CD137 Stimulation Research Articles

CD137 stimulation and p38 MAPK inhibition improve reactivity in an in vitro model of glioblastoma immunotherapy.

Dendritic cell vaccination has become an interesting option for cancer immunotherapy. Tumor-lysate-pulsed dendritic cells (DC) can prime naïve T cells and induce the regression of established tumors including gliomas as shown in various animal models. Despite hopeful results even in clinical studies, the outcome for many patients is still unsatisfying. In the present study, we tested the combination of tumor-lysate-pulsed dendritic cells (TPDC) with a monoclonal antibody against CD137, a monoclonal antibody against CD25 (daclizumab) and a specific p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (SB203580) for improving immunostimulation in an in vitro model of immunotherapy for human gliomas. We observed a higher secretion of interferon gamma by TPDC-primed peripheral blood mononuclear cells (PBMC) that were incubated with an antibody against CD137 or the p38 MAPK inhibitor. In addition, we observed higher specific lysis of tumor cells after incubation of PBMC with the p38 MAPK inhibitor or the anti-CD137 antibody. In contrast, incubation of TPDC-primed PBMC with the anti-CD25 antibody did enhance neither interferon gamma secretion nor cellular cytotoxicity. Cell depletion experiments demonstrated that the immune reaction induced by TPDC is strongly dependent on CD4-positive and CD8-positive cells. Incubation of DC during maturation and antigen loading with the anti-CD137 antibody did not enhance cytotoxicity and interferon gamma secretion in comparison with application of the anti-CD137 antibody during priming. In conclusion, our data suggest that p38 MAPK inhibition and anti-CD137 antibodies can enhance the immune response against glioblastoma cells.

Role of the CD137 ligand (CD137L) signaling pathway during Mycobacterium tuberculosis infection

The role of the CD137–CD137 ligand (CD137L) signaling pathway in T cell co-stimulation has been well established. Dysregulated CD137 or CD137L stimulation can lead to pathological conditions such as inflammatory diseases or cancer. However, the contribution of CD137–CD137L interaction to the control of infectious diseases has not been extensively studied, with the few available reports focusing mainly on viral infections. Here we investigated the role of the CD137–CD137L interactions during Mycobacterium tuberculosis infection. Using CD137L-deficient mice, we found that absence of the CD137L-mediated signaling pathway during M. tuberculosis infection resulted in delayed activation of CD4+ T cells in the draining lymph nodes. This finding was supported by an in vitro mixed lymphocyte reaction assay that revealed impaired priming of T cells by CD137L-deficient dendritic cells upon mycobacterial infection. In addition, greater numbers of CD4+ T cells and antigen presenting cells were measured in the lungs of CD137L-deficient mice. Strikingly, the lung cytokine production profile was profoundly altered in M. tuberculosis-infected CD137L-deficient mice with lower levels of TNF-α, IL-12 and IL-6 and elevated concentrations of IL-17 compared to their wild type counterparts. However and surprisingly, these tangible immunological disorders translated only into a mild and transient increase in the bacterial loads and a higher number of granulomatous lesions with impaired architecture in the lungs of the CD137L-deficient infected mice. Together, while our data support the engagement of the CD137L signaling pathway during M. tuberculosis infection, they underscore the functional redundancy and robustness of the host defense arsenal deployed against mycobacterial infection.

T Cell Costimulation with Anti-CD137 Monoclonal Antibodies Is Mediated by K63–Polyubiquitin-Dependent Signals from Endosomes

