Expression of CD134 (OX40) on activated CD4+ T cells has been observed in acute graft-versus-host disease (GVHD) after human and rat allogeneic bone marrow transplantation (BMT). We investigated the role of interaction between CD134 and CD134 ligand (CD134L) in a murine model of acute GVHD by using a newly established monoclonal antibody (mAb) against murine CD134L. Acute GVHD was induced by transfer of bone marrow cells and spleen cells into lethally irradiated recipients in a parent (C57BL/6) to first filial generation (C57BL/6 crossed with DBA/2) BMT. Administration of anti-CD134L mAb significantly reduced the lethality of acute GVHD and other manifestations of the disease, such as loss of body weight, hunched posture, diarrhea, and patchy alopecia. The survival rate 80 days after BMT in mice treated with the mAb was about 70%, whereas all mice treated with control antibodies died within 43 days. Histologic examinations revealed that inflammatory changes in target organs such as the liver, gut, and skin were also ameliorated in mice treated with the mAb compared with control mice. An in vitro assay of T-cell proliferation showed a marked hyporesponsiveness to host alloantigen in samples from mice treated with anti-CD134L mAb. In addition, low levels of interferon γ and transiently elevated levels of interleukin 4 and IgE in serum samples were found in mice treated with anti-CD134L mAb. These results suggest that CD134-CD134L interactions have an important role in the pathogenesis of acute GVHD.

Allogeneic bone marrow transplantation (BMT) is a potentially curative therapy for patients with several disorders of hematopoiesis and certain hematologic malignant diseases.1,2 However, acute graft-versus-host disease (GVHD), one of the major obstacles to allogenic BMT, occurs in up to 75% of recipients of unmanipulated HLA-matched marrow.3Decreasing the number of T cells in the donor marrow by a variety of purging methods can decrease the incidence and the severity of acute GVHD,4-8 but the overall usefulness of T-cell depletion has been questioned because of its association with higher rates of relapse, graft failure, lethal infection, and Epstein-Barr virus–related lymphoproliferative disorders after BMT.9-11Current clinical approaches to decreasing the occurrence of acute GVHD are targeted on selective manipulation of alloreactive T cells. It has been reported that immunotoxin to the interleukin-2 (IL-2) receptor prevented acute GVHD without affecting entire T-cell populations.12-15 

Tumor necrosis factor (TNF) α has been found to participate in the pathogenesis of acute GVHD.16-19 Administration of anti-TNF antibody ameliorated acute GVHD in a murine model.16 Other molecules belonging to the TNF-TNF receptor (TNFR) family are also involved in the pathogenesis of acute GVHD. Blockade of CD40-CD154 (CD40 ligand [L]) interaction by administration of anti-CD154 monoclonal antibody (mAb) ameliorated murine acute and chronic GVHD.20,21 CD95 (Fas, Apo-1)-CD95L interaction was found to be responsible for acute GVHD in studies using functionally CD95L-defective gld mice as donors22-24 and in investigations using anti-CD95L mAb.19 

CD134 (OX40) is also a member of the TNFR family, and its ligand, CD134L, belongs to the TNF family. CD134 is expressed on recently activated CD4+ T cells, and CD134+CD4+ T cells have been reported to increase in human25 and rat26 BMT recipients with acute GVHD. In addition, a study found that anti-CD134 mAb treatment resulted in conversion of chronic GVHD to acute GVHD.27 

In the study described here, we found that administration of anti-CD134L mAb successfully ameliorated lethal acute GVHD in mice after BMT, with induction of hyporesponsiveness to host alloantigen. This treatment also affected the serum cytokine levels in BMT recipients and alleviated various manifestations of GVHD. Anti-CD134L treatment may be useful for selectively manipulating alloreactive T-cell populations in patients with acute GVHD.

Mice

Female 6- to 8-week-old C57BL/6 (B6, H-2b), DBA/2 (H-2d), C3H/HeJ (H-2k), and (C57BL/6 crossed with DBA/2) first filial generation (BDF1, H-2b/d) mice were purchased from Japan SLC (Shizuoka, Japan) and cared for in our animal facilities.

