anti-OX40L mAb Research Articles

Cardiac Allograft Tolerance Induction with Ox40L Costimulatory Blockade

<h3>Purpose</h3> Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group via hematopoietic mixed chimerism with allogeneic bone marrow and kidney co-transplantation. This model has failed without the regulatory mechanisms associated with the donor kidney. We hypothesized that rapamycin and anti-OX40L mAb (KY1005, Kymab Ltd) could substitute for kidney co-transplantation and induce cardiac allograft tolerance by providing a favorable immunologic milieu for regulatory T cells (Tregs). <h3>Methods</h3> Fourteen cynomolgus NHPs underwent mixed chimerism conditioning (ATGAM, total body and thymic irradiation) then organ and bone marrow transplant. Heart Alone recipients received a heart transplant (n=5), Heart/Kidney recipients received a heart and kidney co-transplant (n=7). Heart/OX40L recipients received a heart transplant plus 40 days of anti-OX40L mAb (n=2). All groups underwent a 12 day course of aCD154 mAb. The Heart Alone and Heart Kidney groups received 28 days of cyclosporine while the Heart aOX40L group received 100 days of rapamycin. <h3>Results</h3> All Heart Alone recipients had cellular rejection (ISHLT 3R) by 200 days post bone marrow transplant (pBMT). No Heart/Kidney recipients showed early rejection. Similarly, no Heart/OX40L recipients showed early rejection and one is currently 258 days pBMT without clinical or pathologic evidence of rejection (Figure 1). The other recipient died of viral complications. Mean peak lymphoid chimerism was 10% for the Heart Alone group, 26% for the Heart/Kidney group, and 29% for the Heart/OX40L group. The anti-OX40L protocol also showed an expansion of the Treg population with a peak of 35% Tregs in the CD4+ population. <h3>Conclusion</h3> The OX40L protocol achieved robust lymphoid chimerism with concomitant expansion of Tregs. The surviving Heart/OX40L recipient achieved longer freedom from allograft rejection when compared to the Heart Alone group. OX40L costimulatory blockade may allow for heart allograft tolerance without kidney co-transplantation.

Blockade of OX40/OX40L pathway combined with ethylene-carbodiimide-fixed donor splenocytes induces donor-specific allograft tolerance in presensitized recipients.

Memory T cells (Tms) are the major barrier preventing long-term allograft survival in presensitized transplant recipients. The OX40/OX40L pathway is important in the induction and maintenance of Tms. In this study, we added anti-OX40L mAb to ethylene-carbodiimide-fixed donor splenocytes (ECDI-SPs)-a method which is effective in inducing allograft tolerance in non-presensitized mouse heart transplant model. Recipient mice received heart transplantation after 6 weeks of donor skin presensitization and were treated with anti-OX40L mAb, ECDI-SPs or anti-OX40L mAb + ECDI-SPs, respectively. Our data showed that the combination of ECDI-SPs and anti-OX40L mAb induced donor-specific tolerance in skin-presensitized heart transplant recipients, with the mechanism for this being associated with suppression of Tms and upregulation of CD4+CD25+Foxp3+ T regulatory cells (Tregs). Importantly, CD25+ T-cell depletion in the combined therapy-treated recipients broke the establishment of allograft tolerance, whereas adoptive transfer of presensitization-derived T cells into tolerant recipients suppressed Tregs expansion and abolished established tolerance. Blockade of OX40/OX40L pathway in combination with ECDI-SPs appears to modulate the Tms/Tregs imbalance so as to create a protective milieu and induce graft tolerance in presensitized recipients.

Regulation of Th1/Th2 balance through OX40/OX40L signalling by glycyrrhizic acid in a murine model of asthma.