Agonist anti-CD137 (4-1BB) mAbs enhance CD8-mediated antitumor immunity. Agonist anti-human CD137 mAbs binding to four distinct epitopes on the CD137 glycoprotein costimulated T cell activation irrespective of the engaged epitope or its interference with CD137L binding. CD137 perturbation with all these agonist mAbs resulted in Ag and Ab internalization toward an endosomal vesicular compartment. Internalization was observed in activated T lymphocytes from humans and mice, not only in culture but also in Ab-injected living animals. These in vivo experiments were carried out upon systemic i.v. injections with anti-CD137 mAbs and showed CD137 internalization in tumor-infiltrating lymphocytes and in activated human T cells transferred to immunodeficient mice. Efficient CD137 internalization required K63 polyubiquitination and endocytosed CD137-containing vesicles recruited TNFR-associated factor (TRAF) 2 and were decorated with K63 polyubiquitins. CD137 stimulation activates NF-κB through a K63-linked polyubiquitination-dependent route, and CD137-associated TRAF2 becomes K63 polyubiquitinated. Consistent with a role for TRAF2 in CD137 signaling, transgenic mice functionally deficient in TRAF2 showed delayed immunotherapeutic activity of anti-CD137 mAbs. As a whole, these findings advance our knowledge of the mechanisms of action of anti-CD137 immunostimulatory mAbs such as those currently undergoing clinical trials in cancer patients.

CD137 Is Induced by the CD40 Signal on Chronic Lymphocytic Leukemia B Cells and Transduces the Survival Signal via NF-κB Activation

CD137 is a member of the tumor necrosis factor receptor family that is expressed on activated T cells. This molecule provides a co-stimulatory signal that enhances the survival, and differentiation of cells, and has a crucial role in the development of CD8 cytotoxic T cells and anti-tumor immunity. Here we report that CD137 expression is also induced on normal or malignant human B cells by CD40 ligation by its ligand CD154. This CD137 induction was more prominent in chronic lymphocytic leukemia (CLL) cells than in other types of B cells. CD137 stimulation on B cells by its ligand induced the nuclear translocation of p52 (a non-canonical NF-κB factor). In agreement with this finding, expression of the survival factor BCL-XL was upregulated. Consequently, the CD137 signal augmented the survival of CD154-stimulated CLL B cells in vitro. This unexpected induction of CD137 on B cells by CD40 signal may influence the clinical course of CLL.

CD137 Expressed on Neutrophils Plays Dual Roles in Antibacterial Responses against Gram-Positive and Gram-Negative Bacterial Infections

Severe sepsis and septic shock caused mainly by bacterial infections are life-threatening conditions that urge the development of novel therapies. However, host responses to and pathophysiology of sepsis have not been clearly understood, which remains a major obstacle for the development of effective therapeutics. Recently, we have shown that stimulation of a costimulatory molecule, CD137, enhanced survival of mice infected with the Gram-positive (G(+)) intracellular bacterium Listeria monocytogenes but decreased survival in a polymicrobial sepsis model. Herein, we report that CD137 deficiency or blocking of CD137 signaling decreased antibacterial responses of mice infected with G(+) bacteria (Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis) but increased these responses in mice infected with Gram-negative (G(-)) bacteria (Escherichia coli, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium). Consistent with these findings, stimulation of CD137 by administration of agonistic antibody enhanced responses against G(+) bacteria, whereas it decreased these responses against G(-) bacteria. Neutrophils were responsible for CD137-mediated opposite roles in control of G(+) and G(-) bacterial infections. Stimulation of CD137 enhanced activities of neutrophils against S. aureus but decreased these activities against E. coli, while CD137 blocking produced opposite results with the stimulation of CD137 in vivo and in vitro. Furthermore, we found that combined signaling of CD137 and Toll-like receptor 2 (TLR2) induced synergistic production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) by neutrophils, but combined signaling of CD137 and TLR4 did not. Our data strongly suggest that CD137 may play a dual role in sepsis in association with TLRs.

