VLA-4 Research Articles

Integrin Binding Dynamics Modulate Ligand-Specific Mechanosensing in Mammary Gland Fibroblasts.

SummaryThe link between integrin activity regulation and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently, generate high forces, and display nuclear YAP on soft collagen-coated substrates, resembling the soft mammary gland tissue. We describe that loss of the integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modeling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of collagen-binding integrin α11β1. Thus integrin activity regulation and α11β1 play a role in collagen-specific mechanosensing in MSFs.

Vedolizumab therapy in common variable immune deficiency associated enteropathy: A case series

A number of gastrointestinal complications occur in common variable immunodeficiency (CVID). Infections are one cause, but various forms of severe non-infectious enteropathy also lead to substantial morbidity. The presence of T cell lymphocytic infiltrates in the mucosa have suggested that vedolizumab, a humanized monoclonal antibody which binds to alpha4 beta7 integrin and inhibits the migration of effector T-lymphocytes into gastrointestinal tissues, would be an effective treatment. A previous report of 3 CVID cases suggested benefit in 2 subjects. In this study 7 CVID patients with severe enteropathy were treated with vedolizumab. Four of the 7 completed vedolizumab induction therapy but 3 subjects had acute decompensation during induction and treatment was stopped. While one subject showed improvement, 6 of the 7 patients were withdrawn from therapy. While vedolizumab may be of use in some CVID subjects, it was not ultimately found helpful in most of these patients.

Abstract WP142: Targeting VCAM1 to Reduce Acute and Chronic Neuroinflammation After Stroke

Background: Ischemia-induced neuroinflammmation is associated with worse outcomes after stroke. The innate immune response can mediate infarct expansion and impair short-term recovery, while adaptive responses lead to chronic neuroinflammation, neurodegeneration remote to the ischemic lesion, and consequent vascular dementia. Vascular cell adhesion molecule 1 (VCAM1) is an endothelial protein that facilitates vascular-immune cell interactions by binding very late antigen-4 (VLA-4). Soluble VCAM1 plasma levels are elevated in stroke patients and in aging people and mice. In aging mice, blocking VCAM1 ameliorates age-induced neuroinflammation and cognitive impairment. Objective: To determine if acute anti-VCAM1 treatment in mice would reduce microgliosis and astrogliosis, while delayed treatment would reduce B and T lymphocyte infiltration. Methodology: Adult (3-month-old) C57BL/6 mice (N=10/group) underwent permanent distal middle cerebral artery occlusion and were dosed with anti-VCAM1 antibody 4h post-stroke, then sacrificed at 72h. Microgliosis and astrogliosis were quantified as percent area immunostained in the infarct border by Iba1 and GFAP, respectively. Results: Acute treatment reduced microgliosis by 30% (p=0.0476) and astrogliosis 39% (p<0.03). Additionally, adult mice (N=10/group) were treated with anti-VCAM1 immediately (4h) or later (4d) post-stroke and euthanized 3 weeks later. B cell infiltration was quantified as percent stroke core immunostained by B220, while T lymphocyte infiltration was quantified as CD3+ cells in the core. Delayed anti-VCAM1 significantly reduced B and T cell infiltration by approximately 25% (p=0.0015) and 50% (p=0.0192), respectively. In contrast, early anti-VCAM1 had no effect on B/T cell infiltration. Conclusions: Together, these findings establish VCAM1 as a possible therapeutic target to prevent stroke-induced neuroinflammation in both acute and chronic settings.

Shp2 activation in bone marrow microenvironment mediates the drug resistance of B-cell acute lymphoblastic leukemia through enhancing the role of VCAM-1/VLA-4