Monoclonal antibodies

Rat antimouse CD134L mAb (RM134L, rat IgG2bκ) was produced in our laboratory as described previously.28Purified antimouse-Thy 1.2 mAb (30-H12), control rat IgG, fluorescein isothiocyanate-conjugated (FITC) anti-CD19 (1D3), anti-CD4 (RM4-5), anti-H-2Kb (AF6-88.5), phycoerythrin (PE)-conjugated anti-CD3 (145-2C11), anti-CD8 (53-6.7), anti-H-2Kd (SF1-1.1), and PE-conjugated avidin were obtained from PharMingen (San Diego, CA). Biotinylated antimouse CD134 mAb (MRC OX86) was prepared as described previously.28 

BMT

Recipient BDF1 mice (10 mice in each group) were lethally irradiated (12 Gy) by using a cobalt 60 irradiator (MBR 1505 R2; Hitachi, Japan). Bone marrow (BM) cells were flushed from the shafts of femurs and tibias of donor mice, and single-cell suspensions were prepared. The BM cells were treated with anti-Thy1.2 mAb and rabbit complement for preparation of T-cell–depleted BM (TCD-BM) cells. Single-cell suspensions of spleen cells from donor mice were used as the source of GVHD effector cells. Recipients received 2 × 107 BM cells with 2.5 × 107 spleen cells (BMS) in 0.5 mL of phosphate-buffered saline administered into the tail vein. To create GVHD-negative controls, TCD-BM cells were transplanted without spleen cells. The day of the BMT was designed as day 0. For the anti-CD134L mAb treatment, 1 mg of the mAb was administered intraperitoneally on the day before BMT (day −1) and on day 0, and then 0.5 mg of the mAb was given intraperitoneally twice a week until day 14 after BMT. We concluded that this dose and treatment period would be the most optimal after titrating the mAb from 0.25 mg per mouse to 1 mg per mouse or extending the treatment period up to 4 weeks after BMT. Rat IgG was also administered to GVHD-positive control recipients. All BMT recipients were given sterilized water containing antibiotics (2 mg/mL of neomycin sulfate) from 2 days before BMT to 14 days afterward. The mice were monitored for clinical signs of GVHD, including death, weight loss, diarrhea, alopecia, and hunched posture. Representative mice were killed at various times after BMT to obtain tissues for histologic examinations.

Histopathologic assessment

Liver, intestine, and skin tissue obtained from mice BMT recipients were fixed in 10% buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and examined microscopically.

Flow cytometric analysis

Spleen cells were obtained from BDF1 mice, recipients of B6 TCD-BM, recipients of B6 BMS plus rat IgG, and recipients of B6 BMS plus anti-CD134L mAb on days 14 and 28 after BMT. Cells were stained with FITC-conjugated CD19, CD4, H-2Kb, PE-conjugated CD3, CD8, H-2Kd, and biotinylated anti-CD134 followed by PE-conjugated avidin and analyzed by means of fluorescence-activated cell sorter (FACScan and Cell Quest program; Becton Dickinson, San Jose, CA). Recipient and donor lymphocytes were identified as H-2Kb+d+ and H-2Kb+d, respectively.

Mixed lymphocyte reaction (MLR)

Responder spleen cells (2 × 105/well) obtained from recipients of TCD-BM, GVHD controls, and recipients of anti-CD134L 14 days after BMT were cultured with irradiated (30 Gy) stimulator DBA/2 (H-2d) or C3H/HeJ (H-2k) spleen cells (2 × 105/well) in 96-well round-bottomed microtiter plates at 37°C in a 5% carbon dioxide humidified atmosphere. Cells were also cultured in the presence of 200 U/mL of recombinant interleukin 2 (IL-2) (Shionogi, Osaka, Japan) to examine their reactivity to alloantigen. After 5 days, the cultures were pulsed for 18 hours with 0.019 MBq per well of tritium thymidine and harvested (Micro 96 Harvester; Skartron, Lier, Norway). Incorporated radioactivity was measured in a β counter (Micro Beta Plus; Wallac, Turku, Finland).

Enzyme-linked immunosorbent assay (ELISA)

Blood samples were obtained from the retro-orbital plexus of mice under ether anesthesia on representative days after BMT. Serum samples were individually divided into aliquots and stored at −40°C until testing. Serum IgE levels were determined by solid-phase ELISA using rat antimouse IgE mAb as described previously.29 Serum interferon γ (IFN-γ) and serum interleukin 4 (IL-4) were evaluated with commercial ELISA kits (PharMingen, San Diego, CA, and Amersham, Buckinghamshire, England, respectively).

Statistical analyses

Data were analyzed by using the Statview program (Brainpower Inc, Calabasas, CA). Comparison assessments included analysis of variance and the Student t test. P values < .05 were considered to represent statistical significance.

Effect of anti-CD134L mAb treatment on acute GVHD-induced mortality and weight loss

Lethal acute GVHD was induced by intravenous inoculation of BMS cells (2 × 107 B6 BM cells plus 2.5 × 107 B6 spleen cells) into lethally irradiated BDF1 mice. As shown in Figure 1A, all recipients given control IgG died within 43 days. In these mice, clinical symptoms of acute GVHD, such as weight loss, hunched posture, diarrhea, and patchy alopecia, were apparent beginning 10 days after BMT. Administration of anti-CD134L mAb significantly improved the survival of BMT recipients; the survival rate among mice given the was 70% on day 80 (Figure 1A). Recovery of body weight in this group was slightly impaired compared with that in recipients of TCD-BM (Figure1B).

