirring. The resultant cell suspension was passed through a sterile cell strainer and was washed twice with RPMI/FCS.
OX40 surface expression was determined using mAb OX86 (5) with secondary FITC-conjugated goat anti-rat IgG (Sigma). Rat IgG (Sigma) was used as a control. Briefly, 400 µl OX86 supernatant or rat IgG (1/50 dilution in PBS containing 0.1% NaN3) was incubated with 500,000 cells for 60 min on ice. Cells were washed once with PBS, pH 7.4, and resuspended in FITC-conjugated secondary Ab (1/50 dilution in PBS/NaN3 containing 4% normal mouse serum) for 30 min on ice. Cells were washed again and resuspended in 1% paraformaldehyde/PBS for counting. Single color flow cytometry was conducted using a FACScan (Becton Dickinson Immunocytometry Systems, Oxford, U.K.).
Statistics
The significance of differences between means was determined using the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine the expression of OX40 on activated T cells, flow
cytometry using the mAb OX86 was performed on splenocytes stimulated
for 60 h with Con A. A peak for OX40-positive cells was observed,
while no expression was detectable on resting cells. There was a double
peak for stimulated cells, suggesting a population of cells with high
and low expression of OX40 (Fig. 1
A). OX40L and OX40 fusion
proteins were then examined for their effect on mitogen activation of
splenocytes and MLN cells (Fig. 1B). Cross-linking of OX40
using the OX40L fusion protein was shown to be costimulatory, enhancing
proliferation by up to 100% in both splenocytes and MLN cells at all
concentrations examined (Fig. 1C). The OX40 fusion protein
inhibited proliferation by up to 50% at a concentration of 50 µg/ml
for MLN and spleen cells. The dose response indicated that OX40-IgG had
a reduced effect on stimulation at lower concentrations (Fig. 1C). Addition of OX40-IgG 15 h before harvesting also
resulted in an inhibition of proliferation (data not shown). No effect
with either fusion protein or control hIgG was observed on resting
cells.
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To examine the effects of OX40-IgG and OX40L-IgG on an Ag-specific
response, mice were immunized with 100 µg KLH in 200 µl CFA s.c.,
and the in vitro proliferation of draining LN cells and splenocytes
challenged with KLH was examined. Ag-induced proliferation was reduced
by using the OX40 fusion protein at 50 µg/ml, whereas OX40L-IgG was
costimulatory (Fig. 2A). Lower
concentrations of OX40L-IgG were, as before, sufficient to promote
proliferation (Fig. 2B). We wanted to eliminate the
possibility that the OX40-IgG fusion could be causing
complement-mediated lysis of cells expressing OX40L. We therefore
examined the effect of OX40-IgG on B cells as these are the principal
cell type expressing OX40L. Following in vitro Con A-induced activation
of splenocytes in the presence of OX40-IgG there was no diminution of B
cells compared to controls as shown by trypan blue exclusion and FACS
analysis for B cell numbers. In addition, neither freshly isolated
mouse serum nor commercial rabbit complement were capable of lysing B
cells in the presence of OX40-IgG (data not shown).
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Increased transcripts for IL-2, IFN-, and IL-4 were found in
Con A-activated cells. OX40-IgG dramatically reduced cytokine
transcripts. However, despite the much greater proliferative response,
OX40L-IgG did not enhance cytokine production (Fig. 3, A–C). Similar results for
KLH-stimulated cells were also seen (Fig. 3, D–E). IL-2,
IFN-, and IL-4 ELISAs were performed on supernatants from Con
A-stimulated splenocytes in the presence of OX40-IgG, and a similar
reduction in protein compared with controls was observed for each
cytokine. IL-2 was reduced from 9.1 to 2.1 U/ml, IFN- from 10.8 to
<1 ng/ml, and IL-4 from 4.3 to <1 pg/ml (data not shown).