B-cell acute lymphoblastic leukemia (B-ALL) is immune to the chemotherapy-induced apoptosis as a result of the protection of bone marrow mesenchymal stromal cells (BMSCs). However, the precise underlying mechanism of such protection remains unclear so far. In this experiment, protein tyrosine phosphatase 2 (Shp2), which was encoded by the PTPN11 gene, was highly expressed in BMSCs of the newly diagnosed and the recurrent B-ALL patients. The plasmid-induced (including Shp2 E76K) Shp2 activation in BMSCs (Shp2-activated BMSCs) markedly increased the BMSCs-mediated resistance of leukemia cells both in vitro and in vivo. Additionally, studies in vitro suggested that, the expression of vascular cell adhesion molecule 1 (VCAM-1) was markedly up-regulated in Shp2-activated BMSCs, and VCAM-1 expression in BMSCs of B-ALL patients was negatively correlated with Shp2 expression. Down-regulation of VCAM-1 in BMSCs using siRNA reversed the resistance of CCRF-SB cells mediated by the Shp2-activated BMSCs. As for the molecular mechanism, the PI3K/AKT pathway mediated the regulation of VCAM-1 by Shp2. Blocking the very late antigen-4 (VLA-4) by antibodies in CCRF-SB cells dramatically reversed the resistance of CCRF-SB cells mediated by the Shp2-activated BMSCs, and decreased the adhesion effects of both CCRF-SB cells and BMSCs. In conclusion, Shp2 activation in BMSCs up-regulates VCAM-1 expression through increasing the PI3K/AKT phosphorylation level, and targeting the VCAM-1/VLA-4 signaling may serve as a clinically relevant mechanism to overcome the BMSCs-mediated chemoresistance of B-ALL cells.

P701 New onset spondyloarthropathy in vedolizumab treated IBD patients

Abstract Background Patients with IBD may have associated spondyloarthropathy. Vedolizumab is used for IBD with its actions considered to be gut specific but the impact of this treatment on existing arthritis or development of arthritis is uncertain. Integrins and adhesion molecules play a role in the interception of recirculating activated lymphocytes between the gut and the synovial membrane due to the inhibition of the alpha4beta7 integrin homing at the level of the gut. We aimed to describe characteristics and outcomes of spondyloarthropathy in IBD patients treated with vedolizumab. Methods We retrospectively collected data on a multicentre cohort of IBD patients with arthropathy which developed following use of Vedolizumab for IBD. This was a part of the European Crohn`s and Colitis Organisation (ECCO) initiated CONFER (Collaborative Network for Exceptionally Rare case reports) project. Data were recorded on a standardised case report form and analysed with descriptive statistics. Results We report data on 14 cases of new onset spondyloarthropathy and 1 case of worsening of pre-existing arthropathy from 7 contributing centres. Vedolizumab was used for 6 patients with CD and 8 patients with UC. IBD was in remission in all but 3 patients at the time of development of the joint features. Sacroiliitis was the manifestation in 10 patients, 3 of whom had both sacroiliitis and peripheral arthritis. Three patients had peripheral arthritis alone and one patient had peripheral arthritis with enthesitis. One patient had worsening of peripheral arthropathy following use of vedolizumab. Raised inflammatory markers (CRP, ESR or both) were recorded only in one-third of the patients. MRI was abnormal in 9 patients being the marrow oedema in SI joints the commonest feature. One patient had abnormal bone scintigraphy and in another patient plain X-ray and ultrasound was used in confirming diagnosis. Eight of the patients developed arthropathy within days after a single infusion of vedolizumab. five patients needed hospitalisation for treatment The treatments used included systemic steroids (11 patients), Intraarticular steroids (3) NSAID (2 patients), Cox inhibitor (1), methotrexate (2) Hydroxychloroquine (1). Vedolizumab was discontinued in 11 of the patients. In the majority of patients (11/15) it took many months to resolve the arthropathy. On re-challenge with vedolizumab, the arthropathy recurred in 2 out of the 3 patients. Conclusion This case series demonstrates the potential risk for development of spondyloarthropathy with vedlizumab in IBD patients which is unrelated to the activity of IBD and can have significant impact including need for use for systemic steroids and hospitalisation. Larger prospective cohorts are needed to confirm these results.

VCAM-1 secreted from cancer-associated fibroblasts enhances the growth and invasion of lung cancer cells through AKT and MAPK signaling.

