CD40 Signaling Research Articles

Activation of CD137 Signaling Promotes Angiogenesis in Atherosclerosis via Modulating Endothelial Smad1/5-NFATc1 Pathway.

BackgroundExcessive angiogenesis is a key feature of vulnerable atherosclerotic plaques, and is considered an independent predictor of cardiovascular risk. CD137 signaling has previously been shown to be involved in atherosclerosis. However, the possible role of CD137 signaling in regulating angiogenesis has not been reported.Methods and ResultsApolipoprotein E‐deficient (ApoE−/−) mice were used as the in vivo model of atherosclerosis. Masson and immunohistochemical analysis of atherosclerotic plaques and Matrigel plug assay were used to evaluate the angiogenesis. Human umbilical vein endothelial cells and mouse brain microvascular endothelial cells were used as in vitro and ex vivo models to study how CD137 signaling affects angiogenesis. Matrigel tube formation assay, mouse aortic ring angiogenesis assay, and migration and proliferation assay were employed to assess angiogenesis. Western blot was used to detect protein expression. We found increased neovessel formation in atherosclerotic plaques of ApoE−/− mice treated with agonist anti‐CD137 antibody. Activation of CD137 signaling induced angiogenesis, endothelial proliferation, and endothelial cell migration. CD137 signaling activates the pro‐angiogenic Smad1/5 pathway, induces the phosphorylation of Smad1/5 and nuclear translocation of p‐Smad1/5, which in turn promotes the expression and translocation of NFATc1. Blocking CD137 signaling with inhibitory anti‐CD137 antibody could inhibit this activation and attenuated agonist anti‐CD137 antibody‐induced angiogenesis.ConclusionsThese findings suggest that CD137 signaling is a new regulator of angiogenesis by modulating the Smad1/5‐NFATc1 pathway.

CD40–CD40L cross-talk drives fascin expression in dendritic cells for efficient antigen presentation to CD4+ T cells

Fascin is an actin-bundling protein that, among immune cells, is restricted to expression in dendritic cells (DCs). Previous reports have suggested that fascin plays an important role in governing DC antigen presentation to CD4+ T cells. However, no report has clearly linked the receptor-ligand engagement that can direct downstream regulation of fascin expression. In this study, bone marrow-derived DCs from wild-type versus CD40-knockout C57BL/6 mice were used to elucidate the mechanisms of fascin expression and activity upon CD40-CD40 ligand (CD40L) engagement. These investigations now show that CD40 engagement governs fascin expression in DCs to promote CD4+ T-cell cytokine production. Absence of CD40 signaling resulted in diminished fascin expression in DCs and was associated with impaired CD4+ T-cell responses. Furthermore, the study found that loss of CD40-CD40L engagement resulted in reduced DC-T-cell contacts. Rescue by ectopic fascin expression in CD40-deficient DCs was able to re-establish sustained contacts with T cells and restore cytokine production. Taken together, these results show that cross-talk through CD40-CD40L signaling drives elevated fascin expression in DCs to support acquisition of full T-cell responses.

C-Myc dysregulation is a co-transforming event for nuclear factor-κB activated B cells.

While c-Myc dysregulation is constantly associated with highly proliferating B-cell tumors, nuclear factor (NF)-κB addiction is found in indolent lymphomas as well as diffuse large B-cell lymphomas, either with an activated B-cell like phenotype or associated with the Epstein-Barr virus. We raised the question of the effect of c-Myc in B cells with NF-κB activated by three different inducers: Epstein-Barr virus-latency III program, TLR9 and CD40. Induction of c-Myc overexpression increased proliferation of Epstein-Barr virus-latency III immortalized B cells, an effect that was dependent on NF-κB. Results from transcriptomic signatures and functional studies showed that c-Myc overexpression increased Epstein-Barr virus-latency III-driven proliferation depending on NF-κB. In vitro, induction of c-Myc increased proliferation of B cells with TLR9-dependant activation of MyD88, with decreased apoptosis. In the transgenic λc-Myc mouse model with c-Myc overexpression in B cells, in vivo activation of MyD88 by TLR9 induced splenomegaly related to an increased synthesis phase (S-phase) entry of B cells. Transgenic mice with both continuous CD40 signaling in B cells and the λc-Myc transgene developed very aggressive lymphomas with characteristics of activated diffuse large B-cell lymphomas. The main characteristic gene expression profile signatures of these tumors were those of proliferation and energetic metabolism. These results suggest that c-Myc is an NF-κB co-transforming event in aggressive lymphomas with an activated phenotype, activated B-cell like diffuse large B-cell lymphomas. This would explain why NF-κB is associated with both indolent and aggressive lymphomas, and opens new perspectives on the possibility of combinatory therapies targeting both the c-Myc proliferating program and NF-κB activation pathways in diffuse large B-cell lymphomas.