Agonist Antibodies to TNFR Molecules That Costimulate T and NK Cells

Therapy for cancer can be achieved by artificially stimulating antitumor T and natural killer (NK) lymphocytes with agonist monoclonal antibodies (mAb). T and NK cells express several members of the TNF receptor (TNFR) family specialized in delivering a costimulatory signal on their surface. Engagement of these receptors is typically associated with proliferation, elevated effector functions, resistance to apoptosis, and differentiation into memory cells. These receptors lack any intrinsic enzymatic activity and their signal transduction relies on associations with TNFR-associated factor (TRAF) adaptor proteins. Stimulation of CD137 (4-1BB), CD134 (OX40), and glucocorticoid-induced TNFR (GITR; CD357) promotes impressive tumor-rejecting immunity in a variety of murine tumor models. The mechanisms of action depend on a complex interplay of CTL, T-helper cells, regulatory T cells, dendritic cells, and vascular endothelium in tumors. Agonist mAbs specific for CD137 have shown signs of objective clinical activity in patients with metastatic melanoma, whereas anti-OX40 and anti-GITR mAbs have entered clinical trials. Preclinical evidence suggests that engaging TNFR members would be particularly active with conventional cancer therapies and additional immunotherapeutic approaches. Indeed, T-cell responses elicited to tumor antigens by means of immunogenic tumor cell death are amplified by these immunostimulatory agonist mAbs. Furthermore, anti-CD137 mAbs have been shown to enhance NK-mediated cytotoxicity elicited by rituximab and trastuzumab. Combinations with other immunomodulatory mAb that block T-cell checkpoint blockade receptors such as CTLA-4 and PD-1 are also promising.

Abstract B2: T cell costimulation in cancer immunotherapy with anti-CD137 monoclonal antibodies is mediated by K63-polyubiquitin-dependent signals from endosomes.

Abstract Agonist anti-CD137(4-1BB) mAbs enhance CD8-mediated antitumor immunity. Agonist anti-human CD137 mAbs covering four distinct epitopes on the CD137 glycoprotein co-stimulated T cell activation irrespective of the engaged epitope or its interference with CD137L binding. CD137 perturbation with all these agonist mAbs resulted in antigen and antibody internalization towards a vesicular compartment. Internalization was observed in activated T cells from humans and mice, not only in culture but also in antibody-injected living animals. These in vivo experiments were carried out upon systemic intravenous injections with anti-CD137 mAbs and showed internalization in tumor-infiltrating lymphocytes and in activated human T cells transferred to mice. CD137-internalization required K63-polyubiquitination and endocytosed CD137-containing deposits were decorated with K63-polyubiquitins. CD137 stimulation activates NF-kB through a K63-linked polyubiquitination-dependent route and CD137-associated TRAF2 becomes K63-polyubiquitinated. Consistent with a role for TRAF2 in CD137 signalling, transgenic mice functionally deficient in TRAF2 show a delayed immunotherapeutic activity of anti-CD137 mAbs Citation Format: Ivan Martinez-Forero, Arantza Azpilicueta, Elixabet Bolaños-Mateo, Estnislao Nistal-Villan, Asis Palazon, Alvaro Teijeira, Gema Perez-Chacon, Aizea Morales-Kastresana, Oihana Murillo, Maria Jure-Kunkel, Juan Manuel Zapata, Ignacio Melero. T cell costimulation in cancer immunotherapy with anti-CD137 monoclonal antibodies is mediated by K63-polyubiquitin-dependent signals from endosomes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B2.

Combined Blockade of Programmed Death-1 and Activation of CD137 Increase Responses of Human Liver T Cells Against HBV, But Not HCV

In patients with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, antiviral functions of T cells are impaired; these might be increased by blocking T-cell co-inhibitory pathways, such as preventing interaction between the receptor programmed death (PD)-1 and its ligand, PD-L1. We attempted to optimize the restoration of T-cell functions in patients with chronic HBV or HCV infection with a combination of reagents that block PD-1 interaction with PD-L1 and stimulate T-cell signaling via CD137, a member of the tumor necrosis factor-receptor family. We assessed the effects of CD137 stimulation (via CD137L), alone or in combination with antibodies that block PD-1 interaction with PD-L1 (anti-PD-L1), on proliferation and production of interferon-γ and interleukin-2 by intrahepatic and peripheral T cells from patients with chronic HBV or HCV infection. We also analyzed expression of different co-stimulatory molecules on virus-specific CD8+ and forkhead box P3+CD4+ cells by flow cytometry. Incubation of intrahepatic T cells with CD137L and anti-PD-L1 increased their responses to HBV, but not HCV. However, HCV-specific T cells isolated from peripheral blood were sensitive to these reagents. Virus-specific T cells from some, but not all patients, had increased responses to anti-PD-L1 when CD137L was added because in some cases the combination of anti-PD-L1 and CD137L overstimulated T cells, leading to their inhibition. Intrahepatic HBV- and HCV-specific CD8+ T cells had different costimulatory profiles; liver cells from patients with chronic HBV infection had a higher proportion of forkhead box P3+ regulatory T cells, with higher levels of PD-1, compared with liver cells from patients with chronic HCV infection. A combination of reagents that prevent interaction between PD-1 and its ligand and activate CD137 signaling increase responses of intrahepatic HBV-specific T cells and circulating HCV-specific T cells. This strategy might be developed to increase T-cell responses to these viruses in patients with chronic hepatitis B or C, and tailoring the dose of CD137L administered will help optimize results.