Several studies have indicated that cancer-associated fibroblasts (CAFs) could promote cancer progression in many malignancies. However, the mechanism by which CAFs promote the growth and metastasis of lung cancer remains poorly defined. In the present study, CAFs and normal fibroblasts (NFs) were isolated from human lung cancer and adjacent tissue. The data showed that the conditional medium (CM) of CAFs could increase the proliferation, migration and invasion of lung cancer cells. Vascular cell adhesion molecule-1 (VCAM-1) showed a higher expression in CAF-CM than NF-CM, and blocking VCAM-1 in CAF-CM attenuated the proliferation and invasion of cancer cells. Further, the results showed that VCAM-1 secreted from CAFs activated AKT and MAPK signaling via receptor α4β1 integrin (very-late antigen (VLA)-4) in lung cancer cells. Moreover, CAFs promoted VCAM-1 expression and tumor growth in vivo. Additionally, bioinformatics analysis indicated a positive correlation on the CAF marker protein alpha-smooth muscle actin (α-SMA) and VCAM-1 expression, which was associated with a poor prognosis in lung cancer patients. These findings demonstrate that the VCAM-1 secreted from CAFs enhances growth and invasion by activating the AKT and MAPK signaling of lung cancer cells.

A Bistable Mechanism Mediated by Integrins Controls Mechanotaxis of Leukocytes

Recruitment of leukocytes from blood vessels to inflamed zones is guided by biochemical and mechanical stimuli, with the mechanisms only partially deciphered. Here, we studied the guidance by the flow of primary human effector T lymphocytes crawling on substrates coated with ligands of integrins lymphocyte function-associated antigen 1 (LFA-1) (αLβ2) and very late antigen 4 (VLA-4) (α4β1). We reveal that cells segregate in two populations of opposite orientation for combined adhesion and show that decisions of orientation rely on a bistable mechanism between LFA-1-mediated upstream and VLA-4-mediated downstream phenotypes. At the molecular level, bistability results from a differential front-rear polarization of both integrin affinities, combined with an inhibiting cross talk of LFA-1 toward VLA-4. At the cellular level, direction is determined by the passive, flow-mediated orientation of the nonadherent cell parts, the rear uropod for upstream migration, and the front lamellipod for downstream migration. This chain of logical events provides a comprehensive mechanism of guiding, from stimuli to cell orientation.

Integrin crosstalk allows CD4+ T lymphocytes to continue migrating in the upstream direction after flow.

In order to perform critical immune functions at sites of inflammation, circulatory T lymphocytes must be able to arrest, adhere, migrate and transmigrate on the endothelial surface. This progression of steps is coordinated by cellular adhesion molecules (CAMs), chemokines, and selectins presented on the endothelium. Two important interactions are between Lymphocyte Function-associated Antigen-1 (LFA-1) and Intracellular Adhesion Molecule-1 (ICAM-1) and also between Very Late Antigen-4 (VLA-4) and Vascular Cell Adhesion Molecule-1 (VCAM-1). Recent studies have shown that T lymphocytes and other cell types can migrate upstream (against the direction) of flow through the binding of LFA-1 to ICAM-1. Since upstream migration of T cells depends on a specific adhesive pathway, we hypothesized that mechanotransduction is critical to migration, and that signals might allow T-cells to remember their direction of migration after the flow is terminated. Cells on ICAM-1 surfaces migrate against the shear flow, but the upstream migration reverts to random migration after the flow is stopped. Cells on VCAM-1 migrate with the direction of flow. However, on surfaces that combine ICAM-1 and VCAM-1, cells crawl upstream at a shear rate of 800s-1 and continue migrating in the upstream direction for at least 30minutes after the flow is terminated-we call this 'migrational memory'. Post-flow upstream migration on VCAM-1/ICAM-1 surfaces is reversed upon the inhibition of PI3K, but conserved with cdc42 and Arp2/3 inhibitors. Using an antibody against VLA-4, we can block migrational memory on VCAM-1/ICAM-1 surfaces. Using a soluble ligand for VLA-4 (sVCAM-1), we can promote migrational memory on ICAM-1 surfaces. These results indicate that, while upstream migration under flow requires LFA-1 binding to immobilized ICAM-1, signaling from VLA-4 and PI3K activity is required for the migrational memory of CD4+ T cells. These results indicate that crosstalk between integrins potentiates the signal of upstream migration.

