IgG-treated Mice Research Articles

EXTH-70. PRECLINICAL TRIALS OF THE ANTIBODY-DRUG CONJUGATE TISOTUMAB VEDOTIN IN ADULT-TYPE DIFFUSE GLIOMAS

Abstract The gene F3, encoding Tissue Factor (TF), is expressed in many cancers and contributes to their malignancy. Our previous work showed that, among adult-type diffuse gliomas, IDH wild-type (IDHwt) glioblastomas (GBM) express more TF than IDH mutant (IDHmut) gliomas (PMID: 27664011, 30266764, 31222125). Tisotumab vedotin (TisVed), an anti-TF antibody conjugated to monomethyl auristatin E, is a therapeutic designed to target cells expressing TF. We therefore sought to determine the therapeutic potential of TisVed in IDHwt vs. IDHmut gliomas. TisVed was more active against cultured IDHwt GBM cells than IDHmut glioma cells (P<0.0001). This activity was increased by 55% with daily washout of the soluble TF secreted by IDHwt GBM cells. TisVed extended the survival of mice intracranially engrafted with IDHwt GBM by 34% (37.5 days vs. 28 days for IgG control, P=0.006), but not mice with IDHmut glioma (30.5 days, P=0.88). TisVed also reduced the growth of IDHwt GBM flank xenografts by 58% (P=0.045). Unconjugated Tis had no antitumor effect intracranially or in the flank. All 12 flank-engrafted mice treated with Tis, and 9/12 mice treated with TisVed, developed large hematomas in and around the flank xenografts, compared to 0/12 IgG-treated mice (P<0.001). Similarly, a semiquantitative analysis showed that intracerebral hemorrhage was more extensive in Tis/TisVed- treated GBM12 mice than in IgG-treated GBM12 mice (P=0.046). These data indicate that TisVed targets high TF-expressing IDHwt GBM, but not low TF-expressing IDHmut glioma, that its activity is predominately through the vedotin conjugate rather than inhibition of TF signaling, and that it may have some effect against TF-expressing IDHwt GBM, but intracerebral hemorrhage may be a concern.

EXTH-45. PRECLINICAL EVALUATION OF THE ANTIBODY-DRUG CONJUGATE TISOTUMAB VEDOTIN IN ADULT-TYPE DIFFUSE GLIOMAS

Abstract The gene F3, encoding Tissue Factor (TF), is expressed in many cancers and contributes to their malignancy. Our previous work showed that, among adult-type diffuse gliomas, IDH wild-type (IDHwt) glioblastomas (GBM) express more TF than IDH mutant (IDHmut) gliomas (PMID: 27664011, 30266764, 31222125). Tisotumab vedotin (TV), an anti-TF antibody conjugated to monomethyl auristatin E, is a therapeutic designed to target cells expressing TF. We therefore sought to determine the therapeutic potential of TV in IDHwt vs. IDHmut gliomas. TV was more active against cultured IDHwt GBM cells than IDHmut glioma cells (P<0.0001). This activity was increased by 55% with daily washout of the soluble TF secreted by IDHwt GBM cells. TV extended the survival of mice intracranially engrafted with IDHwt GBM by 34% (37.5 days vs. 28 days for IgG control, P=0.006), but not mice with IDHmut glioma (30.5 days, P=0.88). TV also reduced the growth of IDHwt GBM flank xenografts by 58% (P=0.045). Unconjugated tisotumab had no antitumor effect intracranially or in the flank. All 12 flank-engrafted mice treated with tisotumab, and 9/12 mice treated with TV, developed large hematomas in and around the flank xenografts, compared to 0/12 IgG-treated mice (P<0.001). Similarly, a semiquantitative analysis showed that intracerebral hemorrhage was more extensive in tisotumab/TV- treated GBM12 mice than in IgG-treated GBM12 mice (P=0.046). These data indicate that TV targets high TF-expressing IDHwt GBM, but not low TF-expressing IDHmut glioma, that its activity is predominately through the vedotin conjugate rather than inhibition of TF signaling, and that it may have some effect against TF-expressing IDHwt GBM, but intracerebral hemorrhage may be a concern.

Activation of cytotoxic lymphocytes through CD6 enhances killing of cancer cells

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell–cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft mouse models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8 + T cells. In particular, tumor-infiltrating cytotoxic lymphocytes from UMCD6-treated mice expressed higher levels of perforin and were found in higher proportions than those from IgG-treated mice. Moreover, RNA-seq analysis of human NK-92 cells treated with UMCD6 revealed that UMCD6 up-regulates the NKG2D-DAP10 receptor complex, important in NK cell activation, as well as its downstream target PI3K. Our results now describe the phenotypic changes that occur on immune cells upon treatment with UMCD6 and further confirm that the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.

Blocking the CTLA-4 and PD-1 pathways during pulmonary paracoccidioidomycosis improves immunity, reduces disease severity, and increases the survival of infected mice.