Regulation of Inflammation by Bidirectional Signaling through CD137 and Its Ligand

Although the majority of research on CD137 has been directed to T cells, it is becoming clear that this molecule has distinct functions in other lineages of cells, including non-hematopoietic cells. In particular, emerging evidence suggests that the CD137-its ligand (CD137L) network involving immune cells and non-immune cells, directly or indirectly regulates inflammation in both positive and negative manners. Bidirectional signaling through both CD137 and CD137L is critical in the evolution of inflammation: 1) CD137L signaling plays an indispensible role in the activation and recruitment of neutrophils by inducing the production of proinflammatory cytokines and chemokines in hematopoietic and non-hematopoietic cells such as macrophages, endothelial cells and epithelial cells; 2) CD137 signaling in NK cells and T cells is required for their activation and can influence other cells participating in inflammation via either their production of proinflammatory cytokines or engagement of CD137L by their cell surface CD137: 3) CD137 signaling can suppress inflammation by controlling regulatory activities of dendritic cells and regulatory T cells. As recognition grows of the role of dysregulated CD137 or CD137L stimulation in inflammatory diseases, significant efforts will be needed to develop antagonists to CD137 or CD137L.

Abstract 4740: Agonist anti-CD137 mAb act on tumor endothelial cells to enhance recruitment ofactivated T lymphocytes

Abstract Agonist monoclonal antibodies to the immune co-stimulatory molecule CD137, also known as 4-1BB, are presently in clinical trials for cancer treatment based on their co-stimulatory effects on primed T cells and perhaps other cells of the immune system. Here we provide evidence that CD137 is selectively expressed on the surface of tumor endothelial cells. Hypoxia upregulated CD137 on murine endothelial cells. Treatment of tumor-bearing immunocompromised Rag-/- mice with agonist CD137 mAb did not elicit any measurable anti-angiogenic effects. In contrast, agonist mAb stimulated tumor endothelial cells, increasing cell surface expression of the adhesion molecules ICAM-1, VCAM-1 and E-selectin. When adoptively transferred into mice, activated T lymphocytes derived from CD137-deficient animals entered more avidly into tumor tissue after treatment with agonist mAb. This effect could be neutralized with anti-ICAM-1 and anti-VCAM-1 blocking antibodies. Thus, stimulation of CD137 not only enhanced T cell activation but also augmented their trafficking into malignant tissue, through direct actions on the blood vessels that irrigate the tumor. Our findings identify an additional mechanism of action that can explain the immunotherapeutic effects of agonist CD137 antibodies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4740. doi:10.1158/1538-7445.AM2011-4740

Abstract 4567: Polyubiquitin K63-related CD137 signalsomes in T cells stimulated with agonist anti-CD137 monoclonal antibodies