Role of caspases in CD95-induced biphasic activation of acid sphingomyelinase.

Acid sphingomyelinase (A-SMase) plays an important role in the initiation of CD95 signaling by forming ceramide-enriched membrane domains that enable clustering and activation of the death receptors. In TNF-R1 and TRAIL-R1/R2 signaling, A-SMase also contributes to the lysosomal apoptosis pathway triggered by receptor internalization. Here, we investigated the molecular mechanism of CD95-mediated A-SMase activation, demonstrating that A-SMase is located in internalized CD95-receptosomes and is activated by the CD95/CD95L complex in a biphasic manner.Since several caspases have been described to be involved in the activation of A-SMase, we evaluated expression levels of caspase-8, caspase-7 and caspase-3 in CD95-receptosomes. The occurrence of cleaved caspase-8 correlated with the first peak of A-SMase activity and translocation of the A-SMase to the cell surface which could be blocked by the caspase-8 inhibitor IETD.Inhibition of CD95-internalization selectively reduced the second phase of A-SMase activity, suggesting a fusion between internalized CD95-receptosomes and an intracellular vesicular pool of A-SMase. Further analysis demonstrated that caspase-7 activity correlates with the second phase of the A-SMase activity, whereas active caspase-3 is present at early and late internalization time points. Blocking caspases-7/ -3 by DEVD reduced the second phase of A-SMase activation in CD95-receptosomes suggesting the potential role of caspase-7 or -3 for late A-SMase activation.In summary, we describe a biphasic A-SMase activation in CD95-receptosomes indicating (I.) a caspase-8 dependent translocation of A-SMase to plasma membrane and (II.) a caspase-7 and/or -3 dependent fusion of internalized CD95-receptosomes with intracellular A-SMase-containing vesicles.

CD40 agonist converting CTL exhaustion via the activation of the mTORC1 pathway enhances PD-1 antagonist action in rescuing exhausted CTLs in chronic infection

Expansion of PD-1-expressing CD8+ cytotoxic T lymphocytes (CTLs) and associated CTL exhaustion are chief issues for ineffective virus-elimination in chronic infectious diseases. PD-1 blockade using antagonistic anti-PD-L1 antibodies results in a moderate conversion of CTL exhaustion. We previously demonstrated that CD40L signaling of ovalbumin (OVA)-specific vaccine, OVA-Texo, converts CTL exhaustion via the activation of the mTORC1 pathway in OVA-expressing adenovirus (AdVova)-infected B6 mice showing CTL inflation and exhaustion. Here, we developed AdVova-infected B6 and transgenic CD11c-DTR (termed AdVova-B6 and AdVova-CD11c-DTR) mice with chronic infection, and assessed a potential effect of CD40 agonist on the conversion of CTL exhaustion and on a potential enhancement of PD-1 antagonist action in rescuing exhausted CTLs in our chronic infection models. We demonstrate that a single dose of anti-CD40 alone can effectively convert CTL exhaustion by activating the mTORC1 pathway, leading to CTL proliferation, up-regulation of an effector-cytokine IFN-γ and the cytolytic effect in AdVova-B6 mice. Using anti-CD4 antibody and diphtheria toxin (DT) to deplete CD4+ T-cells and dendritic cells (DCs), we discovered that the CD40 agonist-induced conversion in AdVova-B6 and AdVova-CD11c-DTR mice is dependent upon host CD4+ T-cell and DC involvements. Moreover, CD40 agonist significantly enhances PD-1 antagonist effectiveness in rescuing exhausted CTLs in chronic infection. Taken together, our data demonstrate the importance of CD40 signaling in the conversion of CTL exhaustion and its ability to enhance PD-1 antagonist action in rescuing exhausted CTLs in chronic infection. Therefore, our findings may positively impact the design of new therapeutic strategies for chronic infectious diseases.

Preferentially expanding Vγ1+ γδ T cells are associated with protective immunity against Plasmodium infection in mice.

