Primary and malignant cholangiocytes undergo CD40 mediated Fas dependent apoptosis, but are insensitive to direct activation with exogenous Fas ligand

Introduction

Cholangiocarcinoma is a rare malignancy of the biliary tract, the incidence of which is rising, but the pathogenesis of which remains uncertain. No common genetic defects have been described but it is accepted that chronic inflammation is an important contributing factor. We have shown that primary human cholangiocyte and hepatocyte survival is tightly regulated via co-operative interactions between two tumour necrosis family (TNF) receptor family members; CD40 and Fas (CD95). Functional deficiency of CD154, the ligand for CD40, leads to a failure of clearance of biliary tract infections and a predisposition to cholangiocarcinoma implying a direct link between TNF receptor-mediated apoptosis and the development of cholangiocarcinoma.

Aims

To determine whether malignant cholangiocytes display defects in CD40 mediated apoptosis. By comparing CD40 and Fas-mediated apoptosis and intracellular signalling in primary human cholangiocytes and three cholangiocyte cell lines.

Results

Primary cholangiocytes and cholangiocyte cell lines were relatively insensitive to direct Fas-mediated killing with exogenous FasL when compared with Jurkat cells, which readily underwent Fas-mediated apoptosis, but were extremely sensitive to CD154 stimulation. The sensitivity of cells to CD40 activation was similar in magnitude in both primary and malignant cells and was STAT-3 and AP-1 dependent in both.

Conclusions

1) Both primary and malignant cholangiocytes are relatively resistant to Fas–mediated killing but show exquisite sensitivity to CD154, suggesting that the CD40 pathway is intact and fully functional in both primary and malignant cholangiocytes 2) The relative insensitivity of cholangiocytes to Fas activation demonstrates the importance of CD40 augmentation of Fas dependent death in these cells. Agonistic therapies which target CD40 and associated intracellular signalling pathways may be effective in promoting apoptosis of malignant cholangiocytes.     

CD40-dependent activation of phosphatidylinositol 3-kinase/Akt pathway mediates endothelial cell survival and in vitro angiogenesis

CD40 has been involved in tumor and inflammatory neoangiogenesis. In this study we determined that stimulation of endothelial CD40 with sCD154 induced resistance to apoptosis and in vitro vessel-like formation by human microvascular endothelial cells (HMEC). These effects were determined to be mediated by CD40-dependent signaling because they were inhibited by a soluble CD40-muIg fusion protein. Moreover, apoptosis of HMEC was associated with an impairment of Akt phosphorylation, which was restored by stimulation with sCD154. The anti-apoptotic effect as well as in vitro vessel-like formation and Akt phosphorylation were inhibited by treatment of HMEC with two unrelated pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002. CD40 stimulation induced a rapid increase in Akt enzymatic activity that was not prevented by cycloheximide, an inhibitor of protein synthesis. The enhanced Akt activity induced by stimulation of endothelial CD40 was temporarily correlated with the association of CD40 with TRAF6, c-Cbl, and the p85 subunit of PI3K. Expression of negative-dominant Akt inhibited the activation of endogenous Akt through CD40 stimulation, despite the observation that association of CD40 with TRAF6, c-Cbl, and PI3K was intact. The defective activation of Akt abrogated not only the anti-apoptotic effect of CD40 stimulation but also the proliferative response, the enhanced motility, and the in vitro formation of vessel-like tubular structures by CD40-stimulated HMEC. In conclusion, these results suggest that endothelial CD40, through activation of the PI3K/Akt signaling pathway, regulates cell survival, proliferation, migration, and vessel-like structure formation, all steps considered critical for angiogenesis.     