Neutrophils promote VLA-4–dependent B cell antigen presentation and accumulation within the meninges during neuroinflammation

The success of B cell depletion therapies and identification of leptomeningeal ectopic lymphoid tissue (ELT) in patients with multiple sclerosis (MS) has renewed interest in the antibody-independent pathogenic functions of B cells during neuroinflammation. The timing and location of B cell antigen presentation during MS and its animal model experimental autoimmune encephalomyelitis (EAE) remain undefined. Using a new EAE system that incorporates temporal regulation of MHCII expression by myelin-specific B cells, we observed the rapid formation of large B cell clusters in the spinal cord subarachnoid space. Neutrophils preceded the accumulation of meningeal B cell clusters, and inhibition of CXCR2-mediated granulocyte trafficking to the central nervous system reduced pathogenic B cell clusters and disease severity. Further, B cell-restricted very late antigen-4 (VLA-4) deficiency abrogated EAE dependent on B cell antigen presentation. Together, our findings demonstrate that neutrophils coordinate VLA-4-dependent B cell accumulation within the meninges during neuroinflammation, a key early step in the formation of ELT observed in MS.

Clinical Challenge and Call for Research on Keloid Disorder: Meeting Report from The 3rd International Keloid Research Foundation Symposium, Beijing 2019

Clinical Challenge and Call for Research on Keloid Disorder: Meeting Report from The 3rd International Keloid Research Foundation Symposium, Beijing 2019

Expression of melanoma cell adhesion molecule-1 (MCAM-1) in natalizumab-treated multiple sclerosis

The objectives were to study the expression of very late antigen (VLA)-4, melanoma cell adhesion molecule-1 (MCAM-1) and activated leukocyte cell adhesion molecule (ALCAM) on CD4+ T cells during natalizumab treatment and to investigate the association with disease activity.We find that subgroups of autoreactive T cells are retained in peripheral blood, in particular MOG-reactive CD4+ T cells expressing MCAM-1. The expression of MCAM-1 or ALCAM on CD4+ T cells was, however, not clearly associated with disease activity (clinical or MRI) during natalizumab treatment. We confirm upregulation of MCAM-1 on CD4+ T cells during natalizumab treatment while VLA-4 is downregulated.

Angiopoietin-Tie Signaling Pathway in Endothelial Cells: A Computational Model.

SummaryThe angiopoietin-Tie signaling pathway is an important vascular signaling pathway involved in angiogenesis, vascular stability, and quiescence. Dysregulation in the pathway is linked to the impairments in vascular function associated with many diseases, including cancer, ocular diseases, systemic inflammation, and cardiovascular diseases. The present study uses a computational signaling pathway model validated against experimental data to quantitatively study various mechanistic aspects of the angiopoietin-Tie signaling pathway, including receptor activation, trafficking, turnover, and molecular mechanisms of its regulation. The model provides mechanistic insights into the controversial role of Ang2 and its regulators vascular endothelial protein tyrosine phosphatase (VE-PTP) and Tie1 and predicts synergistic effects of inhibition of VE-PTP, Tie1, and Tie2 cleavage on enhancing the vascular protective actions of Tie2.