Fig. 1.

Prevention of lethal acute graft-versus-host disease (GVHD) by anti-CD134L monoclonal antibody (mAb).

Lethal acute GVHD was induced by transfer of B6 bone marrow (BM) cells with 2.5 × 107 spleen cells (B6 BMS) into lethally irradiated BDF1 mice. Ten mice in each group were given intraperitoneal injections of 1 mg of anti-CD134L mAb on the day before bone marrow transplantation (BMT) (day −1) and the day of BMT (day 0) and then 0.5 mg of anti-CD134L mAb twice a week until day 14 after BMT. For an acute-GVHD–positive control, control rat IgG was administered instead of anti-CD134L mAb. For an acute-GVHD–negative control, B6 T-cell–depleted BM was transplanted without spleen cells. Administration of anti-CD134L mAb to the B6 BMS group ameliorated both mortality (A) and loss of body weight (B) due to acute GVHD. The survival rate of the recipients treated with anti-CD134L mAb was 70% on day 80 after BMT, whereas all mice in the control-IgG group died within 43 days. Pooled results of 3 similar experiments are shown (total n = 30).

Fig. 1.

Prevention of lethal acute graft-versus-host disease (GVHD) by anti-CD134L monoclonal antibody (mAb).

Lethal acute GVHD was induced by transfer of B6 bone marrow (BM) cells with 2.5 × 107 spleen cells (B6 BMS) into lethally irradiated BDF1 mice. Ten mice in each group were given intraperitoneal injections of 1 mg of anti-CD134L mAb on the day before bone marrow transplantation (BMT) (day −1) and the day of BMT (day 0) and then 0.5 mg of anti-CD134L mAb twice a week until day 14 after BMT. For an acute-GVHD–positive control, control rat IgG was administered instead of anti-CD134L mAb. For an acute-GVHD–negative control, B6 T-cell–depleted BM was transplanted without spleen cells. Administration of anti-CD134L mAb to the B6 BMS group ameliorated both mortality (A) and loss of body weight (B) due to acute GVHD. The survival rate of the recipients treated with anti-CD134L mAb was 70% on day 80 after BMT, whereas all mice in the control-IgG group died within 43 days. Pooled results of 3 similar experiments are shown (total n = 30).

Close modal

Effect of anti-CD134L mAb treatment on histopathologic features of acute GVHD

Figure 2 shows a histopathologic analysis of tissues obtained on day 28 after BMT from representative acute-GVHD–positive control mice, mice treated with anti-CD134, and acute-GVHD–negative control mice. In liver tissue from control mice with acute GVHD, a massive infiltration of mononuclear cells was observed mainly in the periportal areas (Figure 2B). In contrast, such inflammatory changes were minimal in liver tissue from mice treated with anti-CD134L (Figure 2C). Skin from control mice with acute GVHD showed severe inflammatory infiltrates with intraepidermal lymphocytes, dyskeratotic cells, ulceration, and loss of hair follicles (Figure 2E). Such changes were not observed in mice given anti-CD134L (Figure 2F). Gut tissue from control mice with acute GVHD showed dilatation, flattening of the villi, and elevation of crypts, all of which are characteristics of intestinal acute GVHD (Figure 2H). Such findings were minimal in mice treated with anti-CD134L (Figure 2I). Recipients of TCD-BM had almost no signs of GVHD in tissues (Figure 2A, 2D, and2G). These results suggest that administration of anti-CD134L mAb prevented development of acute GVHD in recipients of BMS.

Fig. 2.

Histopathologic examination of liver, skin, and intestine tissues from mice with acute GVHD treated with anti-CD134L mAb.

Induction of lethal acute GVHD (B, E, and H) and administration of anti-CD134L mAb (C, F, and I) were performed and acute-GVHD–negative controls (A, D, and G) were generated as described in the legend for Figure 1. On day 28 after BMT, 3 mice in each group were killed. Paraffin sections of the liver (A-C), skin (C-F), and intestine (G-I) were stained with hematoxylin and eosin. The specimens shown are from 3 representative mice in each group with similar histologic features. Original magnification, ×200.

Fig. 2.

Histopathologic examination of liver, skin, and intestine tissues from mice with acute GVHD treated with anti-CD134L mAb.