γδ T cells play a crucial role in controlling malaria parasites. Dendritic cell (DC) activation via CD40 ligand (CD40L)-CD40 signaling by γδ T cells induces protective immunity against the blood-stage Plasmodium berghei XAT (PbXAT) parasites in mice. However, it is unknown which γδ T-cell subset has an effector role and is required to control the Plasmodium infection. Here, using antibodies to deplete TCR Vγ1+ cells, we saw that Vγ1+ γδ T cells were important for the control of PbXAT infection. Splenic Vγ1+ γδ T cells preferentially expand and express CD40L, and both Vγ1+ and Vγ4+ γδ T cells produce IFN-γ during infection. Although expression of CD40L on Vγ1+ γδ T cells is maintained during infection, the IFN-γ positivity of Vγ1+ γδ T cells is reduced in late-phase infection due to γδ T-cell dysfunction. In Plasmodium-infected IFN-γ signaling-deficient mice, DC activation is reduced, resulting in the suppression of γδ T-cell dysfunction and the dampening of γδ T-cell expansion in the late phase of infection. Our data suggest that Vγ1+ γδ T cells represent a major subset responding to PbXAT infection and that the Vγ1+ γδ T-cell response is dependent on IFN-γ-activated DCs.

Amelioration of Inflammatory Cytokines Mix Stimulation: A Pretreatment of CD137 Signaling Study on VSMC.

Previous studies showed little CD137 expressed in normal vascular smooth muscle cells (VSMCs) and it is important to find a valid way to elevate it before studying its function. The level of CD137 was detected by RT-PCR, western blot, and flow cytometry, respectively. CD137 signaling activation was activated by agonist antibody and measured through phenotype transformation indicators and cell functions. Proteins in supernatants were detected by ELISA. The total CD137 elevates under different concentrations of CM treatment. Among these, 25 ng/ml CM treatment increases the CD137 expression mostly. However, flow cytometry demonstrates that 10 ng/ml CM elevates surface CD137 more significantly than other concentrations and reaches the peak at 36 h. At 10 ng/ml, but not 25 ng/ml CM pretreatment, the levels of phenotype related proteins such as SM-MHC, α-SMA, and calponin decrease while vimentin and NFATc1 increase, suggesting that VSMCs undergo phenotype transformation. Transwell, CCK-8 assay, and ELISA showed that the ability of VSMCs viability, migration, and IL-2 and IL-6 secretion induced by CD137 signaling was significantly enhanced by the pretreatment of 10 ng/ml CM. This research suggested that 10 ng/ml CM pretreatment is more reasonable than other concentrations when exploring CD137 function in VSMCs.

Activation of CD137 signaling accelerates vascular calcification in vivo and vitro

ObjectivesVascular calcification is a characteristic feature of atherosclerosis and is considered as an independent predictor of cardiovascular risk. CD137 signaling has previously shown to be involved in atherosclerosis. However, the possible role of CD137 signaling in regulation of vascular calcification has not been reported. In the present study, we investigated the effect of CD137 signaling on vascular calcification in ApoE−/− mice and in vascular smooth muscle cells (VSMCs) of mice. MethodsCalcium deposition and muscle fibers in vivo or vitro were identified by von-Kossa and Masson's trichrome staining respectively. Alkaline phosphatase (ALP) activity was measured by the ALP assay Kit. The presence of bone morphogenic protein 2 (BMP2) and runt-related transcription factor 2 (Runx2) was detected by real-time PCR, Western blot and immunofluorescence in vitro or vivo. ResultsOur data shows that activation of CD137 signaling by intraperitoneal injection of agonist-CD137 antibody increased the areas of vascular calcification. Activation of CD137 signaling also increased the expression of BMP2 and Runx2 in the atherosclerotic plaques. In vitro, activation of CD137 signaling also aggravated VSMC calcification, while blocking CD137 signaling could alleviate agonist-CD137 induced VSMC calcification. In addition, the levels of calcium, BMP2 and Runx2, indicators of calcification, were all significantly elevated in agonist-CD137 group in VSMCs. ConclusionOur data revealed a previously unrecognized role of CD137 signaling in vascular calcification in vivo and vitro and provides a novel target for prevention and treatment of atherosclerosis in the future.

Vitamin D Receptor Expression in Plasmablastic Lymphoma and Myeloma Cells Confers Susceptibility to Vitamin D.

Plasmablastic B-cell malignancies include plasmablastic lymphoma and subsets of multiple myeloma and diffuse large B-cell lymphomaDLBCL. These diseases can be difficult to diagnose and treat, and they lack well-characterized cell line models. Here, immunophenotyping and FOXP1 expression profiling identified plasmablastic characteristics in DLBCL cell lines HLY-1 and SU-DHL-9, associated with CTNNAL1, HPGD, RORA, IGF1, and/or vitamin D receptor (VDR) transcription. We demonstrated VDR protein expression in primary plasmablastic tumor cells and confirmed in cell lines expression of both VDR and the metabolic enzyme CYP27B1, which catalyzes active vitamin D3 production. Although Vdr and Cyp27b1 transcription in normal B cells were activated by interleukin 4 (IL-4) and CD40 signaling, respectively, unstimulated malignant plasmablastic cells lacking IL-4 expressed both VDR and CYP27B1. Positive autoregulation evidenced intact VDR function in all plasmablastic lines, and inhibition of growth by active vitamin D3 was both dependent on MYC protein inhibition and could be enhanced by cotreatment with a synthetic ROR ligand SR-1078. Furthermore, a VDR polymorphism, FOK1, was associated with greater vitamin D3–dependent growth inhibition. In summary, HLY-1 provides an important model of strongly plasmablastic lymphoma, and disruption of VDR pathway activity may be of therapeutic benefit in both plasmablastic lymphoma and myeloma.