Critical role of OX40 in CD28 and CD154-independent rejection

Blocking both CD28 and CD154 costimulatory pathways can induce transplant tolerance in some, but not all, transplant models. Under stringent conditions, however, this protocol often completely fails to block allograft rejection. The precise nature of such CD28/CD154 blockade-resistant rejection is largely unknown. In the present study we developed a new model in which both CD28 and CD154, two conventional T cell costimulatory molecules, are genetically knocked out (i.e., CD28/CD154 double-knockout (DKO) mice) and used this model to examine the role of novel costimulatory molecule-inducible costimulator (ICOS), OX40, 4-1BB, and CD27 in mediating CD28/CD154-independent rejection. We found that CD28/CD154 DKO mice vigorously rejected fully MHC-mismatched DBA/2 skin allografts (mean survival time, 12 days; n = 6) compared with the wild-type controls (mean survival time, 8 days; n = 7). OX40 costimulation is critically important in skin allograft rejection in this model, as blocking the OX40/OX40 ligand pathway, but not the ICOS/ICOS ligand, 4-1BB/4-1BBL, or CD27/CD70 pathway, markedly prolonged skin allograft survival in CD28/CD154 DKO mice. The critical role of OX40 costimulation in CD28/CD154-independent rejection is further confirmed in wild-type C57BL/6 mice, as blocking the OX40/OX40 ligand pathway in combination with CD28/CD154 blockade induced long term skin allograft survival (>100 days; n = 5). Our study revealed a key cellular mechanism of rejection and identified OX40 as a critical alternative costimulatory molecule in CD28/CD154-independent rejection.     

CD40 ligand (CD40L) does not stimulate proliferation of vascular smooth muscle cells

The present study investigates the effects of CD40 ligand (CD40L) on mitogenic signalling, proliferation, and migration of cultured bovine coronary artery smooth muscle cells (SMC). A time- and concentration-dependent phosphorylation of the extracellular signal-regulated kinases-1/2 (ERK-1/2) and the mitogen-activated protein kinase p38 (p38-MAPK) was observed upon stimulation with soluble CD40L (sCD40L). This phosphorylation was inhibited by neutralizing antibodies against the CD40 and CD40L, respectively. Activation of the phosphatidylinositol-3-phosphate (PI-3) kinase pathway by sCD40L, as determined by the measurement of Akt phosphorylation, was not detected. However, there was evidence for direct activation of the NFkappaB system (degradation of IkappaBalpha and nuclear translocation of the p65 NFkappaB subunit) by sCD40L. Accordingly, sCD40L caused a small but significant increase in DNA synthesis. However, sCD40L-induced DNA synthesis was not followed by proliferation (increase in cell number). Furthermore, sCD40L did not potentiate SMC mitogenesis induced by known mitogens such as platelet-derived growth factor-BB, thrombin or serum. The lack of cell proliferation was not caused by a concomitant induction of SMC apoptosis by sCD40L. The possible role of membrane-bound CD40L in SMC mitogenesis was also studied using different membrane preparations (platelets, lymphocytes). However, no mitogenic effects of membrane-bound CD40L were detected. Finally, sCD40L did not induce SMC migration. From these data it is concluded that CD40L activates mitogenic signalling and DNA synthesis but does not contribute to proliferation or migration of vascular SMC.     

CD154 Is Released from T-cells by a Disintegrin and Metalloproteinase Domain-containing Protein 10 (ADAM10) and ADAM17 in a CD40 Protein-dependent Manner

CD154 (CD40 ligand) is a type II transmembrane protein that belongs to the tumor necrosis factor superfamily. The soluble form of CD154 (sCD154), which results from the shedding of membrane-bound CD154, plays a key role in the production of proinflammatory cytokines and has been linked to various autoimmune and vascular disorders. Therefore, elucidating the mechanisms by which CD154 is released from the cell surface following its interaction with its various receptors is of primordial importance. Using co-culture experiments, we show that CD154 is shed predominantly upon its engagement with CD40. Indeed, only CD40 (both membrane-bound and soluble) and not α5β1 or αMβ2 is involved in the cleavage and release of CD154 from Jurkat E6.1 T-cells. Interestingly, CD154 is cleaved independently of the formation of cell surface CD40 homodimers and independently of its association into lipid rafts. In contrast, we found that the protein kinase C (PKC) signaling family and the matrix metalloproteinases ADAM10 and ADAM17 are intimately involved in this process. In conclusion, our data indicate that CD154 is released from T-cells by ADAM10 and ADAM17 upon CD40 ligation. These findings add significant insights into the mechanisms by which CD154 is down-regulated and may lead to the generation of novel therapeutic targets for the treatment of CD154-associated disorders.     

Determination of carbohydrate structures N-linked to soluble CD154 and characterization of the interactions of CD40 with CD154 expressed in Pichia pastoris and Chinese hamster ovary cells.