Induction of lethal acute GVHD (B, E, and H) and administration of anti-CD134L mAb (C, F, and I) were performed and acute-GVHD–negative controls (A, D, and G) were generated as described in the legend for Figure 1. On day 28 after BMT, 3 mice in each group were killed. Paraffin sections of the liver (A-C), skin (C-F), and intestine (G-I) were stained with hematoxylin and eosin. The specimens shown are from 3 representative mice in each group with similar histologic features. Original magnification, ×200.

Close modal

Effect of anti-CD134L mAb treatment on cellular recovery

To assess immune-system recovery in BMT recipients, 3 representative mice in each study group were killed on days 14 and 28 after BMT, and flow cytometry was used to analyze total cell numbers and surface phenotypes (Table 1). The total number of lymphocytes in the spleen of recipients treated with anti-CD134L was lower than that in recipients given TCD-BM. However, the numbers did not decrease progressively and a certain number of CD19+ B cells were preserved. In contrast, in control IgG-treated BMT recipients, the number of spleen cells decreased successively and the decrease in the number of B cells was remarkable. The percentages of CD134+CD4+ T cells were higher in control IgG-treated recipients, suggesting that CD4+ T cells were more activated in that group than in the other 2 groups. Analysis of H-2K haplotype showed greater than 95% of donor haplotype (H-2Kb+d) in BMT recipients treated with anti-CD134L, suggesting that the mAb did not inhibit engraftment of donor cells. The spleen cells harvested on day 14 were also used as responder cells in MLR to assess their alloreactivity.

Effect of anti-CD134L mAb treatment on MLR

To clarify the mechanisms of amelioration of acute GVHD by anti-CD134L mAb, we examined the alloreactivity of lymphocytes from BMT recipients treated with anti-CD134L. Representative mice from each of the 3 study groups were killed 14 days after BMT, and single-cell suspensions of spleen cells were prepared as responder cells in MLR assessments. Spleen cells from DBA/2 (H-2d) and C3H/HeJ (H-2k) mice were also prepared as stimulator cells in MLR assessments as the host alloantigen and the third-party stimulator, respectively (Figure3). Spleen cells from mice with acute GVHD were highly reactive against host alloantigen (H-2d), whereas spleen cells from mice that received TCD-BM showed little response to H-2d. In mice treated with anti-CD134L, the proliferative response to H-2d was significantly suppressed compared with that in acute-GVHD–positive controls, while the response to the third-party H-2k was preserved. These responses were more evident in the presence of exogenous IL-2. These results suggest strongly that treatment with anti-CD134L induced host-alloantigen–specific hyporesponsiveness in our murine model of acute GVHD.

Fig. 3.

Proliferative response to host alloantigen in mice with acute GVHD treated with anti-CD134L mAb.

Representative mice in each indicated group were killed on day 14 after BMT, and single-cell suspensions of spleen cells were prepared for responder cells in a mixed lymphocyte reaction. Responder cells were cultured with irradiated (30 Gy) stimulator DBA/2 (H-2d) or C3H/He (H-2k) spleen cells (2 × 105/well) in the presence or absence of IL-2 (200 U/mL) in 96-well round-bottomed microtiter plates. After 5 days, the cultures were pulsed for 18 hours with tritium thymidine and the incorporated radioactivity was measured in a β counter. Data are the mean ± SD results from triplicate samples. Responder cells from mice with acute GVHD were highly reactive against host alloantigen (H-2d), whereas responder cells from mice treated with anti-CD134L mAb showed little response to H-2d.

Fig. 3.

Proliferative response to host alloantigen in mice with acute GVHD treated with anti-CD134L mAb.

Representative mice in each indicated group were killed on day 14 after BMT, and single-cell suspensions of spleen cells were prepared for responder cells in a mixed lymphocyte reaction. Responder cells were cultured with irradiated (30 Gy) stimulator DBA/2 (H-2d) or C3H/He (H-2k) spleen cells (2 × 105/well) in the presence or absence of IL-2 (200 U/mL) in 96-well round-bottomed microtiter plates. After 5 days, the cultures were pulsed for 18 hours with tritium thymidine and the incorporated radioactivity was measured in a β counter. Data are the mean ± SD results from triplicate samples. Responder cells from mice with acute GVHD were highly reactive against host alloantigen (H-2d), whereas responder cells from mice treated with anti-CD134L mAb showed little response to H-2d.