736 Vedolizumab: Peri-Operative Infection Rates in Patients With IBD

INTRODUCTION: Vedolizumab (VDZ), a humanized monoclonal antibody, binds to the alpha4beta7 integrin and blocks the interaction of mucosal cell adhesion molecule-1 thus inhibiting the migration of memory T-lymphocytes into inflamed GI parenchymal tissue. This interaction is a hallmark of Ulcerative colitis (UC) and Crohn's disease (CD). It has been used as an alternative therapy to anti-tumor necrosis factor (anti-TNF) agents for Induction and maintenance in patients with moderate to severe inflammatory bowel diseases (IBD). Due to its immunosuppressive effects, VDZ maybe associated with increased risks of infections. Therefore, it is imperative to study the infection rate in this population, particularly during the perioperative period. Our objective is to identify perioperative infection rates in patients with Inflammatory Bowel Disease (IBD). METHODS: A retrospective multi-center case-control study was conducted. Patients with IBD in the period from May 2014 to Dec 2017 were included. Two groups were studied (Pt with IBD and on VDZ, Pt with IBD and not on VDZ). The perioperative period was identified as up to 30 days post-index surgery. ICD-10 codes for intra-abdominal surgeries and infections were used to identify population. Patients developing only new infections were included. Data were classified into two cohorts for the purpose of comparison: Cohort 1 included all-infections and Cohort 2 included only intra-abdominal infection. Different models of logistic regression data analysis were performed. RESULTS: Logistic regression data analysis was performed including simple, multiple and mixed model for both cohorts. The mixed model included multiple encounters per person, age, gender, ethnicity, and race. Results of Cohort 1 showed odds ratio (OR) = 1.332 (CI 0.599-2.965, P = 0.4821). Results of Cohort 2: OR = 1.286 (CI 0.426-3.882, P = 0.6551). These findings indicated that there was no statistical significance between increased rates of peri-operative infection rates and VDZ use. CONCLUSION: Our findings suggested that VDZ is relatively safe to use during the perioperative period. There are a few small studies indicating similar results, however, a unique aspect of our study was to include broad-spectrum infection types (sepsis from any source such as pneumonia, UTI) and not only intra-abdominal or surgery-related infections. We do recommend further investigations with more controlled study and larger population coverage to further solidify our findings.

730 Safety, Pharmacokinetics, and Pharmacodynamics of the Novel Oral Peptide Therapeutic PN-10943 (alpha4beta7 Integrin Antagonist) in Normal Healthy Volunteers

INTRODUCTION: Oral gut-restricted α4β7 integrin peptide antagonists represent a new therapeutic strategy for IBD. An example is PTG-100, which in a recent Phase 2 study was associated with higher rates of clinical and histologic remission relative to placebo in ulcerative colitis patients (Sandborn et al, UEGW 2018). PN-10943 is an oral, gut-restricted, potent, and highly selective α4β7 integrin antagonist peptide that has demonstrated preclinical superiority to PTG-100 (Mattheakis et al, DDW 2019). The purpose of this first-in-human Phase 1 study with PN-10943 was to assess safety, tolerability, PK, and PD activity in NHV and confirm previous preclinical observations. METHODS: A randomized, placebo-controlled Phase 1 trial of single (SAD) and multiple (MAD) ascending doses of PN-10943 was conducted in 70 male NHV. MAD regimen consisted of 14 daily doses. Subjects are monitored for safety and tolerability while undergoing serial plasma PK and blood PD evaluations as measured by integrin receptor occupancy (RO) and expression (RE) on circulating α4β7+ lymphocyte subsets. RESULTS: In the SAD portion of the study, 100, 300, 1000, and 1400 mg cohorts were dosed with no serious adverse events or dose-limiting toxicities. All adverse events were of mild to moderate severity and resolved without complication. In the MAD portion of the study doses studied were 100, 300, & 1000 mg for 14 days. Peak plasma concentration (Cmax) and area under the curve increased in a dose proportional manner and plasma pharmacokinetics were also time invariant between day 1 and day 14 in the MAD cohort. There was a significant dose dependent increase in RO of blood memory T cells (reaching >90% in the 1000 mg cohort). RE of α4β7 on blood memory T cells decreased in a dose dependent manner consistent with α4β7 internalization. The PD responses strongly correlated with plasma Cmax, and the binding activity from subject/time-matched RO on α4β7 memory T cells was comparable to that demonstrated in preclinical studies. CONCLUSION: Following oral administration in NHV, PN-10943 was well tolerated. As expected, plasma exposure was very low, and there was significant dose-related PD. The Phase 1 PK/PD relationship confirms previous preclinical observations in which PN-10943 demonstrated superiority to PTG-100. These Phase 1 PD data for PN-10943 together with the previous clinical activity of PTG-100 suggest that PN-10943 may potentially be an effective oral therapy for the treatment of UC.