Immune checkpoint pathways, i.e., coinhibitory pathways expressed as feedback following immune activation, are crucial for controlling an excessive immune response. Cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death protein-1 (PD-1) are the central classical checkpoint inhibitory (CPI) molecules used for the control of neoplasms and some infectious diseases, including some fungal infections. As the immunosuppression of severe paracoccidioidomycosis (PCM), a chronic granulomatous fungal disease, was shown to be associated with the expression of coinhibitory molecules, we hypothesized that the inhibition of CTLA-4 and PD-1 could have a beneficial effect on pulmonary PCM. To this end, C57BL/6 mice were infected with Paracoccidioides brasiliensis yeasts and treated with monoclonal antibodies (mAbs) α-CTLA-4, α-PD-1, control IgG, or PBS. We verified that blockade of CTLA-4 and PD-1 reduced the fungal load in the lungs and fungal dissemination to the liver and spleen and decreased the size of pulmonary lesions, resulting in increased survival of mice. Compared with PBS-treated infected mice, significantly increased levels of many pro- and anti-inflammatory cytokines were observed in the lungs of α-CTLA-4-treated mice, but a drastic reduction in the liver was observed following PD-1 blockade. In the lungs of α-CPI and IgG-treated mice, there were no changes in the frequency of inflammatory leukocytes, but a significant reduction in the total number of these cells was observed. Compared with PBS-treated controls, α-CPI- and IgG-treated mice exhibited reduced pulmonary infiltration of several myeloid cell subpopulations and decreased expression of costimulatory molecules. In addition, a decreased number of CD4+ and CD8+ T cells but sustained numbers of Th1, Th2, and Th17 T cells were detected. An expressive reduction in several Treg subpopulations and their maturation and suppressive molecules, in addition to reduced numbers of Treg, TCD4+, and TCD8+ cells expressing costimulatory and coinhibitory molecules of immunity, were also detected. The novel cellular and humoral profiles established in the lungs of α-CTLA-4 and α-PD-1-treated mice but not in control IgG-treated mice were more efficient at controlling fungal growth and dissemination without causing increased tissue pathology due to excessive inflammation. This is the first study demonstrating the efficacy of CPI blockade in the treatment of pulmonary PCM, and further studies combining the use of immunotherapy with antifungal drugs are encouraged.

Immune Checkpoint Blockade Promotes Thrombosis Via T-Cell and Neutrophil Activation, and Tumor-Cell Associated Tissue Factor (TF) in a Murine Model of Colorectal Cancer

Background: Immune checkpoint blockade (ICB) is a paradigm shift in cancer therapy. Despite clinical benefit, various adverse events are associated with ICB using immune checkpoint inhibitors (ICI). Venous thrombosis is an emerging clinical problem in cancer patients receiving ICI. Little is known about how ICI promotes thrombosis in cancer. To define the underlying mechanisms of ICI-associated thrombosis (IAT), we assessed the ICI impact on blood hypercoagulability and tumor TF expression using a mouse thrombosis model of colorectal cancer. Methods: We developed a CT26 mouse thrombosis model with ICI (aPD-1+aCTLA4) administration. Systemic hypercoagulability was assessed by measurement of circulating nucleosomes (Nu.Q H3.1 Immunoassay), neutrophil extracellular traps (NETs) (CitH3 immunofluorescence), circulating neutrophil-platelet aggregates (immunophenotyping), and levels of thrombin-antithrombin (TAT) complexes in ICI-treated mice. Tumor-associated TF and TF activity in circulating extracellular vesicles (EV) were assessed by western blotting and Factor Xa generation assays. To gain mechanistic insights into TF induction by ICI, tumor cytokines were also profiled (proteome profiler array). The role of cancer cell-derived TF in IAT was determined using mice implanted with CRISPR/Cas9-mediated TF knockout (TFKO) CT26 tumor cells. Results: ICI treated tumor-bearing mice developed larger thrombi than control mice and demonstrated elevated levels of circulating nucleosomes (114 vs 82 ng/ml), NET (15.8 vs 6.8%), platelet-neutrophil aggregates (44.8 vs 22.3%), and TAT complexes (12.6 vs 7.9 ng/ml) compared to IgG-treated mice. Tumor TF expression was increased in tumor extracts. Cytokine profiling suggested enhanced production of IFNɣ (2-fold) and TNFα (5-fold), as well as CXCL11 (6.8-fold), an effector T cell-recruiting chemokine. IFNɣ-increased TF expression in cultured CT26 cells, suggesting that T-cell derived IFNɣ may play a role in enhancing tumor TF expression in vivo. Circulating EV TF antigen and procoagulant activity were increased by ICI. CRISPR/Cas9-mediated TF KO in CT26 cells resulted in complete loss of TF expression, and mice bearing these cells showed reduced tumor and EV-associated TF (Fig. A), associated with a significant decrease in thrombus formation after ICI treatment (24.2 vs 20.5 mg, Fig. B). There was no significant difference in growth of wild-type and TF KO CT26 tumors. Conclusions: Our findings provide insights into cellular and molecular mechanisms associated with ICI-induced thrombosis, and suggest a role for neutrophil and granulocyte activation, and tumor-associated TF. Additional studies are ongoing to further define these pathways in the murine model and in patients.

Increased Interleukin-17-Producing γδT Cells in the Brain Exacerbate the Pathogenesis of Sepsis-Associated Encephalopathy and Sepsis-Induced Anxiety in Mice.

Overactivated microglia play a key role in sepsis-associated encephalopathy (SAE), although the involvement of T cells is unclear. γδT cells in the brain and meninges regulate normal fear responses via interleukin (IL)-17 in healthy mice. In our sepsis model, the mice showed exacerbated anxious behavior at 10 days post-induction (dpi). At 8 dpi, IL-17 mRNA was significantly upregulated in the brains of septic mice compared with those of control mice. Simultaneously, the number of γδT cells increased in the brains of septic mice in a severity-dependent manner. Additionally, IL-17-producing γδT cells, expressing both the C-X-C motif receptor (CXCR) 6 and the C-C motif receptor (CCR) 6, increased in mice brains, dependent on the severity of sepsis. The frequency of γδT cells in the meninges fluctuated similarly to that in the brain, peaking at 8 dpi of sepsis. Behavioral tests were performed on septic mice after the continuous administration of anti-γδTCR (α-γδTCR) or anti-IL-17A (α-IL-17A) antibodies to deplete the γδT cells and IL-17A, respectively. Compared with IgG-treated septic mice, α-γδTCR- and α-IL-17A-treated septic mice showed suppressed microglial activation and improvements in anxious behavior. These results suggested that CCR6+CXCR6+ IL-17-producing γδT cells in the brain and meninges promote the exacerbation of SAE and sepsis-induced psychological disorders in mice.