Abstract Agonist anti-CD137 monoclonal antibodies are employed to enhance CD8-mediated antitumor immunity both in mouse models and in clinical trials. A panel of agonist anti-human CD137 mAb was prepared recognizing three different epitopes on the CD137 surface glycoprotein. All mAb costimulated T cell activation irrespectively of interference or not with natural ligand-binding. Interestingly, CD137 perturbation with agonist mAb results in internalization towards a vesicular endosomal compartment derived from the plasma membrane. Such internalization was observed on CD137 transfectants in 293 cellsas well as on primary activated T cells from humans and mice. In vivo treatment with anti-CD137 mAb of immunodeficient mice harboring activated human or mouse T cells results in internalization of CD137 to this vesicular compartment. CD137 is known to associate with TRAF2 at the plasma membrane. TRAF-2 is an adaptor protein endowed with E3 ubiquitin ligase activity that synthesizes k63-polyubiquitin chains. K63 polyubiquitin, as opposed to K48 polyubiquitin, constitute a docking site for downtream signaling proteins that lead to NF-kB and AP-1 activation. CD137 vesicles are decorated with k63-polyubiquitin and CD137 stimulation leads to the formation of K63-polyub containing complexes. Therefore CD137 stimulation under therapy like conditions with mAbs leads to the assembly of an intracellular signaling compartment that we term as CD137 -signalsomes Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4567. doi:10.1158/1538-7445.AM2011-4567

CD137 stimulation enhances the antilymphoma activity of anti-CD20 antibodies

AbstractAntibody-dependent cell-mediated cytotoxicity (ADCC), which is largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B-cell lymphomas. CD137 is a costimulatory molecule expressed on a variety of immune cells after activation, including NK cells. In the present study, we show that an anti-CD137 agonistic mAb enhances the antilymphoma activity of rituximab by enhancing ADCC. Human NK cells up-regulate CD137 after encountering rituximab-coated tumor B cells, and subsequent stimulation of these NK cells with anti-CD137 mAb enhances rituximab-dependent cytotoxicity against the lymphoma cells. In a syngeneic murine lymphoma model and in a xenotransplanted human lymphoma model, sequential administration of anti-CD20 mAb followed by anti-CD137 mAb had potent antilymphoma activity in vivo. These results support a novel, sequential antibody approach against B-cell malignancies by targeting first the tumor and then the host immune system.

Agonist Anti-CD137 mAb Act on Tumor Endothelial Cells to Enhance Recruitment of Activated T Lymphocytes

Agonist monoclonal antibodies (mAb) to the immune costimulatory molecule CD137, also known as 4-1BB, are presently in clinical trials for cancer treatment on the basis of their costimulatory effects on primed T cells and perhaps other cells of the immune system. Here we provide evidence that CD137 is selectively expressed on the surface of tumor endothelial cells. Hypoxia upregulated CD137 on murine endothelial cells. Treatment of tumor-bearing immunocompromised Rag(-/-) mice with agonist CD137 mAb did not elicit any measurable antiangiogenic effects. In contrast, agonist mAb stimulated tumor endothelial cells, increasing cell surface expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin. When adoptively transferred into mice, activated T lymphocytes derived from CD137-deficient animals entered more avidly into tumor tissue after treatment with agonist mAb. This effect could be neutralized with anti-ICAM-1 and anti-VCAM-1 blocking antibodies. Thus, stimulation of CD137 not only enhanced T-cell activation but also augmented their trafficking into malignant tissue, through direct actions on the blood vessels that irrigate the tumor. Our findings identify an additional mechanism of action that can explain the immunotherapeutic effects of agonist CD137 antibodies.

Anti-CD137 mAb Deletes Both Donor CD4+and CD8+T Cells in Acute Graft-versus-host Disease

We previously demonstrated that in vivo engagement of CD137, a member of TNF receptor superfamily, can delete allorective CD4+ T cells through the induction of activation-induced cell death (AICD) in chronic graft-versus-host disease (cGVHD) and subsequently reverse established cGVHD. In this study, we further showed that agonistic anti-CD137 mAb was highly effective in triggering AICD of donor CD8+ T cells as well as donor CD4+ T cells in the C57BL/6→unirradiated (C57BL/6 × DBA/2)F1 acute GVHD model. Our results suggest that strong allostimulation should facilitate AICD of both alloreactive CD4+ and CD8+ T cells induced by CD137 stimulation. Therefore, depletion of pathogenic T cells using agonistic anti-CD137 mAb combined with potent TCR stimulation may be used to block autoimmune or inflammatory diseases mediated by T cells.