Glycyrrhizic acid (GA) has been reported to have attenuating airway inflammation effects in asthma mouse model. However, the potential molecular mechanisms by which GA exerts anti-inflammatory effects on ovalbumin (OVA)-induced allergic asthma have not been well elaborated. The effect of GA on OVA-sensitized and challenged mice was investigated. The effect of GA on anti-OX40 mAb stimulated splenocytes from asthma mice model was also examined. In OVA-induced asthmatic mice, GA treatment prevented the decrease of T helper1 cytokine (interferon (IFN)-γ) and the increase of T helper2 cytokines (interleukin (IL)-4, IL-5, IL-13) in bronchoalveolar lavage fluid (BALF), reduced serum immunoglobulin (Ig)E and OVA-specific IgE levels, prohibited the protein and mRNA expression of OX40 and OX40 Ligand (OX40L) in lung tissues, and the expression of OX40 in CD4(+) T cells and OX40L in CD11b(+) monocytes and CD19(+) B cells in spleens in a dose-dependent manner compared with the vehicle treatment (all P < 0.05). Moreover, OVA significantly increased the activation of p38 mitogen-activated protein kinase (MAPK) in lung tissues, whereas GA and anti-OX40L mAb markedly reduced phosphorylation of p38 MAPK. In addition, GA could inhibit the T cell proliferation and modulate the balance of Th1/Th2 in anti-OX40 mAb stimulated CD4(+) T cells from asthmatic spleens (all P < 0.05). GA may exert a therapeutic effect on OVA-induced experimental asthma partly by regulating the Th1/Th2 balance through suppressing OX40-OX40L signalling and p38 MAPK activity. GA may be a promising treatment for asthma.

An immunotherapeutic strategy for prolonging graft survival in mice

BackgroundImmunological memory is one barrier to inducing immune tolerance in alloantigen primed transplant models. Alloreactive memory cells induced by primary organ transplant are difficult to suppress and contribute to rejection following a retransplant. Here, we assess the effects of a novel immunosuppressive strategy on graft survival and host immune response. Materials and methodsAlloantigen primed splenocytes were adoptively transferred into naïve B6 mice before heart transplant. Recipient mice were then treated with mitomycin C -pretreated donor-specific transfusion, low dose rapamycin, and anti-OX40L mAb (alone and in combination). ResultsTreatment with donor-specific transfusion+rapamycin+anti-OX40L increased the mean graft survival time from 5.2days (control group) to 42.5days (p<0.05). None of the grafts in the control group survived past 10days, and only 1 in 3 grafts survived at least 50days in the combined treatment group. Compared with the control and single treatment groups, the combination of donor-specific transfusion+rapamycin + anti-OX40L inhibited infiltration of inflammatory cells; reduced the percentages of CD4+ and CD8+ memory T cells in the blood, spleen, and lymph nodes; suppressed inflammatory cytokines; reduced serum IgG levels; and enhanced the percentage of CD4+Foxp3+ T cells. The percentage of regulatory T cells was the highest in the recipients with long-term graft survival. ConclusionsThe combination of donor-specific transfusion+rapamycin+anti-OX40L inhibits graft rejection mediated by memory cells and is likely to contribute to prolonged allograft survival.

Anti-OX40L monoclonal antibody prolongs secondary heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade

BackgroundMemory T cells (Tms) form a barrier against long-term allograft survival; however, CD4+Foxp3+ regulatory T cells (Tregs) can suppress allograft rejection. The OX40/OX40L pathway is critical to the generation of Tms and turns off Treg suppressor function. MethodsB6 mice that rejected BALB/c skin grafts after 4weeks were used as the secondary heart transplant recipients. The skin recipient mice, termed S0, S2 and S3, were treated with the isotype antibodies, anti-CD40L/LFA-1 or anti-OX40L combined with anti-CD40L/LFA-1 mAbs, respectively. The secondary heart recipients, termed H0 and H2, received anti-CD40L/LFA-1 mAbs or not, respectively (Fig. 1). ResultsFour weeks after primary skin transplantation, the Tms in the S3 group that received anti-OX40L with anti-CD40L/LFA-1 mAbs were reduced compared to those in the S2 group (CD4+ Tm: 32.61±2.20% in S2 vs. 25.36±1.16% in S3; CD8+ Tm: 27.76±1.96% in S2 vs. 20.95±1.30% in S3; P<0.01). Meanwhile, the proportions of Tregs in S3 increased compared to those in S2 (P<0.05). The anti-OX40L with anti-CD40L/LFA-1 mAbs group (S3H2) prolonged the mean survival time (MST) following secondary heart transplantation from 9.5days to 21days (P<0.001). Furthermore, allogeneic proliferation of recipient splenic T cells and graft-infiltrating lymphocytes were significantly inhibited in the S3H2 group. Additionally, a higher level of IL-10 was detected in sera and allografts. ConclusionsAnti-OX40L mAb could prolong secondary heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade. The mechanism of protecting allografts using anti-OX40L mAb involved impairing the generation of Tm and up-regulating IL-10 producing Tregs, inhibiting the function of T cells.