CD40 signaling and hepatic steatosis: Unanticipated links

Obesity predisposes to an increased risk of nonalcoholic fatty liver disease (NAFLD). Hepatic steatosis is the key pathological feature of NAFLD and has emerged as a metabolic disorder in which innate and adaptive arms of the immune response play a central role in disease pathogenesis. Recent studies have revealed unexpected relationships between CD40 signaling and hepatic steatosis in high fat diet rodent models. CD154, the ligand of CD40, is a mediator of inflammation and controls several critical events of innate and adaptive immune responses. In the light of these reports, we discuss potential links between CD40 signaling and hepatic steatosis in NAFLD.

CD40 Signaling in Graves Disease Is Mediated Through Canonical and Noncanonical Thyroidal Nuclear Factor κB Activation.

CD40, a tumor necrosis factor receptor, is a major immune-modulating susceptibility gene for Graves disease (GD) as well as for a variety of other autoimmune diseases. Its broad association with autoimmunity underscores its paramount role in the development of a normal adaptive immune response, primarily in coordinating effective antigen presentation. The molecular pathways by which CD40 activation in the thyroid induces GD are unknown. In this study, we investigated whether NF-κB, a ubiquitious family of transcription factors, mediates the downstream effects of thyroid-specific CD40 activation. Cultured primary human thyrocytes, from patients with and without GD, underwent CD40 stimulation. Once stimulated, cytokines and transcription factors specific for either the canonical nuclear factor κB (NF-κB)1 pathway [interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α], which primarily recruits cells for innate immunity, or the noncanonical NF-κB2 pathway [B cell-activating factor of the TNF family, CC chemokine ligand (CCL)21], which directs B cell viability, were analyzed. Significant upregulation in the messenger RNA and protein levels of both canonical and noncanonical pathway cytokines was observed. Western blot analyses of the specific transcription factors for the NF-κB1 and NF-κB2 pathways (p65 and p100/p52, respectively) demonstrated that p65 is constitutively expressed. In contrast, CD40 stimulation robustly increased the expression of the NF-κB2 p52 transcription factor, and the upregulation was significantly more profound in the GD tissue than in the normal thyroid tissue. Our data show that CD40 activity in thyrocytes is prominently mediated via NF-κB and furthermore suggest that the NF-κB1 and NF-κB2 pathways both contribute to the triggering and the progression of GD.

T Cell Transcriptomes from Paroxysmal Nocturnal Hemoglobinuria Patients Reveal Novel Signaling Pathways