Systemic blockade of proBDNF inhibited the expansion and altered the transcriptomic expression in CD3+B220+ cells in MRL/lpr lupus mice

ObjectivesThe overexpansion of CD3+B220+ cells is the hallmark and main pathological mechanism of clinical manifestations of spontaneously developed MRL/lpr mice, which are primarily used as a mouse model of SLE....

Immune Checkpoint Inhibitors (ICI) Promote Neutrophil-Platelet Aggregate and NET Formation in Tumor-Bearing Mice

Introduction: Immune checkpoint inhibitor (ICI) therapy has revolutionized cancer treatment. Despite remarkable clinical benefit, there are numerous immune-related adverse events (irAEs) associated with ICI therapy. Cancer-associated thrombosis (CAT) is a common complication of cancer, and ICI-associated thrombosis (ICI-CAT) is an underappreciated irAE. There is no data available concerning the mechanisms underlying ICI-CAT. To address this issue, we have established a murine model of ICI-CAT in which tumor-bearing mice treated with ICI develop larger thrombi than those treated with control IgG. We have observed that these ICI-treated mice display greater numbers of neutrophils in thrombi, and increased levels of circulating neutrophil extracellular traps (NETs). Methods: ICI (anti-PD-1 and anti-CTLA-4) was infused into Balb/c mice bearing subcutaneous CT26 colorectal tumors. Neutrophils (CD11b+ Ly6G+) as a percentage of total WBC was assessed using an automated hematology analyzer. Neutrophil-platelet doublets and aggregates were determined by flow cytometry analysis in using antibodies for CD41 (GPIIb) and CD62P (P-selectin). Circulating neutrophils were isolated from tumor-bearing mice treated with IgG and ICI 48h after IVC occlusion using an EasySep mouse neutrophil enrichment kit (STEMCELL technologies). For NET staining, isolated neutrophils (5x105 cells/well) were seeded on poly-L lysine coated coverslips, and treated with or without phorbol myristate acetate (PMA, 100 nM) for 4 h in 37°C. Neutrophils were then blocked using 5% FBS and stained, without permeabilization, using rabbit monoclonal anti-histone H3 (citrulline R2+R8+R17) antibody (ab5103, Abcam). Extracellular CitH3 was detected using anti-rabbit Alexa Fluor 488 secondary antibody. Results: Thrombi in ICI-treated mice were larger and contained greater numbers of neutrophils than thrombi from IgG-treated mice. Complete blood counts demonstrated that neutrophil numbers increased 3-fold in tumor bearing (38.2%) vs non-tumor bearing mice (12.8%) (Data not shown). However, neutrophils from ICI-treated tumor-bearing mice demonstrated significantly more spontaneous NET formation than those isolated from mice treated with control IgG (66.7% vs 18.2%). Smaller differences were also noted in response to PMA (Fig. A). Since binding of platelets to neutrophils is one cause of NET release, we also determined the frequency of neutrophil-platelet aggregates circulating in tumor-bearing mice treated with ICI or control IgG. Increased levels of circulating neutrophil-platelet aggregate in ICI-treated mice were present in a doublet CD41+Ly6G+ population (44.8% vs 22.3%); similar results were noted when platelets were stained with the activation marker CD62 (P-selectin) (8.5% vs 1%) (Fig. B, Left). The frequency of larger neutrophil-platelet aggregates followed a similar trend (ICI vs IgG; 75.8% vs 30% for CD41; 48.1% vs 7.7% for CD62P) (Fig. B, Right). Conclusions: Circulating neutrophils from ICI-treated, tumor bearing mice demonstrate increased spontaneous NET release. Moreover, elevated levels of circulating platelet-neutrophil doublets and aggregates are present in ICI-treated mice compared to those treated with control IgG. These results suggest a potential mechanism of ICI-induced thrombosis, and that platelet-neutrophil interactions may stimulate NET release in ICI-treated mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tissue Factor (TF) Is Upregulated in Tumor-Bearing Mice Treated with Immune Checkpoint Inhibitor (ICI)