Blockade of \u03b14 integrins reduces leukocyte\u2013endothelial interactions in cerebral vessels and improves memory in a mouse model of Alzheimer\u2019s disease

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by cognitive decline associated with the deposition of amyloid-β (Aβ) plaques, hyperphosphorylation of tau protein, and neuronal loss. Vascular inflammation and leukocyte trafficking may contribute to AD pathogenesis, and a better understanding of these inflammation mechanisms could therefore facilitate the development of new AD therapies. Here we show that T cells extravasate in the proximity of cerebral VCAM-1+ vessels in 3xTg-AD transgenic mice, which develop both Aβ and tau pathologies. The counter-ligand of VCAM-1 – α4β1 integrin, also known as very late antigen-4 (VLA-4) – was more abundant on circulating CD4+ T cells and was also expressed by a significant proportion of blood CD8+ T cells and neutrophils in AD mice. Intravital microscopy of the brain microcirculation revealed that α4 integrins control leukocyte–endothelial interactions in AD mice. Therapeutic targeting of VLA-4 using antibodies that specifically block α4 integrins improved the memory of 3xTg-AD mice compared to an isotype control. These antibodies also reduced neuropathological hallmarks of AD, including microgliosis, Aβ load and tau hyperphosphorylation. Our results demonstrate that α4 integrin-dependent leukocyte trafficking promotes cognitive impairment and AD neuropathology, suggesting that the blockade of α4 integrins may offer a new therapeutic strategy in AD.

Intravenous infusion of iPSC-derived neural precursor cells increases acid β-glucosidase function in the brain and lessens the neuronopathic phenotype in a mouse model of Gaucher disease.

Gaucher disease (GD) is caused by GBA1 mutations leading to functional deficiency of acid-β-glucosidase (GCase). No effective treatment is available for neuronopathic GD (nGD). A subclass of neural stem and precursor cells (NPCs) expresses VLA4 (integrin α4β1, very late antigen-4) that facilitates NPC entry into the brain following intravenous (IV) infusion. Here, the therapeutic potential of IV VLA4+NPCs was assessed for nGD using wild-type mouse green fluorescent protein (GFP)-positive multipotent induced pluripotent stem cell (iPSC)-derived VLA4+NPCs. VLA4+NPCs successfully engrafted in the nGD (4L;C*) mouse brain. GFP-positive cells differentiated into neurons, astrocytes and oligodendrocytes in the brainstem, midbrain and thalamus of the transplanted mice and significantly improved sensorimotor function and prolonged life span compared to vehicle-treated 4L;C* mice. VLA4+NPC transplantation significantly decreased levels of CD68 and glial fibrillary acidic protein, as well as TNFα mRNA levels in the brain, indicating reduced neuroinflammation. Furthermore, decreased Fluoro-Jade C and NeuroSilver staining suggested inhibition of neurodegeneration. VLA4+NPC-engrafted 4L;C* midbrains showed 35% increased GCase activity, reduced substrate [glucosylceramide (GC, -34%) and glucosylsphingosine (GS, -11%)] levels and improved mitochondrial oxygen consumption rates in comparison to vehicle-4L;C* mice. VLA4+NPC engraftment in 4L;C* brain also led to enhanced expression of neurotrophic factors that have roles in neuronal survival and the promotion of neurogenesis. This study provides evidence that iPSC-derived NPC transplantation has efficacy in an nGD mouse model and provides proof of concept for autologous NPC therapy in nGD.

The Biological Effects of Interleukin-17A on Adhesion Molecules Expression and Foam Cell Formation in Atherosclerotic Lesions.