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Since activated CD4+ T cells play an important role in
both TNBS colitis and IL-2 knockout mice with colitis (19, 21), we
examined the expression of OX40 in vivo in the gut by
immunohistochemistry. In normal BALB/c and C3H mice, OX40-positive
cells were only observed in lymphoid tissue, including Peyer’s patches
and MLN. Positive cells were, however, seen by immunohistochemistry in
the lamina propria of both TNBS (Fig. 4) and IL-2
knockout mice with colitis, but not in controls. Positive cells were
also observed by FACS analysis of cells isolated from the lamina
propria of mice with TNBS colitis (Fig. 5).
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TNBS colitis was induced in female BALB/c mice. The disease profile (not shown) was such that an initial severe acute colitis took place 1–3 days postinduction, followed on day 4 by T cell infiltration, which was maximal at day 7, after which the colitis subsided. Day 4 was chosen to begin treatment with the fusion proteins. The results shown represent one experiment (n = 6/group). Similar results have been shown in two additional experiments in which n = 5/group (data not shown).
MPO activity in TNBS mice was significantly higher than in ethanol
control mice. MPO in mice that had received three daily doses, or one
single dose, of 100 µg of hOX40-IgG was comparable with the ethanol
control. Treatment with the lower dose of 10 µg of hOX40-IgG, or with
OX40L fusion protein at either 10 or 100 µg had no effect (Fig. 6).
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, IL-12, and IFN- in their gut.
OX40-IgG-treated mice showed a dramatic reduction in all of these
cytokines (Fig. 8, A–D).
Transcripts for Th2-type cytokines in the colon of OX40-IgG-treated
mice were not significantly different from normal mice (Fig. 8, E–F). A single dose of 100 µg of OX40-IgG on day 4 had
similar effects to the three daily doses, but mice given 10 µg still
showed marked elevation in proinflammatory and Th1 cytokines (data not
shown). Treatment with mOX40L-IgG did not reduce cytokine transcripts,
and in one to two mice, per experiment, treated with three daily doses
of 100 µg, they were markedly elevated.
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IL-2 knockout mice with colitis were given three 100 µg doses of
OX40-IgG or hIgG once the first signs of deterioration were apparent.
On day 4 posttreatment, the mice were killed. A massive infiltration of
T cells and macrophages takes place in IL-2 knockout mice with colitis
when compared with normal wild-type mice. Counts for CD3-, CD4-, and
CD8-positive cells were all reduced dramatically in mice treated with
hOX40-IgG (Fig. 9). A significant
reduction in peroxidase-containing cells was also observed (Fig. 6
B).
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, IL-12 and proinflammatory cytokines,
IL-1, and TNF- were markedly increased in IL-2 knockout mice with
colitis treated with control IgG, but were reduced significantly in
mice treated with OX40 fusion protein (Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The in vitro enhancement of mitogen-induced proliferation by costimulation through OX40 is in agreement with previous studies using anti-OX40 Abs (6) or OX40L transfectants (7, 8). The use of an OX40 fusion protein to block the OX40-OX40L interaction between purified CD4+ T cells and OX40L transfectants has also been reported (8). In addition, we have shown that the OX40-OX40L interaction is important in an Ag-specific response that is in agreement with studies showing enhanced proliferation in an MLR in the presence of an anti-OX40 Ab (6). Dose dependency was observed for blocking OX40L, and may be indicative of high OX40L expression in a mixed population of cells in contrast to limited expression of OX40 on activated T cells.