Introduction: Venous thromboembolism (VTE) is an important adverse event associated with immune checkpoint inhibitors (ICI) in cancer patients (12-15% annual rate). We have developed a murine model of venous thrombosis associated with ICI therapy (referred to as ICI-CAT). We found that mice bearing CT26 colon carcinoma treated with ICI developed larger thrombi following IVC occlusion compared with mice treated with IgG. ICI did not increase thrombus weight in mice without tumors. Tissue factor (TF) plays an important role in cancer-associated thrombosis (CAT) in patients treated with conventional therapies, and TF+ extracellular vesicles (EV) were associated with the development of VTE in pancreatic cancer. However, little is known about the impact of ICI on TF expression in patients treated with ICI. To address the underlying mechanism of ICI-associated thrombosis (IAT), we have examined the expression of TF in tumor and immune cells, as well as circulating EV, in our murine CT26 model. Methods: A syngeneic colon carcinoma model (CT26) in BALB/c mice was utilized to evaluate ICI-CAT following inferior vena cava ligation (stasis). Tumor tissue was analyzed for TF expression by immunoblotting using an anti-TF antibody (ab189483, Abcam). Tumor tissue was also dissociated, and TF-positive cells were assessed by flow cytometry analysis with CD45+ and CD45- gating using antibodies for CD11b, Ly6C, Ly6G (BioLegend), and TF (R&D). EV were isolated from platelet poor plasma from ICI or control IgG-treated mice by centrifugation (20,000 x g). Procoagulant activity of EV TF was measured using a chromogenic substrate to detect FXa generation following incubation of EV with FVIIa in the presence or absence of an anti-TF antibody. Results: Treatment of tumor-bearing mice with ICI markedly induced TF expression in tumors (Fig. A, Leftupper). Furthermore, levels of EV TF antigen (Fig. A, Left bottom) and EV TF activity (Fig. A, Right) were increased in mice treated with ICI. To explore the cellular origin of TF, we performed flow cytometry with dissociated tumor cells. A greater percentage of TF-positive cells in both the CD45+ (37.8%) and CD45- (74.6%) populations were detected in tumors from ICI treated mice compared to those treated with control IgG (27.9% and 49.8%, respectively), suggesting both tumor and tumor-infiltrating immune cells contribute to the larger thrombi observed in ICI-treated mice (Fig. B). Further analysis of CD45+ TF+ cells by CD11b gating (data not shown) revealed that a TF expression in a monocyte population (CD11b+ Ly6C+) was increased 3-fold by ICI (46.6%) compared to control IgG (14.2%) (Fig. C). We also found that TF expression was increased in cultured CT26 cells in response to interferon-g, but not in response to anti-PD-1 and anti-CTLA-4 antibodies, suggesting that ICIs do not directly upregulate TF and that more complex cellular interactions and inflammatory changes may be involved. Conclusions: ICI treatment of tumor-bearing mice results in increased tumor TF expression and EV TF expression. TF was also upregulated in immune cells in the tumor microenvironment after ICI treatment, including both tumor/stromal cells (CD45-), and tumor-associated monocytes and neutrophils (CD45+). These results suggest that multiple underlying mechanisms and diverse cellular expression underlie TF expression in this model, and that these processes many contribute to the larger thrombi observed in ICI-treated mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Enhanced anti-tumor immunity of vaccine combined with anti-PD-1 antibody in a murine bladder cancer model.

Programmed cell death protein 1 (PD-1) and ligand programmed death ligand 1 (PD-L1) are important immune-suppressive regulators in the tumor microenvironment. A vaccine-induced immune effect on tumor cells is blunted by the immunosuppressive tumor microenvironment. Therefore, we hypothesized that a dendritic cell (DC) vaccine combined with anti-PD-1 (αPD-1) antibodies could elicit a synergistic anti-tumor immunity in bladder cancer. We produced a model of subcutaneous transplantation in C3H/HeJ mice by transplanting murine MBT-2 bladder cancer cells. DCs were isolated from normal C3H/HeJ mice, followed by stimulation against MBT-2 lysate before injection. Two weeks later of MBT-2 inoculation, αPD-1 and stimulated DCs were injected two times at one-week interval intraperitoneally and intravenously, respectively. Tumor-infiltrating immune cells and splenocytes were analyzed using flow cytometry. T-cell-mediated anti-tumor responses were measured by interferon (IFN)-γ ELISPOT and lactate dehydrogenase assays. The mice treated with DC+αPD-1 showed a significant decrease in tumor volume compared to the DC-treated mice and IgG-treated group. Survival of the DC+αPD-1-treated group was improved compared with that of the IgG-treated mice. IFN-γ secretion from splenocytes against tumor cells was significantly increased in the DC+αPD-1 group compared with that of αPD-1-treated mice. The frequency of CD8+ and CD4+ T-cells in spleens was statistically increased in the DC+αPD-1-treated mice compared to those receiving monotherapy (DC- or αPD-1-treated group). Our results support the hypothesis that the combination therapy of a DC vaccine and αPD-1 antibodies could enhance the anti-tumor immune response against bladder cancer.

PD-L1 blockade exhibits anti-tumor effect on brain metastasis by activating CD8+ T cells in hematogenous metastasis model with lymphocyte infusion

Brain metastases are common complication in cancer patients. Immune checkpoint inhibitors show therapeutic benefits also in patients with central nervous system (CNS) metastases. However, their antitumor effects on metastatic tumors and their underlying mechanisms are still poorly understood. In this study we investigated the antitumor effect of anti-programmed death-ligand 1 (PD-L1) antibody on metastatic brain tumors and evaluated immune responses during treatment. We employed a hematogenous brain metastasis xenograft model using immunodeficient mice with murine lymphocyte infusions. A human non-small-cell lung cancer (NSCLC) cell line stably expressing NanoLuc® reporter (Nluc-H1915) was inoculated from the internal carotid artery of SCID mice. After metastases were established (24 days after inoculation), splenocytes prepared from H1915-immunized BALB/c mice were injected intravenously and mouse IgG or anti-PD-L1 antibody treatment was started (day 1). Evaluated by Nluc activity, tumor volume in the brain on day 14 was significantly lower in anti-PD-L1-treated mice than in mouse IgG-treated mice. Furthermore CD8+ cells were primarily infiltrated intratumorally and peritumorally and anti-PD-L1 treatment induced a significantly higher proportion of Granzyme B (GzmB)+ cells among CD8+ T cells. The antitumor effect of anti-PD-L1 antibody on brain metastasis is thought to be achieved by the enhanced activation of infiltrated CD8+ T cells into metastatic brain tumor. These results suggest that anti-PD-L1 antibody-containing regimens may be promising treatment options for cancer patients with brain metastases.