Interleukin-17A (IL-17A), a major effector cytokine secreted by T helper 17 (Th17) cells, is elevated in atherosclerosis lesions. The purpose of this study was to assess the role of IL-17A in the pathogenesis of atherosclerosis. To measure the expression of adhesion molecules, human umbilical vein endothelial cells (HUVECs) and U937 cells were stimulated with IL-17A. Western blot and real-time polymerase chain reaction analyses revealed that intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in HUVECs, and very late antigen-4 (VLA-4), lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (MAC-1) expression in U937 cells was upregulated by IL-17A. Furthermore, IL-17A stimulation resulted in mRNA and protein expression of scavenger receptor (LOX-1) in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. Oil Red O also demonstrated that IL-17A enhanced foam cell formation by PMA-activated U937 cells induced by oxidized low-density lipoprotein (ox-LDL), and this enhancement of ox-LDL-induced foam cell formation in IL-17A-treated U937 cells was downregulated by transfection of LOX-1 siRNA. These results indicated that IL-17A induced the expression of adhesion molecules, promoted the adherence of monocytes to vascular endothelial cells. IL-17A also stimulated ox-LDL-induced foam cell formation via upregulation of LOX-1 in activated macrophages. IL-17A may be responsible for the pathogenesis of atherosclerosis by inducing the adhesion of leukocytes to vascular endothelium and foam cell formation.

Mucin-1 is required for Coxsackie Virus B3-induced inflammation in pancreatitis

The Muc-1 oncoprotein is a tumor-associated mucin often overexpressed in pancreatic cancer. We report that knockout of Muc-1 reduced the degree of pancreatic inflammation that resulted from infection with Coxsackievirus B3 (CVB3) in a mouse model. CVB3-infected Muc-1-deficient (Muc-1KO) mice had significantly reduced infiltration of macrophages into the murine pancreas. We found that Muc-1 signaling through NF-κB increased expression of ICAM-1, a pro-inflammatory mediator that recruits macrophages. Further investigation revealed that bone marrow derived macrophages (BMDM) from the Muc-1KO mice exhibited defective migration properties, in part due to low expression of the C-C motif chemokine receptor (CCR2) and the integrin Very Late Antigen 4 (VLA-4). The results presented here provide novel insight into the role of Muc-1 in regulating the inflammatory response and the cellular microenvironment in pancreatitis.

Inhibiting PGGT1B Disrupts Function of RHOA, Resulting in T-cell Expression of Integrin α4β7 and Development of Colitis in Mice

It is not clear how regulation of T-cell function is altered during development of inflammatory bowel diseases (IBD). We studied the mechanisms by which geranylgeranyltransferase-mediated prenylation controls T-cell localization to the intestine and chronic inflammation. We generated mice with T-cell-specific disruption of the geranylgeranyltransferase type I, beta subunit gene (Pggt1b), called Pggt1bΔCD4 mice, or the ras homolog family member A gene (Rhoa), called RhoaΔCD4 mice. We also studied mice with knockout of CDC42 or RAC1 and wild-type mice (controls). Intestinal tissues were analyzed by histology, multiphoton and confocal microscopy, and real-time polymerase chain reaction. Activation of CDC42, RAC1, and RHOA were measured with G-LISA, cell fractionation, and immunoblots. T cells and lamina propria mononuclear cells from mice were analyzed by flow cytometry or transferred to Rag1-/- mice. Mice were given injections of antibodies against integrin alpha4beta7 or gavaged with the RORC antagonist GSK805. We obtained peripheral blood and intestinal tissue samples from patients with and without IBD and analyzed them by flow cytometry. Pggt1bΔCD4 mice developed spontaneous colitis, characterized by thickening of the intestinal wall, edema, fibrosis, accumulation of T cells in the colon, and increased expression of inflammatory cytokines. Compared with control CD4+ T cells, PGGT1B-deficient CD4+ T cells expressed significantly higher levels of integrin alpha4beta7, which regulates their localization to the intestine. Inflammation induced by transfer of PGGT1B-deficient CD4+ T cells to Rag1-/- mice was blocked by injection of an antibody against integrin alpha4beta7. Lamina propria of Pggt1bΔCD4 mice had increased numbers of CD4+ T cells that expressed RORC and higher levels of cytokines produced by T-helper 17 cells (granulocyte-macrophage colony-stimulating factor, interleukin [IL]17A, IL17F, IL22, and tumor necrosis factor [TNF]). The RORC inverse agonist GSK805, but not antibodies against IL17A or IL17F, prevented colitis in Pggt1bΔCD4 mice. PGGT1B-deficient CD4+ T cells had decreased activation of RHOA. RhoAΔCD4 mice had a similar phenotype to Pggt1bΔCD4 mice, including development of colitis, increased numbers of CD4+ T cells in colon, increased expression of integrin alpha4beta7 by CD4+ T cells, and increased levels of IL17A and other inflammatory cytokines in lamina propria. T cells isolated from intestinal tissues from patients with IBD had significantly lower levels ofPGGT1B than tissues from individuals without IBD. Loss of PGGT1B from T cells in mice impairs RHOA function, increasing CD4+ T-cell expression of integrin alpha4beta7 and localization to colon, resulting in increased expression of inflammatory cytokines and colitis. T cells isolated from gut tissues from patients with IBD have lower levels of PGGT1B than tissues from patients without IBD.