Close modal

Effect of anti-CD134L treatment on serum cytokines and IgE

It has been reported that IFN-γ is responsible for Th1-type responses such as acute forms of GVHD, whereas IL-4 is related to Th2-type responses such as chronic forms of GVHD.30-32 We evaluated IFN-γ and IL-4 levels in serum samples from representative mice killed 7, 14, 28, and 56 days after BMT (Table2). In mice with acute GVHD, IFN-γ levels were elevated early after BMT and decreased gradually until day 28. Serum IFN-γ levels on day 7 were lower in BMT recipients that received anti-CD134L mAb than in those given control antibody (P < .05) and were undetectable in recipients given TCD-BM. Interestingly, serum IL-4 levels were markedly higher on day 28 in BMT recipients given anti-CD134L mAb than in those that received control antibody or TCD-BM.

We also analyzed serum IgE levels as a marker of murine chronic GVHD. Serum IgE levels, similar to IL-4 levels, were increased in BMT recipients given anti-CD134L mAb (Table 3). The recipients treated with anti-CD134L mAb that survived for longer than 80 days after BMT had no typical manifestations of chronic GVHD as described previously.29 These results suggest that anti-CD134L treatment inhibited IFN-γ production and consequently enhanced IL-4 and IgE production transiently.

It has been reported that expression of CD134 on CD4+ T cells is up-regulated in patients with acute GVHD after BMT.25 Tittle et al26 also found that CD134 is expressed on alloreactive CD4+ T cells derived from donor grafts. These observations suggest that CD134-CD134L interaction may be involved in T-cell functions responsible for acute GVHD. In this study, we examined the ameliorating and preventive effects on acute GVHD of a newly established mAb against murine CD134L in a murine model of lethal acute GVHD.

Administration of anti-CD134L mAb ameliorated the lethality of acute GVHD (Figure 1) and the pathologic manifestations of acute GVHD in target tissues such as the liver, intestine, and skin (Figure 2). CD134 is known to be expressed on CD4+ T cells after activation and ligation of CD134 with CD134L further activates these T cells expressing CD134.33,34 Therefore, the mAb against CD134L might inhibit this costimulatory process of CD134+ T cells. Because the anti-CD134L mAb used in the current study did not deplete the target cells or possess the capacity for signaling,28we could conclude that amelioration of acute GVHD by administration of the mAb was due to in vivo blockade of CD134-CD134L interaction. However, we cannot completely exclude the presence of other mechanisms. Studies using monovalent mAb fragments might be required to clarify this issue.

We also demonstrated that host-alloantigen–specific hyporesponsiveness was induced by administration of anti-CD134L mAb (Figure 3). Because the response to the third-party alloantigen was preserved, the mAb specifically inhibited the response to host alloantigen in vivo. The host-alloantigen–specific hyporesponsiveness in BMT recipients treated with anti-CD134L may have been due to cell death rather than anergy of responsible T cells because the number of CD3 T cells decreased and the decreased response observed on MLR assessment could not be overcome by IL-2. Addition of the mAb to the MLR in vitro failed to inhibit the response to host alloantigen (data not shown). Thus, these findings suggest that CD134-CD134L interactions contribute minimally to the direct presentation of host alloantigen in MLR in vitro but contribute to a large extent to the indirect presentation of host alloantigen in vivo. These results indicate that APC has a potentially important role in antigen presentation and that T-cell priming occurs in the early phase of immune responses. In fact, administration of anti-CD134L mAb after BMT failed to ameliorate the lethality of acute GVHD (data not shown). Therefore, it appears that early blockade of CD134-CD134L interactions in the process of host-alloantigen recognition and subsequent activation of donor T cells is required to suppress development of acute GVHD. CD134-CD134L interactions might be involved in the induction of tolerance. We found previously28that CD134L expressed on the surface of activated B cells can provide CD28-independent costimulatory signals to T cells. In addition, CD134L has been reported to be expressed on dendritic cells35; thus, the mAb might block the interaction between CD134+ T cells and CD134L-expressing APCs. This idea is in agreement with studies showing an ameliorating effect of CD134-Ig in both murine experimental allergic encephalomyelitis (EAE) and inflammatory bowel disease models.36,37 Weinberg et al36 demonstrated that CD134L is expressed on CD11b+ APC derived from the central nervous system of mice with active EAE and that CD134-Ig can inhibit the interaction between antigen-specific T cells and CD11b+APCs. However, it should be noted that there are other molecular pairs known to be responsible for the pathogenesis of acute GVHD, such as CD28-CD152 (CTLA4) and their ligands,37-40 and CD40 and its ligand.20,21 40 Thus, the interaction between CD134 and its ligand may play an important role in the pathogenesis or pathophysiologic aspects of acute GVHD either independently or synergistically with other costimulatory molecular pairs, suggesting the presence of complex in vivo interactions between T cells and APCs in disease states.