In vivo the success of OX40-IgG was also dose dependent and was highly efficient at blocking T cell responses in the gut. Although in certain individual mice treated with OX40L-IgG, colitis did appear to be heightened with respect to MPO and cytokine production, the overall result was not significantly greater than hIgG-treated controls. Transcripts for IL-4 and IL-10 in OX40-IgG-treated groups were not increased when compared with normal mice, demonstrating that OX40-IgG therapy did not result in immune deviation toward a Th2 response. In vitro, an increase in cytokine production did not take place with mOX40L-IgG, which, considering the proliferation data, was surprising. It suggests that the OX40L fusion protein was able to enhance proliferation through cross-linkage of OX40, but was not enough to induce cytokine production, or it may be that an upper limit in detection of such high concentrations of cytokine transcripts was reached. In addition, in vivo treatment with OX40L-IgG did not result in increased cell infiltration. It is clear, however, that endogenous OX40-OX40L interactions play a crucial role in regulating T cell-mediated damage. Many recent studies have dealt with the expression and function of OX40L on B cells, dendritic cells, and endothelial cells (7, 8, 9, 10), and from these studies, it is evident that whatever the cell type expressing the ligand, or indeed one cannot not exclude the existence of a soluble form of ligand, all such interactions are crucial for cross-talk with activated T cells.
The OX40-OX40L interaction is a good example of bidirectional signaling. Cross-linking of OX40L on B cells results in B cell proliferation and secretion of all Ig isotypes and is critical for T cell-dependent terminal differentiation of B cells (12). In addition, signaling through OX40L is also important for the proliferation of activated naive dendritic cells (10). Another possible mechanism for OX40-IgG therapy, which is not related to activation, but rather to homing, is the prevention of recruitment of OX40+ cells to sites of inflammation through OX40L expression on endothelial cells. It has been shown that cells from patients with adult T cell leukemia adhere to HUVECs through OX40-OX40L interaction (9). In human IBD, OX40L endothelial cells have been seen, but OX40L is also expressed on as yet unidentified lamina propria cells (H. Souza and J. Spencer, personal communication). The expression of OX40L on murine endothelial cells has not been reported.
IBD in mice is a result of immune dysregulation. Mice with a variety of
T cell defects including mice with a deleted gene for IL-2 (20),
IL-10 (25), TCR or TCRß (26), and Gi2 (27) as well
as T cell-reconstituted tg
26 mice transgenic for the human CD3
gene (28) and mice transgenic for IL-7 (29) develop chronic IBD.
Disease is mediated principally by activated CD4+ T cells.
Therapeutic strategies have aimed either at blocking cytokines produced
by these and accessory cells, as demonstrated by anti-IL-12 (19),
anti-TNF- (30), or IL-10 (31) treatment, or at inhibiting signal
transduction (32), or at preventing costimulatory signals between T
cells and APCs. Many costimulatory interactions are regulated by
molecules that are normally absent or have low expression, but are
up-regulated upon activation. These include CD25, CD28, CD40L, OX40,
and 4-1BB, all considered as markers of T cell antigenic stimulation,
and therefore viable targets in immunotherapy. The CD40-CD40L
interaction, for example, has been blocked in the treatment of
experimental IBD (33). Negative regulation of T cell activation through
CTLA4 has also been used in the down-regulation of aberrant T cell
responses, as shown by the inhibition of the CD28-B7 interaction in
transplantation (34) and experimental multiple sclerosis (35).
There is a plethora of molecules, including OX40, that can play a role in enhancing the efficiency of activation and proliferation of T cells, and it has been demonstrated with genetically mutant mice that not all of these are essential, indicating that a certain degree of compensation can occur. It is clear that different molecules are important at different stages in the pathway to a fully activated T cell, and that cooperation is needed for a fully efficient response. The success of blocking T cell responses by inhibiting these interactions continues to show promise in the treatment of autoimmune disease, graft rejection, and lymphoma. The advantage of using OX40-IgG in therapy lies in the selective expression of OX40 predominantly on activated CD4+ T cells for a transient period in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas T. MacDonald, Department of Paediatric Gastroenterology, St. Bartholomew’s and The Royal London School of Medicine and Dentistry, Suite 31, Dominion House, 59 St. Bartholomew’s Close, London EC1A 7BE, U.K. E-mail address:
3 Abbreviations used in this paper: L, ligand; h, human; IBD, inflammatory bowel disease; KLH, keyhole limpet hemocyanin; m, murine; MLN, mesenteric lymph node; MPO, myeloperoxidase; TNBS, trinitrobenzene sulfonic acid.