Background. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired blood disease, characterized by hemolytic anemia, bone marrow (BM) failure, and venous thrombosis. The etiology of PNH is a somatic mutation in the phosphatidylinositol glycan class A gene (PIG-A) on the X chromosome, which causes deficiency in glycosyl phosphatidylinositol-anchored proteins (GPI-APs). The involvement of T cells in PNH is strongly supported by clinical overlap between PNH and aplastic anemia (AA); the presence of GPI-AP deficient cells in AA associated with favorable response to immunosuppressive therapy; and an oligoclonal T cell repertoire in PNH patients. However, the molecular mechanisms responsible for the aberrant immune responses in PNH patients are not well understood. To identify aberrant molecular mechanisms involved in immune targeting of hematopoietic stem cells in BM, RNA sequencing (RNA-seq) was applied to examine the transcriptome of T cell subsets from PNH patients and healthy controls.Method. Blood samples were obtained after informed consent from 15 PNH patients and 15 age-matched healthy controls. For RNA extraction, freshly isolated peripheral blood mononuclear cells were sorted on the same day of blood draw to obtain four different T cell (CD3+ CD14- CD19- ViViD-) populations [CD4+ naïve (CD45RA+ CD45RO-), CD4+ memory (CD45RA- CD45RO+), CD8+ naïve (CD45RA+ CD45RO-), and CD8+ memory (CD45RA- CD45RO+) T cells] by fluorescence-activated cell sorter . RNA-Seq analysis from three PNH and three healthy controls was performed using the Illumina HiSeq™ 2000 platform. The Ingenuity® Pathway Analysis and Gene set enrichment analysis (GSEA) were employed to elucidate transcriptional pathways. RNA-seq data were validated by flow cytometry and quantitative real-time RT-PCR (RT-qPCR).Results and Discussion. Differentially expressed gene analysis of four T cell subsets showed distinct gene expression signatures in individual T cell subsets. In CD4+ naïve T cells, 11 gene expression levels were significantly different: five upregulated (including SRRM2 and TNFSF8) and six downregulated genes (including GIMAP6) (> 2 fold change, false discovery rate [FDR] < 0.05). In CD4+ memory T cells, 25 gene expression levels were significantly different: 15 upregulated (including JUND and TOB1) and 10 downregulated genes (including GIMAP4). In CD8+ naive T cells, only two gene expression levels were significantly different: upregulated CTSW and downregulated RPL9. In CD8+ memory T cells, seven gene expression levels were significantly different: two upregulated (CTSW and DPP4) and five downregulated genes (including SLC12A7). Further, differentially expressed gene analysis was performed by combining CD4+ naïve, CD4+ memory, CD8+ naïve, and CD8+ memory T cells from PNH or healthy controls, respectively. Out of 55 gene expression levels that were significantly different, 41 were upregulated (including TNFAIP3, JUN, JUND, TOB1, TNFSF8, and CD69) and 14 downregulated (including GIMAP4). By canonical pathways analysis, putative gene network interactions of differentially expressed genes were significantly enriched for canonical pathways of TNFR1, TNFR2, IL-17A, and CD27 signaling. By GSEA, the most significantly upregulated gene sets in CD4+ naïve, CD4+ memory, CD8+ naïve, and CD8+ memory T cells from PNH patients displayed gene signatures related to the "IGF1 pathway", "Pre-NOTCH expression and processing", "AP-1 pathway", and "ATF2 pathway", respectively. For validation of the RNA-seq data, we chose seven genes (TNFAIP3, JUN, JUND, TOB1, TNFSF8, CD69, and CTSW) because these are important mediators involved in regulation for T cells and dysregulation of these genes is associated with autoimmune diseases. Differential expression levels of TNFAIP3, JUN, and TOB1 were validated by RT-qPCR. By flow cytometry, higher expression of CD69 and TNFSF8 was confirmed in CD4+ and CD8+T cells from PNH compared to healthy controls.Conclusion. Using RNA-seq, we identified novel molecular mechanisms and pathways which may underlie the aberrant T cell immune status in PNH. Specific dysregulation of T cell intracellular signaling may contribute to BM failure and the inflammatory environment in PNH. Understanding these pathways may provide new therapeutic strategies to modulate T cell immune responses in BM failure. DisclosuresHosokawa:Aplastic Anemia and MDS International Foundation: Research Funding. Rios:GSK/Novartis: Research Funding. Weinstein:GSK/Novartis: Research Funding. Townsley:GSK/Novartis: Research Funding.

TLR3-Induced Maturation of Murine Dendritic Cells Regulates CTL Responses by Modulating PD-L1 Trafficking.

Targeting TLR3 through formulations of polyI:C is widely studied as an adjuvant in cancer immunotherapy. The efficacy of such targeting has been shown to increase in combination with anti-PD-L1 treatment. Nevertheless, the mechanistic details of the effect of polyI:C on DC maturation and the impact on T-DC interactions upon PD-L1 blockade is largely unknown. Here we found that although DC treatment with polyI:C induced a potent inflammatory response including the production of type I interferon, polyI:C treatment of DCs impaired activation of peptide specific CD8+ T cells mainly due to PD-L1. Interestingly, we found that PD-L1 trafficking to the cell surface is regulated in two waves in polyI:C-treated DCs. One induced upon overnight treatment and a second more rapid one, specific to polyI:C treatment, was induced upon CD40 signaling leading to a further increase in surface PD-L1 in DCs. The polyI:C-induced cell surface PD-L1 reduced the times of contact between DCs and T cells, potentially accounting for limited T cell activation. Our results reveal a novel CD40-dependent regulation of PD-L1 trafficking induced upon TLR3 signaling that dictates its inhibitory activity. These results provide a mechanistic framework to understand the efficacy of anti-PD-L1 cancer immunotherapy combined with TLR agonists.