CD40-CD154 (CD40 ligand) interactions are essential for the development of protective immunity. Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154. However, these studies have not examined the structure or biological function of the carbohydrate on CD154. Human CD154 contains a single N-linked glycosylation site at asparagine 240. We have characterized the interactions between CD40 and soluble (s) CD154 in which sCD154 contains different types of carbohydrates. Detailed carbohydrate analysis revealed high-mannose structures on sCD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates. sCD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10-30 nM, and it functionally induced CD69 and CD95 expression on primary B cells. Together, these results indicate that the presence of varied types of N-linked glycans on asparagine 240 of CD154 does not play a significant role in the CD40-CD154 interactions.     

CD40 amplifies Fas-mediated apoptosis: a mechanism contributing to emphysema

Excessive apoptosis and prolonged inflammation of alveolar cells are associated with the pathogenesis of pulmonary emphysema. We aimed to determine whether CD40 affects alveolar epithelial cells and endothelial cells, with regard to evoking apoptosis and inflammation. Mice were repeatedly treated with agonistic-anti CD40 antibody (Ab), with or without agonistic-anti Fas Ab, and evaluated for apoptosis and inflammation in lungs. Human pulmonary microvascular endothelial cells and alveolar epithelial cells were treated with agonistic anti-CD40 Ab and/or anti-Fas Ab to see their direct effect on apoptosis and secretion of proinflammatory molecules in vitro. Furthermore, plasma soluble CD40 ligand (sCD40L) level was evaluated in patients with chronic obstructive pulmonary disease (COPD). In mice, inhaling agonistic anti-CD40 Ab induced moderate alveolar enlargement. CD40 stimulation, in combination with anti-Fas Ab, induced significant emphysematous changes and increased alveolar cell apoptosis. CD40 stimulation also enhanced IFN-γ-mediated emphysematous changes, not via apoptosis induction, but via inflammation with lymphocyte accumulation. In vitro, Fas-mediated apoptosis was enhanced by CD40 stimulation and IFN-γ in endothelial cells and by CD40 stimulation in epithelial cells. CD40 stimulation induced secretion of CCR5 ligands in endothelial cells, enhanced with IFN-γ. Plasma sCD40L levels were significantly increased in patients with COPD, inversely correlating to the percentage of forced expiratory volume in 1 s and positively correlating to low attenuation area score by CT scan, regardless of smoking history. Collectively CD40 plays a contributing role in the development of pulmonary emphysema by sensitizing Fas-mediated apoptosis in alveolar cells and increasing the secretion of proinflammatory chemokines.     

CD40 signaling activated by Epstein–Barr virus promotes cell survival and proliferation in gastric carcinoma-derived human epithelial cells

CD40 signaling plays a critical role in the survival and proliferation of EBV-infected lymphocytes. Here we show that CD40 is constitutively expressed in the human gastric carcinoma-derived cell lines AGS, MKN28, and MKN74, and expression of CD40L is induced by in vitro infection with EBV. Blocking the interaction between CD40 and CD40L with CD40Ig, a fusion protein of CD40 and IgG, impaired proliferation of EBV-infected AGS cells and enhanced their calcium ionophore-induced apoptosis. These results suggest that CD40 signaling plays a critical role in the survival and proliferation of EBV-infected epithelial cells, as well as in the virus-infected lymphocytes.     

Influence of sCD40L on gastric cancer cell lines

The CD40 signaling pathway plays a key role in tumor cell proliferation, differentiation, and apoptosis. Gastric cancer usually possesses a higher level of CD40 expression than normal tissue. We evaluated inhibition of soluble CD40 ligand (sCD40L) in apoptosis induction of gastric cancer cells. Gastric cancer cells (AGS and BGC-823) were incubated with sCD40L. Cell viability and cell cycle were determined by methyl-tetrazolium (MTT) assay and flow cytometry, respectively. The results showed that sCD40L hindered cell growth, arrested cells at G0/G1 phase and induced apoptosis. In conclusion, sCD40L suppress growth of gastric cancer cells through apoptosis induction and cell cycle quiescence. This work will provided a new approach to gene therapy of gastric cancer.     