Immune Checkpoint Inhibitors (ICI) Enhance Cancer Associated Thrombosis (CAT) in Murine Model of Colon Carcinoma

AbstractBackground:Venous thromboembolism (VTE) is an important immune-related adverse event (irAE) associated with immune checkpoint inhibitors (ICI) cancer therapy. To better understand the pathogenesis of ICI-induced VTE during ICI treatment, we utilized a murine model of cancer associated thrombosis (CAT) to examine the impact of ICI treatment on the development of flow restriction-induced thrombosis.MethodsA syngeneic colon carcinoma murine model (CT26) on BALB/c background was utilized to evaluate venous thrombosis following inferior vena cava (IVC) ligation. Non-tumor-bearing and tumor-bearing animals were treated with therapeutic doses of ICI: anti-programmed cell death receptor 1 antibody (anti-PD1) and cytotoxic T‐lymphocyte-associated protein 4 (anti‐CTLA4) or isotype control antibodies. Mice underwent surgical treatment for IVC ligation followed by surgical retrieval of thrombus. Western blot analysis was performed on plasma cleaved high molecular weight kininogen (cHK), and citrullinated histone H3 (CitH3).ResultsTumor-bearing mice undergoing IVC ligation after anti-PD1 and anti-CTLA4 infusion developed larger thrombi compared to mice treated with isotype control IgG. Thrombus weights in CT26 tumor-bearing mice treated with ICI (19.56±4.41 mg) were significantly increased compared to those in mice treated with isotype control IgG (14.67±2.76 mg) (P=0.043). The weight of thrombi in non-tumor-bearing mice was not affected by ICI treatment (9.25±2.22 mg with control IgG vs 9.33±1.15 mg with ICI). In addition, there was a significant increase in thrombus length in mice treated with ICI (9±1.02 mm vs 7.61±0.99 mm with IgG, P=0.039). Cleaved high molecular weight kininogen (cHK), an indicator of contact activation was increased in plasma pre-IVC occlusion from ICI-treated mice compared to IgG-treated mice (68% vs 38% in HK cleavage). Citrullinated histone H3 (CitH3), a NETosis marker, was elevated in plasma (and in thrombus) post-IVC occlusion from ICI-treated mice compared to that of IgG-treated mice.ConclusionsICI treatment of tumor-bearing mice undergoing flow restriction-induced thrombosis resulted in enhanced clot formation. Larger thrombi in ICI treated mice was accompanied with enhanced contact activation and NETosis marker CitH3. [Display omitted] DisclosuresKhorana: Halozyme: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Anthos: Consultancy, Honoraria. McCrae: Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy; Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria.

CD39 and CD73 Activity Are Protective in a Mouse Model of Antiphospholipid Antibody-Induced Miscarriage

Antiphospholipid antibodies (aPL-ab) including lupus anticoagulant, anticardiolipin (aCL) antibodies and anti-β2-glycoprotein I (β2GP1) antibodies initiate inflammation, tissue factor-activated coagulation, oxidative stress and complement activation. These pathways contribute to the pathophysiology of thrombotic and obstetric manifestations in antiphospholipid syndrome (APS). Not all patients with aPL-ab develop disease phenotype and hence a ‘second hit’ is postulated to be necessary. Extracellular purines (ATP, ADP, and adenosine) are important signalling molecules with the general acceptance that ATP promotes inflammation, ADP is pro platelet-aggregatory, while adenosine has vasodilatory, antithrombotic and anti-inflammatory effects. Extracellular ATP/ADP levels are tightly regulated by CD39 and CD73. CD39 hydrolyses ATP and ADP to AMP, which is then hydrolysed to adenosine by the ecto-5'-nucleotidase CD73.We hypothesised that CD39 and CD73 mitigate both vascular thrombosis as well as inflammatory responses in APS. We developed a murine model of APS-miscarriages by passive infusion of aPL-ab harvested from patients into pregnant mice. Comparison made with infusion of non-immune IgG. We applied this model to wild type (WT), CD39-transgenic mice as well as CD39-null and CD73-null mice. Non-immune IgG administration to WT or CD39-/- C57BL/6 mice did not cause miscarriages, whereas aPL-ab-treated WT mice displayed a mean resorption frequency of 3.67%. In comparison, aPL-ab-treated CD39-/- mice showed a significantly higher mean resorption frequency of 20.1% (p<0.001 vs aPL-ab-treated WT; Fig. 1a). aPL-ab-treated CD73-/- mice also had a significantly increased mean resorption frequency of 10.5% (p<0.05 vs aPL-ab-treated WT).We have previously demonstrated that CD39 over expression in BALB/c transgenic mice is associated with enhanced adenosine generation under prothrombotic conditions. WT BALB/c mice injected with non-immune IgG had a mean resorption frequency of 21.2%, which doubled with aPL-ab injection (39.7%; p<0.05 vs non-immune IgG-treated WT). In transgenic BALB/c mice over-expressing CD39, mean resorption frequencies were significantly lower in the aPL-ab-treated group (11.4% vs 39.7% in aPL-ab-treated WT, p<0.01).QRT-PCR showed 1.6-fold and 2.4-fold increase in TF expression in aPL-ab administered CD39-/- and CD73-/- mice relative to both WT C57BL/6 mice and littermates injected with non-immune sera and. CD39 over expression suppressed TF expression compared with WT BALB/c mice after both non-immune IgG and aPL-ab challenge particularly with 6.3 fold lower TF mRNA levels after aPL-ab infusion. Decidua from aPL-ab injected CD39-/- and CD73-/- C57BL/6 mice showed increased TF immunoreactivity compared to aPL-ab-treated or nonimmune IgG-treated WT mice. C3d deposition was increased by aPL-ab treatment relative to non-immune IgG treatment in all groups except the CD39 transgenic mice (Fig. 3). C3d deposition was most marked in aPL-ab-treated CD39-/- and CD73-/- mice.Placental expression of inflammatory cytokines C57BL/6was measured at the mRNA level by qRT-PCR. TNF-α expression was doubled in CD39-/- and CD73-/- mice relative to non-immune IgG controls. CD39 Tg mice showed suppression of TNF- α after aPL-ab challenge. Consistent with the previously documented role for IL-10 in exacerbating lupus/auto-immune diseases, we noted a significant reduction in IL-10 mRNA in aPL-ab-treated CD39 Tg mice compared to aPL-ab-treated WT mice.Therefore, we demonstrate a protective role for CD39 in APS and provide rationale for the development of soluble CD39 as a novel therapeutic for APS and analysis of perturbations in the purinergic pathway to explain human disease DisclosuresDwyer:Kyowa Kirin Pharmaceutical Development, Inc.: Employment.