CDX-1140, a Novel Agonist CD40 Antibody with Potent Anti-Lymphoma Activity

CD40, a member of the tumor necrosis factor receptor (TNFR) superfamily, is essential for activating both innate and adaptive immune systems. CD40 is expressed by antigen-presenting cells (APC) including dendritic cells and B cells. The engagement by its natural ligand (CD40L) on T cells activates APC thus enhancing immune responses. CD40 is also expressed on many cancer cells, in particular B cell lymphomas, and agonist CD40 antibodies can lead to apoptosis and impaired growth of cancer cells due to direct signaling effects. There are several CD40 agonist antibodies in clinical development, which are differentiated by their level of agonist activity, route of administration, and associated side effect profile. We have generated a panel of CD40-specific fully human antibodies derived from human immunoglobulin transgenic mice with the aim to select a clinical candidate with strong agonist activity and a safety profile that allows for systemic dosing. CDX-1140 is a human IgG2 antibody that stimulates CD40 signaling without the requirement for cross-linking or Fc receptor interactions. CDX-1140 is a potent stimulator of human B cells and dendritic cells in vitro, and has potent direct anti-tumor activity against B cell lymphoma cell lines transplanted into immunocompromised mice. To demonstrate the contribution of immune cell activation to the anti-lymphoma activity in vivo, we included human PBMCs in subsequent xenograft studies. Using human B cell lymphoma cell lines Raji and Ramos as tumor models and severe combined immunodeficiency mice (SCID-beige) as hosts, we found that addition of human PBMC alone had a minor impact on tumor growth (median survival 32 days, compared to 26 days with no treatment, p<0.01). Administration of CDX-1140 alone showed partial anti-tumor activity (median survival 39 days, p< 0.001), while the combination of human PBMCs and CDX-1140 provided a significant increase in survival with all mice alive at day 45. These studies provide evidence that CDX-1140 is able to induce in vivo activation and anti-tumor activity of effector cells derived from injected human PBMC, in addition to the direct signaling effects on lymphoma cells. We are investigating anti-lymphoma efficacy of combining CDX-1140 with other immunotherapies including varlilumab, our CD27 agonist antibody, which is currently in clinical development. Together with our prior preclinical studies characterizing this antibody, these results suggest that CDX-1140 is a promising candidate for clinical development in patients with lymphomas. DisclosuresHe:Celldex Therapeutics, Inc.: Employment. Testa:Celldex Therapeutics, Inc.: Employment. Anna:Celldex Therapeutics, Inc.: Employment. Jeffery:Celldex Therapeutics, Inc.: Employment. Sisson:Celldex Therapeutics, Inc.: Employment. Vitale:Celldex Therapeutics, Inc.: Employment. O'Neill:Celldex Therapeutics, Inc.: Employment. Crocker:Celldex Therapeutics, Inc.: Employment. Widger:Celldex Therapeutics, Inc.: Employment. Goldstein:Celldex Therapeutics, Inc.: Employment. Marsh Jr.:Celldex Therapeutics, Inc.: Employment. Keler:Celldex Therapeutics: Employment, Equity Ownership.

Utility of Fluorodeoxyglucose Positron Emission Tomography(FDG-PET) and Biomarkers, Serum Vitamin B12 and HDL for Assessing Lymphoproliferation in Children and Adults with Autoimmune Lymphoproliferative Syndrome Due to FAS Mutations (ALPS-FAS)