CD40 mediated human cholangiocyte apoptosis requires JAK2 dependent activation of STAT3 in addition to activation of JNK1/2 and ERK1/2

CD40 is critically involved in Fas-mediated cholangiocyte apoptosis during liver inflammation, but the underlying signalling events are poorly understood. Our recent work implicated AP-1 in CD40-induced cholangiocyte apoptosis, but suggested involvement of other signalling pathways. Because STAT3 has been implicated in liver regeneration we investigated this signalling pathway during CD40 mediated cholangiocyte apoptosis. Western immunoblotting, electrophoretic mobility gel shift assays, In situ DNA end labelling and caspase-3 activity were used to investigate intracellular signalling and apoptosis in primary human cholangiocytes following CD40 activation. CD40-activation induced caspase-3 dependent cholangiocyte apoptosis and 3-fold increases in JNK/ERK phosphorylation (concomitant with increased AP-1 binding activity) and 4-fold increases in pSTAT3, which were sustained for up to 24 h. Protein levels of c-Jun, c-Fos and pSTAT3 confirmed the upregulation. Phosphorylation of p38 remained unchanged suggesting that this MAP kinase was not involved in CD40 mediated apoptosis. Increased JAK2 phosphorylation accompanied increased STAT3 phosphorylation after CD40 ligation. Cholangiocytes were also shown to express JAK1 and 3 which was phosphorylated following control stimulation with TNFalpha or IL2 respectively but not after CD40 ligation. JNK, ERK and JAK2 inhibitors partially abrogated apoptosis and when used in combination reduced it to basal levels. In conclusion, induction of CD40-mediated cholangiocyte apoptosis requires JAK2-mediated phosphorylation of STAT3 as well as sustained JNK1/2, ERK1/2 activation. This study demonstrates that STAT3 can function as a proapoptotic factor in primary human liver epithelial cells.     

可溶性三聚化CD40-异亮氨酸拉链融合蛋白的制备及鉴定

以克隆的CD40cDNA为模板,经多步PCR构建羧基端融合异亮氨酸拉链(isoleucine zipper,IZ)三聚化基序和His6标签的可溶性CD40融合蛋白(sCD40IZ)的原核表达载体,在大肠杆菌中获得高效表达,分子量为27kD,与理论大小相符,表达产物主要存在于包涵体中,对包涵体蛋白进行稀释复性和纯化得到可溶性的sCD40IZ重组蛋白,该蛋白在溶液中的分子量为91kD,表明最有可能以三聚体形式存在。活性分析显示该蛋白能够与细胞上的CD40L结合,并且其结合活性与不含IZ基序的可溶性CD40相比明显提高。这些结果表明,在可溶性CD40羧基端融合IZ基序能够促进形成三聚体,并且具有增强的配基结合活性。     

Structure of CD40 ligand in complex with the Fab fragment of a neutralizing humanized antibody

BACKGROUND: CD40 ligand (CD40L or CD154), a member of the tumor necrosis factor (TNF) family, plays a critical role in both humoral and cellular immune responses and has been implicated in biological pathways involving epithelial cells, fibroblasts, and platelets. Such a pathway is T cell-mediated B cell activation, a process that occurs through the interaction of CD40L with CD40 receptor expressed on B cells. It results in various B cell responses, including immunoglobulin isotype switching and B cell differentiation and proliferation. These responses can be inhibited by the monoclonal antibody 5c8, which binds with high affinity to CD40L. RESULTS: To understand the structural basis of the inhibition, we determined the crystal structure of the complex of the extracellular domain of CD40L and the Fab fragment of humanized 5c8 antibody. The structure shows that the complex has the shape of a three-bladed propeller with three Fab fragments bound symmetrically to a CD40L homotrimer. To further study the nature of the antibody-antigen interface, we assessed the ability of 23 site-directed mutants of CD40L to bind to 5c8 and CD40 and analyzed the results in the context of the crystal structure. Finally, we observed via confocal microscopy that 5c8 binding to CD40L on the cell surface results in the formation of patches of clustered complexes. CONCLUSIONS: The structure reveals that 5c8 neutralizes CD40L function by sterically blocking CD40 binding. The antigenic epitope is localized in a region of the surface that is likely to be structurally perturbed as a result of genetic mutations that cause hyper-IgM syndrome. The symmetric trimeric arrangement of the Fab fragments in the complex results in a geometry that facilitates the formation of large clusters of complexes on the cell surface.     