Effects of OX40–OX40 ligand interaction on the levels of ROS and Cyclophilin A in C57BL/6J mice atherogenesis

BackgroundAn increasing amount of evidence shows that the OX40–OX40L interaction serves an important function in atherosclerosis. However, the mechanism of the OX40 signaling pathway remains unclear. This study investigates the effect of OX40–OX40L interaction on the levels of intracellular reactive oxygen species (ROS) and the secretion of Cyclophilin A (CyPA) in C57BL/6J mice atherogenesis. MethodsThe atherosclerotic plaque model was established by placing a rapid perivascular carotid collar on C57BL/6J mice fed with a western-type diet. In vivo, the expressions of CyPA in mouse plaque and lymphocytes were detected by immunohistochemical and Western blot analyses, respectively. In vitro, the expression of CyPA protein in cultured lymphocytes of C57BL/6J mice was assessed by using Western blot analysis. The level of ROS was detected through flow cytometry. ResultsCyPA expression was significantly increased in the atherosclerotic lesions and lymphocytes from C57BL/6J mice. The ROS levels in OX40+-lymphocytes were increased in vitro and in vivo. After stimulating the OX40–OX40L interaction, the ROS and CyPA levels in lymphocytes were obviously increased in vitro, whereas anti-OX40L mAb significantly down-regulated the anti-OX40 mAb-induced ROS generation and inhibited CyPA secretion in lymphocytes. ConclusionThe OX40–OX40L interaction up-regulates intracellular levels of ROS in C57BL/6J mice and increases CyPA secretion in lymphocytes. Increased CyPA secretion may serve an important function in atherosclerotic plaque formation.

OX40 ligand regulates splenic CD8− dendritic cell-induced Th2 responses in vivo

In mice, splenic conventional dendritic cells (cDCs) can be separated, based on their expression of CD8α into CD8− and CD8+ cDCs. Although previous experiments demonstrated that injection of antigen (Ag)-pulsed CD8− cDCs into mice induced CD4 T cell differentiation toward Th2 cells, the mechanism involved is unclear. In the current study, we investigated whether OX40 ligand (OX40L) on CD8− cDCs contributes to the induction of Th2 responses by Ag-pulsed CD8− cDCs in vivo, because OX40–OX40L interactions may play a preferential role in Th2 cell development. When unseparated Ag-pulsed OX40L-deficient cDCs were injected into syngeneic BALB/c mice, Th2 cytokine (IL-4, IL-5, and IL-10) production in lymph node cells was significantly reduced. Splenic cDCs were separated to CD8− and CD8+ cDCs. OX40L expression was not observed on freshly isolated CD8− cDCs, but was induced by anti-CD40 mAb stimulation for 24h. Administration of neutralizing anti-OX40L mAb significantly inhibited IL-4, IL-5, and IL-10 production induced by Ag-pulsed CD8− cDC injection. Moreover, administration of anti-OX40L mAb with Ag-pulsed CD8− cDCs during a secondary response also significantly inhibited Th2 cytokine production. Thus, OX40L on CD8− cDCs physiologically contributes to the development of Th2 cells and secondary Th2 responses induced by Ag-pulsed CD8− cDCs in vivo.

Memory T Cells Mediate Cardiac Allograft Vasculopathy and are Inactivated by Anti-OX40L Monoclonal Antibody.