CD137-Mediated Pathogenesis from Chronic Hepatitis to Hepatocellular Carcinoma in Hepatitis B Virus-Transgenic Mice

Chronic hepatitis B virus (HBV) infection is characterized by sustained liver inflammation with an influx of lymphocytes, which contributes to the development of cirrhosis and hepatocellular carcinoma. The mechanisms underlying this immune-mediated hepatic pathogenesis remain ill defined. We report in this article that repetitive infusion of anti-CD137 agonist mAb in HBV-transgenic mice closely mimics this process by sequentially inducing hepatitis, fibrosis, cirrhosis, and, ultimately, liver cancer. CD137 mAb initially triggers hepatic inflammatory infiltration due to activation of nonspecific CD8(+) T cells with memory phenotype. CD8(+) T cell-derived IFN-γ plays a central role in the progression of chronic liver diseases by actively recruiting hepatic macrophages to produce fibrosis-promoting cytokines and chemokines, including TNF-α, IL-6, and MCP-1. Importantly, the natural ligand of CD137 was upregulated significantly in circulating CD14(+) monocytes in patients with chronic hepatitis B infection and closely correlated with development of liver cirrhosis. Thus, sustained CD137 stimulation may be a contributing factor for liver immunopathology in chronic HBV infection. Our studies reveal a common molecular pathway that is used to defend against viral infection but also causes chronic hepatic diseases.

CD137 stimulation of natural killer cells to enhance the antilymphoma activity of rituximab.

8068 Background: Rituximab, a monoclonal antibody (mAb) directed against CD20, has significantly improved the outcome of patients with lymphoma. Antibody-dependant cell-mediated cytotoxicity (ADCC)...

Treatment with anti-CD137 mAbs causes intense accumulations of liver T cells without selective antitumor immunotherapeutic effects in this organ.

Cancer therapy with agonist anti-CD137 mAbs has been shown to induce immune-mediated tumor rejections in mice, and equivalent agents of this kind are currently being tested in cancer patients. Previous reports indicated that CD137 stimulation induced polyclonal infiltrates of T lymphocytes in the liver. This study characterizes the liver infiltrates and the target dependency of the phenomena and addresses the question of whether tumors nested in the liver are a more favorable target for CD137-based immunotherapy. Liver infiltrates were studied with conventional histology and multiple color flow cytometry of total liver leukocytes. CD137(-/-) mice, mice with a single rearrangement of the TCR (OT-1 mice) and Rag(-/-) mice were used to clarify molecular requirements. Mice implanted with MC38 colon carcinomas either subcutaneously or inside the liver were used for comparative studies under treatment with agonist anti-CD137 mAbs. CD137 treatment caused mononuclear inflammation in the portal spaces of the liver, which gave rise to moderate increases in transaminases without signs of cholestasis. Marked increases in the numbers of CD8+ T cells were observed, including CD8+ T lymphocytes co-expressing CD11c. Infiltrates were absent in CD137(-/-) mice and mitigated in mice harboring a single transgenic TCR on their CD8 T cells. Despite the tumor-independent accumulation of T cells in the liver, immunotherapeutic effects were not more prominent against tumors located in this organ. Target-dependent effects of CD137 stimulation lead to liver infiltration with T cells, but lymphocyte enrichment in this organ does not privilege this site for immunotherapeutic effects against transplanted tumors.

CD137 Expression Is Enhanced in EBV-Infected T or NK Cells by Viral Protein LMP1 and Mediates Anti-Apoptotic Intracellular Signaling through NF-ĸB Activation.