The role of the CD95, CD38 and TGFbeta1 during active human cytomegalovirus infection in liver transplantation

AIM: Human cytomegalovirus (HCMV) has highly evolved mechanisms for avoiding detection by the host immune system. The aim of this study was to analyze the expression levels of TGFbeta1, soluble form of CD95, CD95 ligand (sCD95 and sCD95L, respectively) in plasma and CD95 expression on CD3(+) cells, CD38 expression on CD8(+) cells in liver transplanted recipients with active HCMV infection. METHODS: Blood samples were collected from 15 liver transplanted recipients with active HCMV infection and 15 recipients without HCMV infection. CD95 expression on CD3(+) cells and CD38 expression on CD8(+) cells were quantitatively detected with two-color fluorescence activated cell sorter (FACS) analysis. Lymphocyte surface phenotypes of CD4 and CD8 were detected with FACS analysis. Plasma sCD95, sCD95L and TGFbeta1 levels were determined with enzyme linked-immuno-sorbent assay (ELISA). The results were compared with that from 15 healthy individuals. RESULTS: CD95 expression on CD3(+) T-cells and CD38 expression on CD8(+) cells were significantly increased in active HCMV infection group compared with that in stable group or healthy group (P<0.01). No significant difference was seen between stable group and healthy group (P>0.05). The percentages of CD4(+) T-cell and CD4/CD8 ratio in active HCMV infection group were significantly lower than the values in stable group and healthy group (P<0.05). Plasma levels of TGFbeta1 and sCD95 were significantly increased in active HCMV infection group compared to stable group and healthy group (P<0.05). In contrast, plasma levels of sCD95L in healthy group were not significantly different from that expressed in active HCMV infection group and stable group (P>0.05). CONCLUSION: HCMV suppress proliferation of activated T cells by apoptosis and by releasing immunosuppressive cytokine TGFbeta1. This may provide an important clue to a better understanding of the pathogenesis in liver transplanted recipients with active HCMV infection.     

Expression, regulation, and function of B cell-expressed CD154 in germinal centers.

Activated B cells and T cells express CD154/CD40 ligand in vitro. The in vivo expression and function of B cell CD154 remain unclear and therefore were examined. Tonsillar B and T cells expressed CD154 at a similar density both in situ and immediately ex vivo, whereas a significantly higher percentage of the former expressed CD154. CD154-expressing B cells were most frequent in the CD38positiveIgD+ pre-germinal center (GC)/GC founder, CD38positive GC and CD38-IgD- memory populations, and were also found in the CD38-IgD+ naive and CD38brightIgD+ plasmablast subsets, but not in the CD38brightIgD- plasma cell subset. B cell expression of CD154 was induced by engaging surface Ig or CD40 by signals that predominantly involved activation of AP-1/NF-AT and NF-kappaB, respectively. The functional importance of CD154-mediated homotypic B cell interactions in vivo was indicated by the finding that mAb to CD154 inhibited differentiation of CD38positiveIgD- GC B cells to CD38-IgD- memory cells. In addition, mAb to CD154 inhibited proliferation induced by engaging sIg or CD40, indicating the role of up-regulation of this molecule in facilitating B cell responsiveness. Of note, CD154 itself not only functioned as a ligand but also as a direct signaling molecule as anti-CD154-conjugated Sepharose beads costimulated B cell responses induced by engaging surface Ig. These results indicate that CD154 is expressed by human B cells in vivo and plays an important role in mediating B cell responses.     

TNF-alpha blockade down-regulates the CD40/CD40L pathway in the mucosal microcirculation: a novel anti-inflammatory mechanism of infliximab in Crohn's disease

The CD40/CD40 ligand (CD40L) pathway is involved in Crohn's disease (CD) pathogenesis. In the patients' circulation, soluble CD40L (sCD40L) levels are elevated and surface CD40L is increased in platelets and T cells, whereas in the intestine CD40 is overexpressed in the microvasculature and CD40L in platelets and T cells. The therapeutic effects of infliximab in CD are attributed to its systemic anti-TNF-alpha action, but because TNF-alpha modulates both CD40 and CD40L, we investigated whether infliximab affects the CD40/CD40L pathway in the intestine. Eighteen CD patients were evaluated before and after infliximab therapy. Plasma sCD40L was measured by ELISA and platelet and peripheral blood T cell (PBT) CD40L expression by flow cytometry. Microvascular CD40 and VCAM-1 expression were assessed in mucosal biopsies by immunohistochemistry and by flow cytometry in human intestinal microvascular endothelial cells (HIMEC). Cell cultures were performed in the presence and absence of infliximab. Infliximab treatment significantly reduced plasma sCD40L levels and eliminated CD40 and VCAM-1 from mucosal microvessels. In vitro infliximab prevented TNF-alpha-induced CD40 and VCAM-1 expression by HIMEC, and reduced PBT, but not platelet, surface CD40L expression and sCD40L release. In addition, infliximab decreased T cell-induced VCAM-1 expression in HIMEC by down-regulating CD40L in T cells and promoting T cells apoptosis. These findings point to a novel mechanism of action of infliximab, i.e., the disruption of CD40/CD40L-dependent cognate interactions between intestinal microvessels and T cells. Thus, in addition to neutralizing TNF-alpha and inducing T cell death, the therapeutic effects of infliximab in CD appear to be also mediated by inhibition of vascular inflammation in the gut.     