Cardiac allograft vasculopathy (CAV) is a major complication limiting the long-term survival of cardiac transplants. The role of memory T cells (Tmem) in the pathogenesis of CAV remains elusive. This study investigated the role of Tmem cells in the development of CAV and the therapeutic potential of targeting the OX40/OX40L pathway for heart transplant survival. Tmem cells were generated in Rag-1(-/-) C57BL/6 (B6) mice by homeostatic proliferation (HP) of CD40L null CD3(+) T cells from B6 mice. Rag-1(-/-) B6 mice (H-2(b)) harboring Tmem cells received cardiac allografts from BALB/c mice (H-2(d)), and were either untreated or treated with anti-OX40L monoclonal antibody (mAb) (0.5mg/mouse/day) for 10days. Six weeks after HP, the majority of transferred CD40L(-/-) T cells in Rag-1(-/-) B6 mice were differentiated to CD44(high) and CD62L(low) Tmem cells. BALB/c heart allografts in Rag-1(-/-) B6 recipient mice in the presence of these Tmem cells developed a typical pathological feature of CAV; intimal thickening, 100days after transplantation. However, functionally blocking the OX40/OX40L pathway with anti-OX40L mAb significantly prevented CAV development and reduced the Tmem cell population in recipient mice. Anti-OX40L mAb therapy also significantly decreased cellular infiltration and cytokine (IFN-γ, TNF-α and TGF-β) expression in heart allografts. Tmem cells mediate CAV in heart transplants. Functionally blocking the OX40/OX40L pathway using anti-OX40L mAb therapy prevents Tmem cell-mediated CAV, suggesting therapeutic potential for disrupting OX40-OX40L signaling in order to prevent CAV in heart transplant patients.

Natural OX40L expressed on human T cell leukemia virus type-I-immortalized T cell lines interferes with infection of activated peripheral blood mononuclear cells by CCR5-utilizing human immunodeficiency virus

BackgroundOX40 ligand (OX40L) co-stimulates and differentiates T cells via ligation of OX40 that is transiently induced on T cells upon activation, resulting in prolonged T cell survival and enhanced cytokine production by T cells. This view has led to the targeting of OX40 as a strategy to boost antigen specific T cells in the context of vaccination. In addition, the ligation of OX40 has also been shown to inhibit infection by CCR5-utilizing (R5) but not CXCR4-utilizing (X4) human immunodeficiency virus type-1 (HIV-1) via enhancement of production of CCR5-binding β-chemokines. It was reasoned that human T cell leukemia virus type-I (HTLV-1) immortalized T cell lines that express high levels of OX40L could serve as an unique source of physiologically functional OX40L. The fact that HTLV-1+ T cell lines simultaneously also express high levels of OX40 suggested a potential limitation.ResultsResults of our studies showed that HTLV-1+ T cell lines bound exogenous OX40 but not OX40L, indicating that HTLV-1+ T cell lines express an active form of OX40L but an inactive form of OX40. Anti-OX40 non-blocking monoclonal antibody (mAb), but not blocking mAb, stained HTLV-1+ T cell lines, suggesting that the OX40 might be saturated with endogenous OX40L. Functionality of the OX40L was confirmed by the fact that a paraformaldehyde (PFA)-fixed HTLV-1+ T cell lines inhibited the infection of autologous activated peripheral blood mononuclear cells (PBMCs) with R5 HIV-1 which was reversed by either anti-OX40L blocking mAb or a mixture of neutralizing mAbs against CCR5-binding β-chemokines.ConclusionsAltogether, these results demonstrated that autologous T cell lines immortalized by HTLV-1 can be utilized as a conventional source of physiologically functional OX40L.

OX40-OX40L INTERACTION TARGETS NFATC1 IN APOE−/− MICE DURING ATHEROGENESIS

ObjectivesWe investigated the effect of OX40-OX40L interaction on the nuclear factor of activated T cells c1 (NFATc1) in ApoE−/− mice.MethodsAtherosclerotic plaque was induced by rapid perivascular carotid collar placement in...

CD4 + CD25 + regulatory T cells-induced tolerance of islet transplantation by targeting OX40/ OX40L costimulation pathway