Introduction/Background:The autoimmune lymphoproliferative syndrome (ALPS) is a disorder of impaired lymphocyte apoptosis commonly associated with defects in the FAS gene in the CD95 signaling cascade. Childhood onset lymphadenopathy, splenomegaly and autoimmune cytopenias are characteristic clinical presentation of ALPS. The immunophenotypical hallmark of ALPS is the expansion of "double negative" TCR-__+T lymphocytes (DNTs) that lack both CD4 and CD8 markers and when these cells comprise more than 1.5% of all circulating lymphocytes. Patients with ALPS are at increased risk for B-cell lymphoma.Patients and Methods:We sought to evaluate the lymphoproliferative burden of patients referred to our institution and meeting the diagnostic criteria for ALPS using FDG-PET scans over a 10year period. Readily available biomarkers like serum vitamin B12 and HDL have also been very useful in diagnosis and follow up of patients with ALPS-FAS.Results:We have performed 91 FDG-PET scans in 76 ALPS patients. Whole body PET scans were performed, extending from the pelvis up to the cervical nodal regions, starting at 90 minutes after the FDG injection. The longer uptake times were chosen to account for nonmalignant nature of the condition under study. This included patients with ALPS-FAS (n=71) who had a median standardized uptake value (SUV) of 7.87 (range: 3.8-13.73) and non-FAS patients (n=20) with a median SUV of 7.47 (range: 4.21-14.41); none of these patients had lymphoma on clinical evaluation and further follow-up. In addition, 3 ALPS-FAS patients with lymphoma who underwent an FDG-PET scan prior to therapy for lymphoma had SUVs of 11.78, 15.24, and 26.86, respectively. Here we share the SUVMax data from PET imaging and associated biomarker data (serum vitamin B12, n=64 and serum HDL, n=21) available from 64 patients with ALPS-FAS (Table 1). FDG-PET SUVMax did not statistically correlate with high serum vitamin B12 levels and low serum HDL (n=21) (Correlation coefficient -0.14 and -0.02 respectively). Younger ALPS-FAS patients with more prolific lymphoproliferation were noted to have higher serum vitamin B12 values and more FDG avid lymphadenopathy in our series (Correlation coefficient Ð 0.56 and 0.29 respectively).Conclusions:1. FDG accumulates in the lymph nodes of patients affected by ALPS, but we postulate that FDG may accumulate in higher concentrations in lymph nodes from ALPS patients that have undergone malignant transformation.2. Clinical correlation including history of sudden onset focal enlargement of nodes and systemic symptoms is essential to suspect a diagnosis of lymphoma in ALPS-FAS patients as FDG-PET scans by themselves have limited utility in patients with background of nonmalignant lymphoproliferative disorders.3. FDG-PET/CT may aid the early detection of lymphoma in this at-risk population, as ALPS patients have a predisposition to develop malignancies, and choosing the most FDG avid lymph nodes for biopsy may reduce the need for repeat biopsies in response to lymph node size changes commonly observed in ALPS patients.4. For diagnosis as well as assessing lymphoproliferative burden during long term follow up of ALPS-FAS patients both high serum vitamin B12 levels and lipid profile (low serum HDL) can be used as surrogate biomarkers even though they do not correlate very well with FDG avidity of their lymph nodes (Figure1) in the limited series presented here*.*Reference: Moraitis AG, Freeman LA, Shamburek RD, Wesley R, Wilson W, Grant CM, Price S, Demosky S, Thacker SG, Zarzour A, Hornung RL, Pucino F, Csako G, Yarboro C, McInnes IB, Kuroiwa T, Boumpas D, Rao VK, Illei GG, Remaley AT. Elevated interleukin-10: a new cause of dyslipidemia leading to severe HDL deficiency.J Clin Lipidol. 2015 Jan-Feb;9(1):81-90. PMID: 25670364. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Acute Virus Control Mediated by Licensed NK Cells Sets Primary CD8+ T Cell Dependence on CD27 Costimulation.

NK cells represent a critical first-line of immune defense against a bevy of viral pathogens, and infection can provoke them to mediate supportive and suppressive effects on virus-specific adaptive immunity. In mice expressing MHC class I Dk (Dk), a major murine CMV (MCMV) resistance factor and self-ligand of the inhibitory Ly49G2 (G2) receptor, licensed G2+ NK cells provide essential host resistance against MCMV infection. Additionally G2+ NK cell responses to MCMV increase the rate and extent of dendritic cell (DC) recovery, as well as early priming of CD8+ T cell effectors in response to MCMV. However, relatively little is known about the NK cell effect on costimulatory ligand patterns displayed by DCs or on ensuing effector and memory T cell responses. In this study, we found that CD27-dependent CD8+ T cell priming and differentiation are shaped by the efficiency of NK responses to virus infection. Surprisingly, differences in specific NK responses to MCMV in Dk-disparate mice failed to distinguish early DC costimulatory patterns. Nonetheless, although CD27 deficiency did not impede licensed NK-mediated resistance, CD70 and CD27 were required to efficiently prime and regulate effector CD8+ T cell differentiation in response to MCMV, which eventually resulted in biased memory T cell precursor formation in Dk mice. In contrast, CD8+ T cells accrued more slowly in non-Dk mice and eventually differentiated into terminal effector cells regardless of CD27 stimulation. Disparity in this requirement for CD27 signaling indicates that specific virus control mediated by NK cells can shape DC costimulatory signals needed to prime CD8+ T cells and eventual T cell fate decisions.

CD40L is transferred to antigen-presenting B cells during delivery of T-cell help.

The delivery of T-cell help to B cells is antigen-specific, MHC-restricted, and CD40L (CD154) dependent. It has been thought that when a T cell recognizes an antigen-presenting B cell, CD40L expressed on the T-cell surface engages with CD40 on the surface of B cells as long as the cells remain conjugated. By adding fluorescently labeled anti-CD40L antibody during overnight incubation of antigen-presenting B cells with antigen-specific T cells, we discovered that CD40L does not remain on the surface of the T cell, but it is transferred to and endocytosed by B cells receiving T-cell help. In the presence of anti-CD40L antibody, transferred CD40L is nearly absent on bystander B cells that are not presenting antigen, and the bystander cells do not become activated. Because transfer of CD40L to B cells correlates with B-cell activation, we speculate that persistence of helper T-cell-derived CD40L on or in B cells could permit sustained CD40 signaling enabling survival and proliferation of antigen-presenting B cells following brief interactions with helper T cells in vivo in germinal centers.