Ligation or cross-linking of CD40 has different effects on AGS gastric cancer cells

A gastric cancer (GC) cell line, AGS, has high-level expression of CD40, a tumor necrosis factor receptor (TNFR) family member. CD40 is present on the surfaces of a large variety of cells, including B cells, endothelial cells, dendritic cells and some carcinoma cells, and delivers signals regulating diverse cellular responses, such as proliferation, differentiation, growth suppression, and cell death. In this research, we studied the effects of different forms of CD40 stimulation on AGS cells by flow cytometry, Western blotting and siRNA transfection. We found that different forms of CD40 stimulation, either recombinant soluble CD40L (sCD40L, ligation) or agonist anti-CD40 antibody (cross-linking), induced different effects in AGS gastric cancer cells, proliferation or apoptosis. We also showed that VEGF provided a significant contribution to sCD40L-induced proliferation, while agonist anti-CD40 antibody induced GADD45 upregulation and promoted apoptosis.     

Membrane-anchored CD40 is processed by the tumor necrosis factor-alpha-converting enzyme. Implications for CD40 signaling

The soluble form of CD40 (sCD40), which co-exists with the membrane-anchored form (mCD40), is a natural antagonist of mCD40/CD154 interaction. However, the mechanism leading to the production of sCD40 has never been investigated. Here, we show that the engagement of mCD40 on the surface of B lymphocytes by anti-CD40 antibody led to enhanced sCD40 release associated with decreased amounts of mCD40. This sCD40 production was not affected by vesicular traffic inhibitors but was completely blocked by a broad-spectrum synthetic metalloproteinase (MP) inhibitor (GM6001) or a membrane-anchored MP-specific inhibitor (dec-RVKR-cmk). Recombinant MP disintegrin tumor necrosis factor-alpha converting enzyme (TACE) cleaved the purified CD40 ectodomain/Fc chimeric protein in vitro, giving rise to an sCD40 form similar to that shed from B cell cultures. Moreover, spontaneous production of sCD40 by mCD40-transfected human embryonic kidney cells (constitutively expressing TACE) was enhanced by the overexpression of TACE and abrogated by co-transfection with a dominant-negative TACE mutant. These results provide strong evidence that sCD40 production is an active process regulated by the engagement of mCD40 and its proteolytic cleavage by TACE or a related MP disintegrin. Given the antagonistic activity of sCD40 on the CD40/CD154 interaction, this shedding mechanism might represent an important negative feedback control of CD40 functions.     

Critical, but conditional, role of OX40 in memory T cell-mediated rejection

Memory T cells can be a significant barrier to the induction of transplant tolerance. However, the molecular pathways that can regulate memory T cell-mediated rejection are poorly defined. In the present study we tested the hypothesis that the novel alternative costimulatory molecules (i.e., ICOS, 4-1BB, OX40, or CD30) may play a critical role in memory T cell activation and memory T cell-mediated rejection. We found that memory T cells, generated by either homeostatic proliferation or donor Ag priming, induced prompt skin allograft rejection regardless of CD28/CD154 blockade. Phenotypic analysis showed that, in contrast to naive T cells, such memory T cells expressed high levels of OX40, 4-1BB, and ICOS on the cell surface. In a skin transplant model in which rejection was mediated by memory T cells, blocking the OX40/OX40 ligand pathway alone did not prolong the skin allograft survival, but blocking OX40 costimulation in combination with CD28/CD154 blockade induced long-term skin allograft survival, and 40% of the recipients accepted their skin allograft for >100 days. In contrast, blocking the ICOS/ICOS ligand and the 4-1BB/4-1BBL pathways alone or combined with CD28/CD154 blockade had no effect in preventing skin allograft rejection. OX40 blockade did not affect the homeostatic proliferation of T cells in vivo, but markedly inhibited the effector functions of memory T cells. Our data demonstrate that memory T cells resisting to CD28/CD154 blockade in transplant rejection are sensitive to OX40 blockade and suggest that OX40 is a key therapeutic target in memory T cell-mediated rejection.