Objective To observe the role of OX40/OX40L costimulation pathway in the CD4+CD25 + regulatory T (Treg) cells-induced tolerance of islet transplantation.Methods Diabetes mellitus was induced in C57BL/6 mice as recipients,and islets from DBA/2 mice were transplanted into the C57BL/6 mice.The recipients were divided into three groups (n =8):treated with IgG as controls,antiOX40L mAb (RM134L) + anti-CD154 mAb (MR1),and RM134L + MR1 + anti-CD25 mAb.The mean survival time (MST) of allograft was observed.The expression of OX40 in T effector cells,Treg cells of wide type and CD154 knock-out mice was detected.The expression of Foxp3 gene in allograft in the absence and absence of anti-OX40L mAb and anti-CD154 mAb was detected.T effector cells and Treg cells were co-cultured at different ratio,and the cell proliferation was assayed.Results The MST in RM134L +MR1 group was ＞ 150 days (n =8),significantly longer than in IgG group ( MST + 19 days,n =5 ) and RM134L + MR1 + anti-CD25 mAb (23 days,n =4) (P ＜0.05).Anti-OX40L mAb and anti-CD154 mAb treatment could successfully induce tolerance,and also induce donor specific tolerance,but the tolerance was blocked by anti-CD25 mAb treatment (P ＜ 0.05 ).OX40 was expressed on the Treg cells surface,whereas the T effector cells had no OX40 expression.The expression level of Foxp3 gene in the allograft treated with anti-OX40L mAb and anti-CD154 mAb was 123 AU,significantly higher than without treatment (21 AU) and syngenic control ( 8 AU) ( P ＜ 0.05 ).After Treg cells were co-cultured with T effector cells at different ratio ( 1∶2 and 1∶ 4),the mean count per minute (CPM) of T effector cells was 63 x 103and 41 × 103 respectively (P ＜0.05),and after addition of OX40,the CPM was 128 × 103 and 135 × 103respectively ( P ＞ 0.05 ).Conclusion CD4 + CD25 + Treg cells were critical to tolerance induction in mice islet transplant recipients through targeting OX40/OX40L costimulation pathway. Key words: Islet transplantation; Regulatory T cells; Tolerance; OX40

Suppression of CCR5-Tropic HIV Type 1 Infection by OX40 Stimulation via Enhanced Production of β-Chemokines

To elucidate the immunological role for the costimulatory molecule OX40 against the early stage of HIV-1 infection, fresh peripheral blood mononuclear cells (PBMCs) from normal donors were stimulated with immobilized anti-CD3 monoclonal antibody (mAb) together with soluble anti-CD28 mAb for 24 h, infected with CCR5-tropic (R5) HIV-1, and then cocultured in the presence or absence of OX40 ligand (OX40L). Results of these studied showed that OX40 stimulation led to a marked reduction in levels of p24, the frequency of intracellular p24(+) cells, as well as HIV-1-mediated syncytium formation. The suppression was reversed by anti-OX40L mAb. The mechanism underlying the R5 HIV-1 suppression was shown to be mediated in part by the CCR5-binding β-chemokines RANTES, MIP-1α, and MIP-1β, since the effect of the OX40 stimulation was reversed by a neutralizing antibody mixture against these three β-chemokines. Thus, OX40 stimulation enhanced the production of these CCR5-binding β-chemokines by the activated PBMCs and subsequently down-modulated CCR5 expression on the activated CD4(+) T cells. Taken together, the present data revealed a new role for OX40 in HIV-1 infection and documents the fact that OX40 stimulation suppresses the infection of primary activated PBMCs with R5 HIV-1 via enhanced production of R5 HIV-1 suppressing β-chemokines.

Cryptomerione induces Th1 cell polarization via influencing IL-10 production by cholera toxin-primed dendritic cells

Dendritic cells play an important role in the initiation and regulation of immune response. Dendritic cells have a key influence in the differentiation of naïve T cells into Th1, Th2 or Th17 effector cells. Cryptomerione is terpene isolated from the heartwood of Cryptomeria japonica. In this study, we investigated the effects of Cryptomerione on the phenotypic and functional maturation of human monocyte-derived dendritic cells in vitro. Human monocytes were exposed to either Cryptomerione alone, or in combination with lipopolysaccaride (LPS) or cholera toxin (CT) and thereafter co-cultured with naïve T cells. We found no enhanced CD1a, CD80, CD83, CD86 and HLA-DR expression on Cryptomerione-primed dendritic cells. However, Cryptomerione augmented T cell stimulatory capacity in an allogeneic mixed lymphocyte reaction to CT-primed dendritic cells and influenced the production of interleukin (IL)-10 and IL-12p70 by CT-primed dendritic cells, but not LPS-primed dendritic cells. Cryptomerione also inhibited Th2 cell polarization induced by CT-primed dendritic cells, but enhanced IFN-γ secretion by naïve T cells co-cultured with CT-primed dendritic cells. Cytokine production by CT-primed dendritic cells alone, or in combination with Cryptomerione was also influenced following treatment with anti-IL-10 mAb and anti-OX40L mAb. Thus, the potential mechanisms underlying the enhancement of Th1 polarization in CT-primed dendritic cells induced by Cryptomerione appeared to depend on IL-10 secretion and OX40L. These results suggest that Cryptomerione modulates human dendritic cells function in a fashion that favors Th1/Th2 cell polarization.