In this study, anti-CD134L mAb inhibited IFN-γ production in the early phase after BMT and elevated IL-4 and IgE production in the relatively late phase, suggesting that the Th1-type response that normally develops in mice with acute GVHD was suppressed and might have been transiently converted to a Th2-type response. However, it has been reported that ligation of CD134 expressed on CD4+ T cells with CD134L can induce a Th2-type response.41 These results disagree with our finding that administration of the mAb suppressed the Th1-type response and led to a transient Th2-type response. If our mAb had blocked the induction of the Th2-type response, acute GVHD would have been exacerbated. At present, we do not have direct evidence that CD134-CD134L interaction induces Th1-type responses in our acute-GVHD model. Whether the apparently opposite results were caused by different assay systems (ie, in vivo compared with vitro) or different mouse strains (C57BL/6 or DBA/2 compared with BALB/c or C57BL/6) remains to be determined.

Studies have found that acute GVHD is ameliorated by administration of interleukin 10,42 which is known to be one of the Th2-type cytokines, or by transfer of CD4+ or CD8+ T cells polarized to type 2.43 It has also been reported that administration of interleukin 12, which is known to be one of the Th1-type cytokines, converted Th2-type chronic GVHD to Th1-type acute GVHD.44 On the other hand, Murphy et al,45 in a study using mice deficient in IFN-γ or IL-4 as donors, clearly demonstrated that donor T cells lacking IFN-γ accelerated acute GVHD, whereas those lacking IL-4 prevented acute GVHD. According to their results, IFN-γ prevents the occurrence of acute GVHD and IL-4 exacerbates acute GVHD, contrary to our speculation. It must still be determined whether the decreased IFN-γ and increased IL-4 levels in the mice in our study that were given anti-CD134L were the primary cause of amelioration of acute GVHD or a secondary consequence of the ameliorating effect.

Imura et al46 reported that CD134L is expressed on vascular endothelial cells and related to tissue infiltration of CD134-expressing adult T-cell leukemia-lymphoma cells. It has also been reported that expression of CD134L on vascular endothelial cells is related to various skin diseases.47 Thus, it is possible that administration of the mAb could suppress the infiltration of T cells into CD134L-expressing tissue.

In summary, we believe that anti-CD134L treatment could ameliorate acute GVHD by (1) inhibition of antigen presentation and T-cell priming, (2) modulation of balance between Th1 and Th2 immune responses, (3) inhibition of T-cell migration to target tissue, or (4) a combination of these mechanisms. Our results should help to reveal additional aspects of the complex regulatory mechanisms of acute GVHD and contribute to development of a novel strategy for treatment of this disease.

We thank T. Hirano, H. Miyajima, and Y. Hayakawa for technical assistance and helpful suggestions.

Supported by grants from the Ministry of Education, Science and Culture, and the Ministry of Health, Japan.