Abstract 5010: Potentiating immune checkpoint blockade therapeutic efficacy using a small molecule activator of integrin cell adhesion receptors

Abstract Background: Immune checkpoint blockade (ICB) has therapeutic benefit in several human cancers, but in many patients, ICB - induced T cells do not infiltrate tumors, preventing clinical benefit. Intratumoral T cell accumulation requires firm adhesion mediated by the integrins very late antigen-4 (VLA-4) and lymphocyte function-associated antigen-1 (LFA-1) on activated T cells. The integrin class of cell adhesion receptors also play critical roles in multiple phases of tumor immune responses. Blocking the LFA-1 interaction with its ligand Intercellular adhesion molecule 1 (ICAM-1) abrogates CD8+ tumor infiltration after anti-CTLA4 therapy. Here, we evaluated the effect of an integrin activator, 7HP349, on promoting intratumoral T cell accumulation to potentiate CTLA-4 and PD-L1 checkpoint blockade anti-tumor activity. Experimental Procedure: To evaluate the effect of 7HP349 in promoting ICB therapeutic activity, we combined 7HP349 with anti-CTLA-4 or anti-PD-L1 antibodies in preclinical murine models for melanoma and colon carcinoma. Results: In cell adhesion assays, 7HP349 increased by >100-fold the number of VLA-4 or LFA-1 expressing T cells that bind to vascular cell adhesion molecule 1 (VCAM-1) and ICAM-1, respectively. 7HP349 facilitated chemo-attraction of T cells across matrices of VCAM-1 or ICAM-1, induced by the chemokine SDF-1α. In a PDL-1 negative B16/BL6 melanoma model, 7HP349 was dosed twice weekly at 1 mg/Kg intratumorally either alone as a single agent compared to vehicle control (13% vs 0%, p<0.01) or in combination with anti-CTLA-4 (44% vs 21%, p<0.01). After intraperitoneal administration, 7HP349 increased the complete response in combinations with anti-CTLA-4 (77% vs 27%, p<0.01). In addition, 7HP349 increased median survival in mice with CT-26 colon cancer when dosed either as a single agent versus control (21 vs 15 days, P<0.0012) or in combination with anti-PD-L1 (24 vs 21 days, P<0.02). 7HP349 may also increase the effectiveness of anti-CD137 without increasing liver toxicity. Levels of CD8 effector T cells (Teff) detected in tumor were significantly higher in mice treated with anti-CTLA-4 and 7HP349 than anti-CTLA-4 and vehicle. Some of these Teff showed specificity to p15E, an endogenous retroviral epitope expressed on B16. Mice treated with anti-CTLA-4 and 7HP349 showed increased depigmentation (vitiligo) suggesting immunity to melanocyte differentiation antigens. Conclusion: 7HP349 described here represents a first-in-concept agent that positively regulate VLA-4 and LFA-1 function. Activation of integrin cell adhesion molecules with 7HP349 is a promising approach to enhance the anti-cancer activity of checkpoint blockade therapy with antibodies against CTLA-4 and PD-(L)1. Citation Format: Yared Hailemichael, Peter Vanderslice, Robert V. Market, Ronald J. Biediger, Darren G. Woodside, Upendra K. Marathi, Willem W. Overwijk. Potentiating immune checkpoint blockade therapeutic efficacy using a small molecule activator of integrin cell adhesion receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5010.