Differential roles for OX40 and IL‐7 on homeostasis of effector memory CD4 + T cells

Homeostatic proliferation of T cells is critically involved in the generation and maintenance of memory T cells. Recent studies have focused much attention on the crucial roles of MHC-TCR interaction and cytokines, such as IL-7 and IL-15, in the homeostatic control of memory CD4+ T cells. However, the role for costimulatory signals on the homeostatic proliferation of CD4+ T cells remains controversial although costimulatory signals contribute to the optimal proliferation and survival of T cells. To address the roles for signals through a T cell costimulatory molecule, OX40 on the homeostatic proliferation of effector memory CD4+ T cells, we have set up several experimental settings, in which polyclonal effector memory (CD44highCD62Llow) CD4+ T cells were transferred into lymphopenic recipient mice. Treatment with blocking anti-OX40L mAb during homeostatic proliferation of effector memory CD4+ T cells specifically suppressed the rapidly-proliferating MHC II-dependent cells. In contrast, inhibition of IL-7 signals by administration of blocking anti-IL-7Rα mAb during homeostatic proliferation demonstrated a preferential reduction of the slowly-proliferating population. Furthermore, simultaneous blockade of both OX40 and IL-7 signals completely inhibited the homeostatic proliferation of effector memory CD4+ T cells by suppressing both the rapid and slow homeostatic proliferation. Collectively, OX40 signals contribute to the homeostasis of effector memory CD4+ T cells in an IL-7-independent manner. This work was supported in part by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science.

Blockade of the OX40 ligand prolongs corneal allograft survival

Although corneal transplantation is one of the most common tissue transplantations and is known to have a high graft acceptance rate, occasional corneal graft rejection remains a cause of blindness. OX40, a member of the TNF receptor superfamily, is expressed on activated T cells, and transmits a costimulatory signal by binding to OX40 ligand (OX40L) expressed on several cells with antigen-presenting functions. Using a blocking monoclonal antibody (mAb) against murine OX40L, we investigated the role of OX40 in a murine model of corneal transplantation. C3H/He mouse corneas were transplanted to BALB/c mice orthotopically. Administration of anti-OX40L mAb significantly reduced allograft rejection, and increased graft survival rate to 40% at 8 weeks after transplantation, while all corneas were rejected within 5 weeks in control IgG-treated mice. Similar reduced rejection was observed when wild-type donor corneas were transplanted to OX40L-deficient recipients. In vitro study revealed that the anti-OX40L mAb treatment reduced proliferative response and IFN-gamma production of draining lymph node cells in response to stimulation with donor alloantigen. These results demonstrate that OX40L blockade is effective for prolongation of corneal allograft survival by inhibiting recipient T cell activation.

Effects of costimulatory pathway OX40/OX40L on the pathogenesis of allergic asthma in mice