Received for publication May 8, 1998. Accepted for publication September 21, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. Mol. Cell. Biol. 18:558. i2-deficient mice. Nat. Genet. 10:143.[Medline]
B abrogates established experimental colitis in mice. Nat. Med. 2:998.[Medline]
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 T. Totsuka, T. Kanai, K. Uraushihara, R. Iiyama, M. Yamazaki, H. Akiba, H. Yagita, K. Okumura, and M. Watanabe Therapeutic effect of anti-OX40L and anti-TNF-alpha MAbs in a murine model of chronic colitis Am J Physiol Gastrointest Liver Physiol, April 1, 2003; 284(4): G595 - G603. [Abstract] [Full Text] [PDF]
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S. Fillatreau and D. Gray T Cell Accumulation in B Cell Follicles Is Regulated by Dendritic Cells and Is Independent of B Cell Activation J. Exp. Med., January 20, 2003; 197(2): 195 - 206. [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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L. Taylor, M. Bachler, I. Duncan, S. Keen, R. Fallon, C. Mair, T. T. McDonald, and H. Schwarz In vitro and in vivo activities of OX40 (CD134)-IgG fusion protein isoforms with different levels of immune-effector functions J. Leukoc. Biol., September 1, 2002; 72(3): 522 - 529. [Abstract] [Full Text] [PDF]
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K. W. Chan, C. D. Hopke, S. M. Krams, and O. M. Martinez CD30 Expression Identifies the Predominant Proliferating T Lymphocyte Population in Human Alloimmune Responses J. Immunol., August 15, 2002; 169(4): 1784 - 1791. [Abstract] [Full Text] [PDF]
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 L. M. Gaetke, H. S. Oz, W. J. S. de Villiers, G. W. Varilek, and R. C. Frederich The Leptin Defense against Wasting Is Abolished in the IL-2-Deficient Mouse Model of Inflammatory Bowel Disease J. Nutr., May 1, 2002; 132(5): 893 - 896. [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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T. De Smedt, J. Smith, P. Baum, W. Fanslow, E. Butz, and C. Maliszewski Ox40 Costimulation Enhances the Development of T Cell Responses Induced by Dendritic Cells In Vivo J. Immunol., January 15, 2002; 168(2): 661 - 670. [Abstract] [Full Text] [PDF]
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A. Kotani, T. Ishikawa, Y. Matsumura, T. Ichinohe, H. Ohno, T. Hori, and T. Uchiyama Correlation of peripheral blood OX40+(CD134+) T cells with chronic graft-versus-host disease in patients who underwent allogeneic hematopoietic stem cell transplantation Blood, November 15, 2001; 98(10): 3162 - 3164. [Abstract] [Full Text] [PDF]
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L. C. Ndhlovu, N. Ishii, K. Murata, T. Sato, and K. Sugamura Critical Involvement of OX40 Ligand Signals in the T Cell Priming Events During Experimental Autoimmune Encephalomyelitis J. Immunol., September 1, 2001; 167(5): 2991 - 2999. [Abstract] [Full Text] [PDF]
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V. Malmstrom, D. Shipton, B. Singh, A. Al-Shamkhani, M. J. Puklavec, A. N. Barclay, and F. Powrie CD134L Expression on Dendritic Cells in the Mesenteric Lymph Nodes Drives Colitis in T Cell-Restored SCID Mice J. Immunol., June 1, 2001; 166(11): 6972 - 6981. [Abstract] [Full Text] [PDF]
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 J C Hoffmann, K Peters, S Henschke, B Herrmann, K Pfister, J Westermann, and M Zeitz Role of T lymphocytes in rat 2,4,6-trinitrobenzene sulphonic acid (TNBS) induced colitis: increased mortality after {gamma}{delta} T cell depletion and no effect of {alpha}{beta} T cell depletion Gut, April 1, 2001; 48(4): 489 - 495. [Abstract] [Full Text] [PDF]