Abstract 1929Poster Board I-952Epstein-Barr virus (EBV) can infect not only B cells but also T or NK cells uncommonly and causes lymphoid malignancies, such as extranodal NK/T-cell lymphoma nasal type (ENKL), aggressive NK-cell leukemia, and EBV-positive T/NK-cell lymphoproliferative disease (EBV-T/NK-LPD), which is also known as chronic active EBV infection. However, why and how EBV infects T or NK cells and the mechanism of action responsible for these EBV-induced malignancies have not been elucidated to date. To clarify the molecular mechanism underlying development of EBV-T/NK-LPD, we focused on costimulatory receptor CD137, which is expressed on the surface of activated T cells and plays a pivotal role in their proliferation, survival, and differentiation. We investigated CD137 expression on the surface of EBV-infected T/NK cells (EB-T/NK cells) by flow cytometry. First, three EBV-positive T and NK cell lines, SNT8, SNK6, and SNT16, were obtained for examination. These cell lines had been established from primary lesions of ENKL patients (SNT8 and SNK6) and peripheral blood of an EBV-T/NK-LPD patient (SNT16). CD137 expression was confirmed on the cell surface of these cells, whereas the EBV-negative T and NK cell line, Jurkat and KHYG1 cells, respectively, were negative for CD137. Next, we investigated expression on the surface of EB-T/NK cells derived from EBV-T/NK-LPD patients. EBV-T/NK-LPD was diagnosed according to the following criteria: presence of persistent infectious mononucleosis-like symptoms, elevation of EBV-DNA titer in the peripheral blood (PB), and detection of EBV-infected T or NK cells. To detect the infected cells, we isolated peripheral mononuclear cells and divided them into CD19-, CD4-, CD8-, or CD56-positive fractions using antibody-conjugated magnetic beads. Next, we measured the EBV-DNA titer of each fraction by quantitative RT-PCR. Nine patients (aged 8–41 years; 4 male, 5 female; 4 T and 5 NK cell types) were diagnosed with EBV-T/NK-LPD. Then, we examined surface CD137 expression of the infected cells of each patient. Expression was detected in 7 of 9 patients. Control cells (PB mononuclear cells of a healthy donor, who was negative for EBV-DNA titer in the PB) did not express the molecule. We also examined transcription of CD137 mRNA by RT-PCR assay and detected it in all the 12 EB-T/NK-cell samples described above. From these results we concluded that CD137 expression was induced at the level of both mRNA and protein in EB-T/NK cells. To investigate the molecular mechanism of CD137 overexpression in EBV-T/NK cells, we examined the influence of viral proteins on CD137 expression. EB-T/NK cells express EBV-encoded proteins, including LMP1, LMP2A, LMP2B, and EBNA1 (latency type 2). We cotransfected expression plasmids for these proteins with a luciferase reporter plasmid containing the CD137 gene promoter in Jurkat cells and performed a luciferase assay. LMP1 significantly upregulated the CD137 promoter activity, although the other molecules did not. Furthermore, in a transient expression assay of these viral proteins using Jurkat cells, transcription of endogenous mRNA of CD137 was upregulated only in the LMP1 transfectant. These results indicate that LMP1 may transactivate CD137 transcription and expression in EBV-T/NK cells. Next, we investigated the role of CD137 in developing EBV-T/NK-LPD. We cultured the above-mentioned CD137-expressing EBV-T/NK cells on CHO cells that stably express human CD137L on the cell surface. NF-ĸB activation was detected in CD137-positive EBV-T/NK cells that were cocultured with CD137L-expressing CHO cells. We confirmed that both p50 and p52 translocated to the nucleus, indicating that both canonical and non-canonical pathways for NF-ĸB activation were activated downstream of CD137. Finally, we investigated the role of CD137-mediated NF-ĸB activation in the development of EBV-T/NK-LPD. We cocultured EB-T/NK cells on CHO-wt or CHO-CD137L with VP-16 for 48 h and determined apoptosis by measuring DiCO6 uptake. We noted that stimulation of CD137 significantly suppressed VP-16-induced apoptosis of these cells. Together, these results indicate that EBV-infected T/NK-cells express CD137 on the cell surface, which may be induced by LMP1 and activate the anti-apoptotic intracellular signaling pathway through NF-ĸB activation. This pathway may contribute to immortalization of the infected cells and development of EBV-T/NK-LPD. Disclosures:No relevant conflicts of interest to declare.