Allergic asthma is thought to be mediated by CD4+ T lymphocytes producing the Th2-associated cytokines, which play a critical role in the development of the airway hyper-responsiveness and the eosinophilic inflammatory response. The costimulatory pathway CD28/B7 has been shown to play an important role in CD4+ T cell activation in allergic asthma. This study was conducted to evaluate the role of another costimulatory pathway OX40/OX40 ligand (L) in the pathogenesis of allergic asthma in BALB/c mice. An allergic asthma model in BALB/c mice was established. Thirty-six BALB/c mice were randomly divided into three groups with 12 in each. Mice in treatment group (group B) were treated with neutralizing anti-OX40L monoclonal antibody (mAb, 300 microg per mouse) during the sensitization period. Mice in two control groups, asthma model group (group A) and IgG antibody group (group C) were treated with normal saline (NS) and control IgG respectively instead of anti-OX40L mAb. Bronchoalveolar lavage fluid (BALF) was collected from the mice of each group for counting the total number of white blood cells (including neutrophil granulocyte, lymphocyte, monocyte and eosinophil granulocyte) and the proportions of these cells. The levels of IL-4 and INF-gamma in BALF were measured by ELISA. Lungs were removed for morphological examination after HE and PAS staining, and expression of OX40 in lungs was evaluated by immunohistochemical method. (1) The count of total number of white blood cells in BALF (x10(6)/ml) was lower in group B than that of group A and group C (26.6 +/- 4.6 vs. 36.8 +/- 5.2 and 34.3 +/- 6.9, respectively), the difference between the treatment group (group B) and two control groups (groups A and C) was significant; The proportions of eosinophils and lymphocytes in the BALF (%) were lower in group B than those in group A and group C (eosinophils 15.1 +/- 2.6 vs. 20.0 +/- 4.1 and 19.9 +/- 3.9, respectively; lymphocytes 7.0 +/- 0.9 vs. 8.9 +/- 1.6 and 8.6 +/- 1.8, respectively), the difference between the treatment group and two control groups was significant. (2) The IL-4 level in BALF (pg/ml) was lower in group B than that in group A and group C (672 +/- 58 vs. 809.57 +/- 106.00 and 784 +/- 58, respectively), but the INF-gamma levels in BALF (pg/ml) were higher than those in group A and group C (0.86 +/- 0.09 vs. 0.69 +/- 0.15 and 0.67 +/- 0.13 respectively), and all the differences were statistically significant. (3) The expression of OX40 in the lungs of mice in group B were at a lower level than that of group A and group C, and the morphological changes of asthma were ameliorated in the mice of the treatment group. The signs of mice in treatment group were obviously ameliorated as compared to the two control groups. Blocking the costimulatory pathway by administering the neutralizing anti-OX40L mAb during the sensitization period of allergic asthma model could balance the Th1/Th2 responses, inhibit lung inflammation and ameliorate the signs of mice model of asthma.

Short-Term Immunosuppression Facilitates Induction of Mixed Chimerism and Tolerance after Bone Marrow Transplantation without Cytoreductive Conditioning

Induction of mixed chimerism and tolerance usually requires cytoreduction or transplantation of high numbers of bone marrow cells (BMC). However, such protocols have only a suboptimal success rate and, more importantly, equivalent numbers of BMC cannot be routinely obtained in the clinical setting. The authors therefore evaluated whether a short-course of immunosuppression (IS) given in addition to co-stimulation blockade would facilitate chimerism induction and allow reduction of the minimally required number of BMC without cytoreduction. B6 mice received 200, 100, or 50 x 10 unseparated BMC from Balb/c donors plus an anti-CD40L monoclonal antibody (mAb) and CTLA4Ig (without irradiation or cytotoxic drugs). Some groups were treated additionally with IS (rapamycin, methylprednisolone, and mycophenolate mofetil for 4 weeks after bone marrow transplantation), donor-specific transfusion (DST), or anti-OX40L mAb, as indicated. IS led to long-term multilineage chimerism in 9 of 10 mice receiving 200 x 10 BMC (without IS, 1 of 4; P<0.05), in all mice (n=10) receiving 100 x 10 (without IS, 6 of 9; P<0.05), and notably in 9 of 10 mice treated with 50 x 10 BMC (without IS, 4 of 10; P<0.05). With transient IS, donor skin grafts were accepted longer than 170 days in 9 of 10 mice receiving 200 x 10 (without IS, 0 of 5 mice; P<0.05), all mice receiving 100 x 10 (without IS, 6 of 9; P<0.05), and 6 of 11 mice receiving 50 x 10 BMC (without IS, 4 of 10). The use of DST or anti-OX40L mAb had no beneficial effect. Transient IS significantly improves rates of chimerism and donor skin graft survival, and allows lasting mixed chimerism after transplantation of only 50 x 10 BMC. Thus, IS might help in the further development of noncytoreductive chimerism protocols.

Costimulation through OX40 is crucial for induction of an alloreactive human T-cell response.