Club Cell Protein 16 Attenuates CD16brightCD62dim Immunosuppressive Neutrophils in Damaged Tissue upon Posttraumatic Sepsis-Induced Lung Injury.

Background Recently, identification of immunosuppressive polymorphonuclear leukocytes (PMNL) that were traditionally described as proinflammatory cells emerged in the field of posttraumatic immunity. To understand their local and remote distribution after trauma, PMNL-subsets and the impact of immunomodulatory Club Cell protein (CC)16 that correlates with pulmonary complications were assessed. Methods C57BL/6N mice were divided into three groups, receiving isolated blunt chest trauma (TxT), undergoing TxT followed by cecal ligation and puncture (CLP, TxT + CLP) after 24 h, or sham undergoing analgosedation (n = 18/group). Further, each group was subdivided into three groups receiving either no treatment (ctrl) or intratracheal neutralization of CC16 by application of anti-CC16-antibody or application of an unspecific IgG control antibody (n = 6/group). Treatment was set at the time point after TxT. Analyses followed 6 h post-CLP. PMNL were characterized via expression of CD11b, CD16, CD45, CD62L, and Ly6G by flow cytometry in bone marrow (BM), blood, spleen, lung, liver, and bronchoalveolar and peritoneal lavage fluid (BALF and PL). Apoptosis was assessed by activated (cleaved) caspase-3. Results from untreated ctrl and IgG-treated mice were statistically comparable between all corresponding sham, TxT, and TxT + CLP groups. Results Immature (CD16dimCD62Lbright) PMNL increased significantly in BM, circulation, and spleen after TxT vs. sham and were significantly attenuated in the lungs, BALF, PL, and liver. Classical-shaped (CD16brightCD62Lbright) PMNL increased after TxT vs. sham in peripheral tissue and were significantly attenuated in circulation, proposing a trauma-induced migration of mature or peripheral differentiation of circulating immature PMNL. Immunosuppressive (CD16brightCD62Ldim) PMNL decreased significantly in the lungs and spleen, while they systemically increased after TxT vs. sham. CLP in the TxT + CLP group reduced immunosuppressive PMNL in PL and increased their circulatory rate vs. isolated TxT, showing local reduction in affected tissue and their increase in nonaffected tissue. CC16 neutralization enhanced the fraction of immunosuppressive PMNL following TxT vs. sham and decreased caspase-3 in the lungs post-CLP in the TxT + CLP group, while apoptotic cells in the liver diminished post-TxT. Posttraumatic CC16 neutralization promotes the subset of immunosuppressive PMNL and antagonizes their posttraumatic distribution. Conclusion Since CC16 affects both the distribution of PMNL subsets and apoptosis in tissues after trauma, it may constitute as a novel target to beneficially shape the posttraumatic tissue microenvironment and homeostasis to improving outcomes.

The pathogenic role of circulating Hashimoto's Thyroiditis-derived TPO-positive IgG on fetal loss in naïve mice.

Antibody-mediated autoimmune diseases, such as autoimmune thyroid diseases (ATD), systemic lupus erythematosus (SLE), and antiphospholipid syndrome (APS), often are associated with recurrent fetal loss. One of the ATD is Hashimoto's thyroiditis which recently showed association with complications of pregnancy with increased levels of circulating autoantibodies reactive with epitopes on thyroid tissue such as thyroid peroxidase (anti-TPO). In retrospective study of sera analyses in patients with Hashimoto's thyroiditis, all patients had mainly elevated circulating anti-TPO autoantibodies. We assessed the potential of human anti-TPO highly positive IgG, derived from patients with Hashimoto's thyroiditis sera associated with complications of pregnancy, to cause directly complications of pregnancy in murine model. Naïve ICR female mice, infused intravenously with 100μg of anti-TPO-positive IgG, showed increased fetal loss and embryo small for date (P<.001) in comparison with mice passively transferred with commercial IgG or PBS. Moreover, we observed embryos small for date in the mice passively transferred with anti-TPO-positive IgG, exemplified by reduced weight of embryos and placentae (P=.001). Histopathological examination revealed delay in fetal development in 50% cases of anti-TPO-positive IgG-treated mice. Importantly, pathological changes in the transition zone, state of glycogen cells, and significant structural changes in the labyrinth part of placenta were observed in all anti-TPO-positive IgG samples. The current study shows in the first time, a direct proof of concept, on the association of human TPO-positive IgG from Hashimoto's thyroiditis patients on fetal loss induction in murine model.