Abstract B066: The hexavalent CD40 agonist APG1233 is a superior inducer of immune cell activation compared to trimeric formats or agonistic anti-CD40 antibodies

Abstract Introduction: The co-stimulatory receptor CD40 is strongly expressed on B cells, monocytes and antigen-presenting cells (APC). By promoting their maturation, activation and survival, CD40 signaling greatly contributes to anti-tumor responses of the immune system. The HERA-Technology developed by Apogenix is a powerful engineering platform for the production of modular fusion proteins targeting the TNF-receptor superfamily. Structurally, these novel ligands comprise a single-chain arrangement of three TNFSF protomers forming one TNFSF-receptor binding domain (RBD) with specific linkers and the Fc-part of human IgG as dimerization scaffold. The result is a stable and defined hexavalent receptor agonist with high clustering capacity for the cognate receptor. Here we compared the efficacy of different CD40 agonist formats, including the novel hexavalent scCD40L-RBD-Fc (APG1233), and the functional consequences of differential receptor clustering. In addition, the in vivo pharmacokinetics of the fully human APG1233 is compared with a chimeric mouse/human surrogate molecule APG1274. Materials and Methods: Immune cells were isolated from healthy-donor blood samples and profiled by multicolor flow cytometry (MC-FC). Subsequently, immune cells were cultured in growth media containing various forms of CD40 agonists. Upregulation of activation markers on B cells and monocytes (e.g. CD69, CD86, HLA-DR) and T cell-induced killing of tumor cells in direct co-culture was assessed by MC-FC and employing a real-time cell analysis system (xCELLigence), respectively. Secretion of cytokines in response to CD40 ligation and the pharmacokinetic properties of the fully human APG1233 and the chimeric murine/human APG1274 were determined by ELISA. Results: In vivo stability of APG1233 was demonstrated in a single dose mouse PK study revealing a terminal half-life of 84 hours. The human CD40L, however, does not bind murine CD40. Therefore, we used APG1274, a chimeric protein comprising the murine CD40L fused to human IgG and binding to mouse CD40, to determine serum pharmacokinetics. As a result of functional binding of APG1274 to murine CD40, the surrogate molecule is eliminated much quicker (t1/2 of 4 hours) demonstrating the specificity of both compounds. In vitro only the hexavalent APG1233 displayed activity in terms of stimulation of B cells, monocytes and PBMCs. In contrast, neither trimeric CD40L nor an agonistic antibody against CD40 were able to upregulate expression of activation markers. Similarly, the secretion of proinflammatory cytokines such as IL-12, CD95L and IFNγ by PBMCs was only stimulated after exposure to APG1233 and not in the presence of other CD40 agonists. In functional co-culture assays, after exposure to APG1233, in vitro generated M2-macrophages underwent conversion and acquired M1-type surface markers which strongly enhanced proliferation of naïve CD4+ T cells. Induction by M2-macrophages in this setup was much lower. Consistent with these data, only the hexavalent CD40 agonist APG1233 efficiently increased direct cytotoxic activity of immune cells against tumor cells measured by a real-time cell analysis assay. Conclusion: The CD40 agonist APG1233 is a member of a novel class of hexavalent TNFRSF agonists which binds its target with high specificity, exhibits excellent in vivo stability and superior biological activity over other agonistic formats (e.g. agonistic antibodies). Citation Format: Christian Merz, Jaromir Sykora, Meinolf Thiemann, David M. Richards, Thamara Beyer, Stefanie Kühn, Harald Fricke, Christian Gieffers, Oliver Hill. The hexavalent CD40 agonist APG1233 is a superior inducer of immune cell activation compared to trimeric formats or agonistic anti-CD40 antibodies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B066.

GW27-e0292 Activation of CD137 signaling accelerates vascular calcification in vivo and vitro

GW27-e0292 Activation of CD137 signaling accelerates vascular calcification in vivo and vitro

GW27-e0248 Effects of CD137 Signaling on the expression of NFATc1 in mice VSMCs through TRAF6/JNK/AP-1 pathway

GW27-e0248 Effects of CD137 Signaling on the expression of NFATc1 in mice VSMCs through TRAF6/JNK/AP-1 pathway