The alloreactive immune response is a series of events initiated by the interaction of T cells with allogeneic dendritic cells (DCs), involving alloantigen recognition and costimulatory signals. In this study, we investigated the role of OX40 in alloreactivity in vitro. We first demonstrated that anti-OX40 ligand (anti-OX40L) monoclonal antibody (mAb) could markedly suppress the mixed leucocyte reaction (MLR) of peripheral blood mononuclear cells (PBMC). To further define the contribution of the OX40/OX40L system to the MLR, we set up a co-culture system of CD4+ T cells and allogeneic monocyte-derived dendritic cells (DCs). After 2 days, OX40 expression was induced on CD4+ T cells and this induction was strongly inhibited by the addition of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-Fc fusion protein, suggesting that the expression of OX40 during alloreaction is dependent on CD28 signalling. Next we examined the effects of anti-OX40L mAb, CTLA-4-Fc fusion protein and anti-human leucocyte antigen (HLA)-DR mAb on the proliferative response of CD4+ T cells to allogeneic DCs. The proliferation of T cells was almost completely suppressed by anti-OX40L mAb, which was comparable with that of CTLA-4-Fc. Measurement of interleukin-2 (IL-2) production in the culture supernatants showed that suppression of a proliferative response was at least in part ascribed to reduced IL-2 production. Furthermore, purified OX40L- allogeneic DCs could induce considerable proliferation of CD4+ T cells, which was suppressed by anti-OX40L mAb. These results suggest that the OX40/OX40L system is crucial for induction of the allogeneic T-cell response and the OX40/OX40L system is subsequent to and dependent on CD28 signalling, but is crucial for the end outcome of the human alloreactive T-cell response.

Critical role for OX40 ligand in the development of pathogenic Th2 cells in a murine model of asthma.

Bronchial asthma is characterized by massive infiltration of eosinophils and airway hyperreactivity (AHR), which are caused by overproduction of Th2 cytokines (IL-4, IL-5, and IL-13) by allergen-specific T cells. We recently demonstrated a critical contribution of OX40 ligand (OX40L) to the development of Th2-mediated experimental leishmaniasis. In this study, we have examined the role of OX40L in the development of Th2-mediated pulmonary inflammation by utilizing OX40L-deficient mice and a neutralizing anti-OX40L mAb in a murine model of asthma. Sensitization and airway challenge with ovalbumin in wild-type BALB/c mice induced a typical allergic asthma characterized by AHR, accumulation of eosinophils, increased mucus production, and high levels of Th2 cytokines in the lung. All these asthmatic responses were not induced in OX40L-deficient BALB/c mice. Administration of neutralizing anti-OX40L mAb in wild-type BALB/c mice during the sensitization period also abolished the induction of asthmatic responses. In contrast, administration of anti-OX40L mAb during the challenge period did not inhibit the asthmatic responses. These results indicate a critical role for OX40L in the induction phase, which leads to the development of pathogenic Th2 cells, but not in the effector phase, which includes migration and activation of pathogenic Th2 cells in the lung.

Therapeutic effect of anti-OX40L and anti-TNF-alpha MAbs in a murine model of chronic colitis.

Interaction of OX40 (CD134) on T cells with its ligand (OX40L) on antigen-presenting cells has been implicated in pathogenic T cell activation. This study was performed to explore the involvement of OX40/OX40L in the development of T cell-mediated chronic colitis. We evaluated both the preventive and therapeutic effects of neutralizing anti-OX40L MAb on the development of chronic colitis in SCID mice induced by adoptive transfer of CD4(+)CD45RB(high) T cells as an animal model of Crohn's disease. We also assessed the combination of anti-OX40L and anti-TNF-alpha MAbs to improve the therapeutic effect. Administration of anti-OX40L MAb markedly ameliorated the clinical and histopathological disease in preventive and therapeutic protocols. In vivo treatment with anti-OX40L MAb decreased CD4(+) T cell infiltration in the colon and suppressed IFN-gamma, IL-2, and TNF-alpha production by lamina propria CD4(+) T cells. The combination with anti-TNF-alpha MAb further improved the therapeutic effect by abolishing IFN-gamma, IL-2, and TNF-alpha production by lamina propria CD4(+) T cells. Our present results suggested a pivotal role of OX40/OX40L in the pathogenesis of T cell-mediated chronic colitis. The OX40L blockade, especially in combination with the TNF-alpha blockade, may be a promising strategy for therapeutic intervention of Crohn's disease.