NK cells modulate T cell responses via interaction with dendritic cells in Chlamydophila pneumoniae infection

Protective immune response to chlamydial infection is largely dependent on cell-mediated immune responses with IFN-γ production. Recent studies have shown the critical role of NK cells in bridging innate and adaptive immune responses. In this study, we investigated the effect of NK cells on T cell responses during Chlamydophila pneumoniae (Cpn) lung infection. The results showed that NK cells play a protective role in Cpn infection and influence T cell immunity largely though modulating dendritic cells (DCs) function. Specifically, we found that NK depletion significantly impaired type 1 T cell responses, but enhanced FOXP3+Treg cells and IL-10-producing CD4+T cells. The alteration of T cell responses was associated with more disease severity and higher chlamydial growth in the lung. Further analysis of DC phenotype and cytokine profile found that DCs from NK cell-depleted mice expressed lower levels of co-stimulatory molecules and produced higher levels of IL-10 than those from control IgG-treated mice. More importantly, the adoptive transfer of DCs from NK cell-depleted mice induced a much lower degree of type 1 T cell responses but a higher amount of FOXP3+ Treg cells and IL-10-producing CD4+T cells in the recipient mice than DCs from IgG-treated mice. In contrast to the strong protective effect observed in recipients of DCs from IgG-treated mice, the recipients of DCs from NK cell-depleted mice failed to be protected against Cpn infection. The data suggest that NK cells play a critical role in coordinating innate and adaptive immunity in Cpn lung infection by modulating the DC function to influence T cell responses.

A major role of TWEAK/Fn14 axis as a therapeutic target for post-angioplasty restenosis

BackgroundTumor necrosis factor-like weak inducer of apoptosis (Tnfsf12; TWEAK) and its receptor Fibroblast growth factor-inducible 14 (Tnfrsf12a; Fn14) participate in the inflammatory response associated with vascular remodeling. However, the functional effect of TWEAK on vascular smooth muscle cells (VSMCs) is not completely elucidated. MethodsNext generation sequencing-based methods were performed to identify genes and pathways regulated by TWEAK in VSMCs. Flow-citometry, wound-healing scratch experiments and transwell migration assays were used to analyze VSMCs proliferation and migration. Mouse wire injury model was done to evaluate the role of TWEAK/Fn14 during neointimal hyperplasia. FindingsTWEAK up-regulated 1611 and down-regulated 1091 genes in VSMCs. Using a gene-set enrichment method, we found a functional module involved in cell proliferation defined as the minimal network connecting top TWEAK up-regulated genes. In vitro experiments in wild-type or Tnfrsf12a deficient VSMCs demonstrated that TWEAK increased cell proliferation, VSMCs motility and migration. Mechanistically, TWEAK increased cyclins (cyclinD1), cyclin-dependent kinases (CDK4, CDK6) and decreased cyclin-dependent kinase inhibitors (p15lNK4B) mRNA and protein expression. Downregulation of p15INK4B induced by TWEAK was mediated by mitogen-activated protein kinase ERK and Akt activation.Tnfrsf12a or Tnfsf12 genetic depletion and pharmacological intervention with TWEAK blocking antibody reduced neointimal formation, decreasing cell proliferation, cyclin D1 and CDK4/6 expression, and increasing p15INK4B expression compared with wild type or IgG-treated mice in wire-injured femoral arteries. Finally, immunohistochemistry in human coronary arteries with stenosis or in-stent restenosis revealed high levels of Fn14, TWEAK and PCNA in VSMCs enriched areas of the neointima as compared with healthy coronary arteries. InterpretationOur data define a major role of TWEAK/Fn14 in the control of VSMCs proliferation and migration during neointimal hyperplasia after wire injury in mice, and identify TWEAK/Fn14 as a potential target for treating in-stent restenosis. FundISCiii-FEDER, CIBERCV and CIBERDEM.