Reprints:Tetsuji Kobata, Division of Immunology, Institute for Medical Science, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, Japan; e-mail:tkobata@dokkyomed.ac.jp.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Thomas
ED
Karnofsky memorial lecture. Marrow transplantation for malignant disease.
J Clin Oncol.
1
1983
517
2
Bortin
MM
Rimm
AA
Increasing utilization of bone marrow transplantation.
Transplantation.
42
1986
229
3
Herve
P
Cahn
JY
Beatty
PG
Graft-versus-host disease after bone marrow transplantation from donors other than HLA-identical siblings.
Graft-Versus-Host Disease.
Burakoff
SJ
Deeg
HJ
Ferrara
J
Atkins
K
1990
425
Dekker
New York, NY
4
Wagner
JE
Donnenberg
AD
Noga
SJ
et al
Lymphocyte depletion of donor bone marrow by counterflow centrifugal elutriation: results of a phase I clinical trial.
Blood.
72
1988
1168
5
Young
JW
Papadopoulos
EB
Cunningham
I
et al
T-cell-depleted allogeneic bone marrow transplantation in adults with nonlymphocytic leukemia in first remission.
Blood.
79
1992
3380
6
Filipovich
AH
Vallera
D
McGlave
P
et al
T cell depletion with anti-CD5 immunotoxin in histocompatible bone marrow transplantation. The correlation between residual CD5 negative T cells and subsequent graft-versus-host disease.
Transplantation.
50
1990
410
7
Hale
G
Cobbold
S
Waldmann
H
T cell depletion with CAMPATH-1 in allogeneic bone marrow transplantation.
Transplantation.
45
1988
753
8
Soiffer
RJ
Fairclough
D
Robertson
M
et al
CD6-depleted allogeneic bone marrow transplantation for acute leukemia in first complete remission.
Blood.
89
1997
3039
9
Marmont
AM
Horowitz
MM
Gale
RP
et al
T-cell depletion of HLA-identical transplants in leukemia.
Blood.
78
1991
2120
10
Goldman
JM
Gale
RP
Horowitz
MM
et al
Bone marrow transplantation for chronic myelogenous leukemia in chronic phase: increased risk for relapse associated T-cell depletion.
Ann Intern Med.
108
1988
806
11
Mitsuyasu
RT
Champlin
RE
Gale
RP
et al
Treatment of donor bone marrow with monoclonal anti-T-cell antibody and complement for the prevention of graft-versus-host disease. A prospective, randomized, double-blind trial.
Ann Intern Med.
105
1986
20
12
Herve
P
Wijdenes
J
Bergerat
JP
et al
Treatment of corticosteroid resistant acute graft-versus-host disease by in vivo administration of anti-interleukin-2 receptor monoclonal antibody (B-B10).
Blood.
75
1990
1017
13
Anasetti
C
Martin
PJ
Hansen
JA
et al
A phase I-II study evaluating the murine anti-IL-2 receptor antibody 2A3 for treatment of acute graft-versus-host disease.
Transplantation.
50
1990
49
14
Blaise
D
Olive
D
Hirn
M
et al
Prevention of acute GVHD by in vivo use of anti-interleukin-receptor monoclonal antibody (33B31.1).
Bone Marrow Transplant.
8
1991
105
15
Anasetti
C
Hansen
JA
Waldmann
TA
et al
Treatment of acute graft-versus-host disease with humanized anti-Tac: an antibody that binds to the interleukin-2 receptor.
Blood.
84
1994
1320
16
Piguet
PF
Grau
GE
Allet
B
Vassalli
P
Tumor necrosis factor/cachectin is an effector of skin and gut lesions of the graft-vs.-host disease.
J Exp Med.
166
1987
1280
17
Speiser
DE
Bachmann
MF
Frick
TW
et al
TNF receptor p55 controls early acute graft-versus-host disease.
J Immunol.
158
1997
5185
18
Hattori
K
Hirano
T
Ushiyama
C
et al
A metalloproteinase inhibitor prevents lethal acute graft-versus-host disease in mice.
Blood.
90
1997
542
19
Hattori
K
Hirano
T
Miyajima
H
et al
Differential effects of anti-Fas ligand and anti-tumor necrosis factor α antibodies on acute graft-versus-host disease pathologies.
Blood.
91
1998
4051
20
Blazar
BR
Taylor
PA
Panoskaltsis-Mortari
A
et al
Blockade of CD40 ligand-CD40 interaction impairs CD4+ T cell-mediated alloreactivity by inhibiting mature donor T cell expansion and function after bone marrow transplantation.
J Immunol.
158
1997
29
21
Durie
FH
Aruffo
A
Ledbetter
J
et al
Antibody to the ligand of CD40, gp39, blocks the occurrence of the acute and chronic forms of graft-versus-host disease.
J Clin Invest.
94
1994
1333
22
Braun
MY
Lowin
B
French
L
Acha-Orbea
H
Tschopp
J
Cytotoxic T cells deficient in both functional Fas ligand and perforin show residual cytolytic activity yet lose their capacity to induce lethal acute graft-versus-host disease.
J Exp Med.
183
1996
657
23
Via
CS
Nguyen
P
Shustov
A
Drappa
J
Elkon
KB
A major role for the Fas pathway in acute graft-versus-host disease.
J Immunol.
157
1996
5387
24
Baker
MB
Altman
NH
Podack
ER
Levy
RB
The role of cell-mediated cytotoxicity in acute GVHD after MHC-matched allogeneic bone marrow transplantation in mice.
J Exp Med.
183
1996
2645
25
Weinberg
AD
Bourdette
DN
Sullivan
TJ
et al