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C. Nohara, H. Akiba, A. Nakajima, A. Inoue, C.-S. Koh, H. Ohshima, H. Yagita, Y. Mizuno, and K. Okumura Amelioration of Experimental Autoimmune Encephalomyelitis with Anti-OX40 Ligand Monoclonal Antibody: A Critical Role for OX40 Ligand in Migration, But Not Development, of Pathogenic T Cells J. Immunol., February 1, 2001; 166(3): 2108 - 2115. [Abstract] [Full Text] [PDF]
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J. Kjærgaard, J. Tanaka, J. A. Kim, K. Rothchild, A. Weinberg, and S. Shu Therapeutic Efficacy of OX-40 Receptor Antibody Depends on Tumor Immunogenicity and Anatomic Site of Tumor Growth Cancer Res., October 1, 2000; 60(19): 5514 - 5521. [Abstract] [Full Text]
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I. Gramaglia, A. Jember, S. D. Pippig, A. D. Weinberg, N. Killeen, and M. Croft The OX40 Costimulatory Receptor Determines the Development of CD4 Memory by Regulating Primary Clonal Expansion J. Immunol., September 15, 2000; 165(6): 3043 - 3050. [Abstract] [Full Text] [PDF]
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N. Tsukada, H. Akiba, T. Kobata, Y. Aizawa, H. Yagita, and K. Okumura Blockade of CD134 (OX40)-CD134L interaction ameliorates lethal acute graft-versus-host disease in a murine model of allogeneic bone marrow transplantation Blood, April 1, 2000; 95(7): 2434 - 2439. [Abstract] [Full Text] [PDF]
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H. Akiba, Y. Miyahira, M. Atsuta, K. Takeda, C. Nohara, T. Futagawa, H. Matsuda, T. Aoki, H. Yagita, and K. Okumura Critical Contribution of Ox40 Ligand to T Helper Cell Type 2 Differentiation in Experimental Leishmaniasis J. Exp. Med., January 17, 2000; 191(2): 375 - 380. [Abstract] [Full Text] [PDF]
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K. Murata, N. Ishii, H. Takano, S. Miura, L. C. Ndhlovu, M. Nose, T. Noda, and K. Sugamura Impairment of Antigen-Presenting Cell Function in Mice Lacking Expression of Ox40 Ligand J. Exp. Med., January 17, 2000; 191(2): 365 - 374. [Abstract] [Full Text] [PDF]
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P. Lane Role of Ox40 Signals in Coordinating Cd4 T Cell Selection, Migration, and Cytokine Differentiation in T Helper (Th)1 and Th2 Cells J. Exp. Med., January 17, 2000; 191(2): 201 - 206. [Full Text] [PDF]
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S. D. Pippig, C. Pena-Rossi, J. Long, W. R. Godfrey, D. J. Fowell, S. L. Reiner, M. L. Birkeland, R. M. Locksley, A. N. Barclay, and N. Killeen Robust B Cell Immunity but Impaired T Cell Proliferation in the Absence of CD134 (OX40) J. Immunol., December 15, 1999; 163(12): 6520 - 6529. [Abstract] [Full Text] [PDF]
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H S Souza, C C S Elia, J Spencer, and T T MacDonald Expression of lymphocyte-endothelial receptor-ligand pairs, alpha 4beta 7/MAdCAM-1 and OX40/OX40 ligand in the colon and jejunum of patients with inflammatory bowel disease Gut, December 1, 1999; 45(6): 856 - 863. [Abstract] [Full Text] [PDF]
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L. S.K. Walker, A. Gulbranson-Judge, S. Flynn, T. Brocker, C. Raykundalia, M. Goodall, R. Forster, M. Lipp, and P. Lane Compromised Ox40 Function in Cd28-Deficient Mice Is Linked with Failure to Develop Cxc Chemokine Receptor 5-Positive Cd4 Cells and Germinal Centers J. Exp. Med., October 18, 1999; 190(8): 1115 - 1122. [Abstract] [Full Text] [PDF]
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