057 Interleukin-6 regulates psoriasis-like dermatitis induced by blockade of programmed cell death 1 signaling

We recently reported that serum levels of interleukin (IL)-6 were increased in melanoma patients who developed neutrophil-rich psoriasiform dermatitis during immunotherapies with anti-programmed cell death 1 (PD-1) antibodies. To identify the roles of IL-6 in its pathogenesis, a murine model of imiquimod (IMQ)-induced psoriasiform dermatitis was used, by which we analyzed the difference between PD-1 deficient (PD-1-/-) mice and wild-type (WT) mice. PD-1-/- mice presented significantly more severe psoriasiform dermatitis with thicker ear swelling and higher PASI score, representing the severities of erythema, scaling and skin thickness, than WT mice (5.6 ± 0.24 v.s. 2.6 ± 0.24, P < 0.01). Histological investigations showed that more severe epidermal hyperplasia, and greater epidermal infiltration of both neutrophils and CD8 T cells in the ear samples of PD-1-/- mice than those of WT mice. PD-1-/- mice showed significantly higher mRNA levels of IL-6 and interferon (IFN)-γ in CD45-positive cells infiltrating into the epidermis, and CXCL9 in keratinocytes than WT mice. Flow cytometry analysis revealed that IL-6 productions of CD8 T cells and neutrophils were not enhanced by PD-1 deficiency. These results indicate that PD-1 deficiency enhances the infiltrating cell numbers of CD8 T cells and neutrophils, resulting in enhance of inflammation and increase of IL-6 production in the skin. The dermatitis exhibited by anti-IL-6 receptor antibody (MR16-1) treated PD-1-/- mice was significantly milder than that of isotype control IgG-treated PD-1-/- mice (PASI score; 4.3 ± 0.29 v.s. 7.3 ± 0.31 , P < 0.01), and about equal to that of isotype control IgG-treated WT mice (PASI score; 3.5 ± 0.27). In addition, MR16-1 treated PD-1-/- mice showed significantly suppressed mRNA levels in the ear skins and serum levels of psoriasis-related cytokines including IL-6, IFN-γ, IL-17A and IL-23 than those of control PD-1-/- mice. Hence, IL-6 blockade is a potential therapy to control anti-PD-1 antibody-induced psoriasisiform dermatitis.

Anti-β2GPI antibodies enhance atherosclerosis in ApoE-deficient mice

Accelerated atherosclerosis often occurs in patients with antiphospholipid syndrome (APS), and auto-antibodies to β2 glycoprotein I (anti-β2GPI) are confirmed as pathogenic antibodies to APS. Our previous studies have demonstrated that the conversion of mouse peritoneal macrophages into foam cells could be enhanced by co-existence of β2GPI and anti-β2GPI IgG, but this phenomenon has not been explored in vivo. Here, we present a mouse model to observe the effect of anti-β2GPI IgG in the development of atherosclerosis. Male ApoE-deficient mice were intraperitoneally injected with anti-β2GPI IgG (100 μg/mouse) and homologous control IgG (100 μg/mouse) every week for 16 weeks. Plasma lipid composition, magnetic resonance imaging (MRI) and histological staining were used to evaluate vascular inflammation, lumen stenosis and plaque stability. The results showed that the levels of total cholesterol, triglycerol and low-density lipoprotein-cholesterol in plasma were not changed in all mice fed with high-fat diet, but the level of high-density lipoprotein-cholesterol was lower and the atherosclerosis index was significantly increased in HD + anti-β2GPI group than in other high-fat diet groups. In addition, compared with NR IgG-treated mice, anti-β2GPI IgG-treated mice showed more lipid deposition in the carotid artery, markedly narrowed arteriolar lumen as well as higher MMP-9 expression, more macrophages and fewer collagen fibers in the aortic arch root. Furthermore, the aortic mRNA levels of TNF-α, IL-1β, and MCP-1 were significantly increased in anti-β2GPI IgG-treated mice. Together, these data indicate that anti-β2GPI IgG increases vascular inflammation, aggravates atherosclerosis and promotes the formation of vulnerable plaque in ApoE-deficient mice.

FGF23 Neutralizing Antibody Partially Improves Bone Mineralization Defect of HMWFGF2 Isoforms in Transgenic Female Mice.

Mice overexpressing high molecular weight FGF2 isoforms (HMWTg) in osteoblast lineage phenocopy human X-linked hypophosphatemic rickets (XLH) and a Hyp murine model of XLH demonstrating increased FGF23/FGF receptor signaling and hypophosphatemic rickets/osteomalacia. Because HMWFGF2 was upregulated in bones of Hyp mice and abnormal FGF23 signaling is important in XLH, HMWTg mice were used to examine the effect of the FGF23 neutralizing antibody (FGF23Ab). Eight-week-old female Vector control mice and HMWTg mice were treated with FGF23Ab or control IgG. A single injection of FGF23Ab rescued abnormal hypophosphatemia in HMWTg. The decreased type II sodium-dependent phosphate co-transporter (Npt2a) was rescued by FGF23Ab treatment. Inappropriately low serum 1,25(OH)2 D in HMWTg mice was normalized by FGF23Ab treatment, which is accompanied by increased anabolic vitamin D hydroxylase Cyp27b1 and decreased catabolic vitamin D hydroxylase Cyp24 mRNA in kidney. Long-term treatment with FGF23Ab normalized femur length and significantly increased vertebrae BMD and BMC, and femur BMC in HMWTg mice compared to IgG-treated HMWTg mice. Micro-computed tomography (μCT) revealed increased cortical porosity and decreased cortical apparent density in the HMWTg-IgG group compared with the Vector-IgG group; however, FGF23Ab treatment rescued defective cortical mineralization, decreased porosity, and increased apparent density in HMWTg mice. Bone histomorphometry analysis showed FGF23Ab treatment decreased osteoid volume, increased intra-label thickness, mineralization apposition rate, and bone formation rate in HMWTg mice. FGF23Ab improved disorganized double labeling in femurs from HMWTg mice. Quantitative real-time PCR analysis of tibia shafts showed FGF23Ab treatment normalized the osteocalcin (Ocn) mRNA expression in HMWTg mice, but further increased expression of SIBLING protein-related and pyrophosphate-related genes that are important in matrix mineralization, suggesting that HMWFGF2 modulates these genes independent of FGF23. We conclude that FGF23Ab partially rescued hypophosphatemic osteomalacia in HMWTg. However, long-term treatment with FGF23Ab further increased SIBLING protein-related genes and pyrophosphate-related genes in bone that could contribute to incomplete rescue of the mineralization defect. © 2018 American Society for Bone and Mineral Research.