CD137/4-1BB Is Inducibly Expressed On CLL B Cells through CD40 Signaling.

Abstract 1266Poster Board I-288CD137/4-1BB, a TNFR family member is expressed on activated T cells as a co-stimulator. It transduces the signal for cell survival and differentiation and plays a crucial role in CD8 cytotoxic T cells. Recently, an increasing number of reports have indicated its important role on tumor immunity, and the studies of immunotherapy targeting CD137 are on going. Therefore, it is important to know the expression of CD137 and CD137L on malignant cells for the establishment of immunotherapy. As a first step, we examined CD137 expression on PBMCs from healthy donors and CLL patients. When PBMCs from healthy donors were stimulated with PMA and ionomycin, CD137 expression was induced not only on T cells but also on activated B cells. However, when PBMCs from CLL patients were stimulated in the same way, we could not detect CD137 induction on CLL B cells. Since more than 90% of lymphocytes in the patients were CLL B cells, it is conceivable that activated T cells were required to induce CD137 on B cells. To test this hypothesis we next co-cultured CLL cells with T cells activated with anti-CD3/CD28 antibody-coated beads. In this co-culture CD137 was induced on CLL B cells, and this induction was diminished by anti-CD154 blocking antibody. Furthermore, CD137 was inducibly expressed on CLL B cells after co-culture with HeLa cells transfected with CD154 gene. The induction of CD137 mRNA was also clearly detected by RT-PCR after this stimulation. This CD137 induction was more significantly observed on CLL B cells (n=14, MFIR 11.5±6.9) as compared with B cells from healthy donors (n=4, MFIR 3.7±0.6, p=0.001) or non-CLL B cell malignancies (n=9, MFIR 4.2±3.43, p=0.003). Stimulation of CD137 expressed on BJAB transfectants by co-culture with CD137L-transfected CHO cells induced a conspicuous nuclear translocation of p52, a non-canonical NF-κB factor. In agreement with this activation, the expression of survival factor BCL-XL was upregulated. Consequently, the CD137 signal augmented the survival of CD154 stimulated CLL B cells in vitro. CD40 ligation can induce anti-CLL immunity and reduce CLL cells clinically. These data suggest that the inducibly expressed CD137 may diminish the effectiveness. It is possible that the fine adjustment of these co-stimulators can lead more effective immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

Blockade of CD137 Signaling Counteracts Polymicrobial Sepsis Induced by Cecal Ligation and Puncture

Sepsis, a leading cause of death worldwide, involves proinflammatory responses and inefficient bacterial clearance. Previously, we have shown that CD137 (4-1BB), a member of the tumor necrosis factor receptor superfamily, plays critical roles in eradicating infective Listeria monocytogenes, a gram-positive bacterium, and that stimulation of CD137 protects mice from sepsis-induced death. In this study, we unexpectedly found that CD137 activation aggravated polymicrobial sepsis due to mixed gram-positive and gram-negative bacterial infection induced by cecal ligation and puncture (CLP). CD137-deficient (CD137(-/-)) mice showed significantly lower mortality than CD137-sufficient (CD137(+/+)) mice in the CLP model. Administration of an agonistic anti-CD137 monoclonal antibody (MAb) to CD137(+/+) mice decreased their survival in this infection model, while administration of a blocking anti-CD137 ligand MAb (TKS-1) to such mice increased their survival. CD137(-/-) mice and TKS-1-treated CD137(+/+) mice had lower levels of chemokines/proinflammatory cytokines (monocyte chemoattractant protein 1, interleukin-6 [IL-6], tumor necrosis factor alpha, IL-12) and an anti-inflammatory cytokine (IL-10), exhibited improved bacterial clearance in the peritoneum, liver, and blood, and had greater numbers of infiltrated peritoneal neutrophils and macrophages in the CLP model than control mice. Our data suggest that CD137 activation aggravates polymicrobial sepsis induced by CLP.