Selective depletion of myelin-reactive T cells with the anti-OX40 antibody ameliorates autoimmune encephalomyelitis.
Nat Med.
2
1996
183
26
Tittle
TV
Weinberg
AD
Steinkeler
CN
Maziarz
RT
Expression of the T-cell activation antigen, OX-40, identifies alloreactive T cells in acute graft-versus-host disease.
Blood
89
1997
4652
27
Durie
FH
Fanslow
WC
Zappone
J
et al
Conversion of chronic graft-versus-host disease to that of the acute phenotype using in vivo administration of an antibody to OX40 [abstract].
FASEB J.
10
1996
1435
28
Akiba
H
Oshima
H
Takeda
K
et al
CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.
J Immunol.
162
1999
7058
29
Ushiyama
C
Hirano
T
Miyajima
H
Okumura
K
Ovary
Z
Hashimoto
H
Anti-IL-4 antibody prevents graft-versus-host disease in mice after bone marrow transplantation.
J Immunol.
154
1995
2687
30
Allen
RD
Staley
TA
Sidman
CL
Differential cytokine expression in acute and chronic murine graft-versus-host disease.
Eur J Immunol.
23
1993
333
31
Krenger
W
Ferrara
JL
Graft-versus-host disease and the Th1/Th2 paradigm.
Immunol Res.
15
1996
50
32
Tanaka
J
Imamura
M
Kasai
M
et al
The important balance between cytokines derived from type 1 and type 2 helper T cells in the control of graft-versus-host disease.
Bone Marrow Transplant.
19
1997
571
33
Baum
PR
Gayle
RB
III
Ramsdell
F
et al
Molecular characterization of murine and human OX40/OX40 ligand systems: identification of a human OX40 ligand as the HTLV-1-regulated protein gp34.
EMBO J.
13
1994
3992
34
Godfrey
WR
Fagnoni
FF
Harara
MA
Buck
D
Engleman
EG
Identification of human OX-40 ligand, a costimulator of CD4+ T cells with homology to tumor necrosis factor.
J Exp Med.
180
1994
757
35
Ohshima
Y
Tanaka
Y
Tozawa
H
Takahashi
Y
Maliszewski
C
Delespesse
G
Expression and function of OX40 ligand on human dendritic cells.
J Immunol.
159
1997
3838
36
Weinberg
AD
Wegmann
KW
Funatake
C
Whitham
RH
Blocking OX40/0X-40 ligand interaction in vivo and in vitro leads to decreased T cell function and amelioration of experimental allergic encephalomyelitis.
J Immunol.
162
1999
1818
37
Higgins
LM
McDonald
SAC
Whittle
N
Crockett
N
Shields
JG
McDonald
TT
Regulation of T cell activation in vitro and in vivo by targeting the OX40-OX40 ligand interaction: amelioration of ongoing inflammatory bowel disease with an OX40-IgG fusion protein, but not with an OX40 ligand-IgG fusion protein.
J Immunol.
162
1999
486
38
Blazer
BR
Taylor
PA
Linsley
PS
Vallera
DA
In vivo blockade of CD28/CTLA4: B7/BB1 interaction with CTLA4-Ig reduces lethal murine graft-versus-host disease across the major histocompatibility complex barrier in mice.
Blood.
83
1994
3815
39
Saito
K
Azuma
M
Hashimoto
H
Yagita
H
Okumura
K
Effect of CD80 and CD86 blockade and anti-interleukin-12 treatment on mouse acute graft-versus-host disease.
Eur J Immunol.
26
1996
3098
40
Saito
K
Sakurai
J
Ohta
J
et al
Involvement of CD40 ligand-CD40 and CDLA4-B7 pathways in murine acute graft-versus-host disease induced by allogeneic T cells lacking CD28.
J Immunol.
160
1998
4225
41
Flynn
S
Toellner
KM
Raykundalia
Goodall
M
Lane
P
CD4 T cell cytokine differentiation: the B cell activation molecule OX40 ligand, instructs CD4 T cells to express interleukin 4 and upregulates expression of the chemokine receptor, Blr-1.
J Exp Med.
188
1998
297
42
Smith
SR
Terminelli
C
Pennline
KJ
Kenworthy-Bott
L
Donkin
J
Calzetta
A
Inhibitory effects of recombinant human interleukin 10 on disease manifestation in a P→F1 model of acute graft versus host disease.
Transplantation.
59
1995
890
43
Krenger
W
Snyder
KM
Byon
JCH
Falzarano
G
Ferrara
JLM
Polarized type 2 alloreactive CD4+ and CD8+ donor T cells fail to induce experimental acute graft-versus-host disease.
J Immunol.
155
1995
585
44
Via
CS
Rus
V
Gately
MK
Finkelman
FD
IL-12 stimulates the development of acute graft-versus-host disease in mice that normally would develop chronic, autoimmune graft-versus-host disease.
J Immunol.
153
1994
4040
45
Murphy
WJ
Welniak
LA
Taub
DD
et al
Differential effect of the absence of interferon-g and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice.
J Clin Invest.
102
1998
1742
46
Imura
A
Hori
T
Imada
K
et al
OX40 expressed on fresh leukemic cells from adult T-cell leukemia patients mediates cell adhesion to vascular endothelial cells: implication for the possible involvement of OX40 in leukemic cell infiltration.
Blood.
89
1997
2951
47
Matsumura
Y
Imura
A
Hori
T
Uchiyama
T
Imamura
S
Localization of OX40/gp34 in inflammatory skin disease: a clue to elucidate the interaction between activated T cells and endothelial cells in infiltration.
Arch Dermatol Res.
289
1997
653
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