Cell Trafficking Research Articles

Abstract 2823: Development of an in vitro blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system

Abstract Pediatric T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological malignancy, is characterized by abnormal thymocyte proliferation, frequently with mutations of the Notch-1 gene and has an overall 10-year survival rate of ~27%. A marker of poor prognosis is the presence of T-ALL within the central nervous system (CNS), which is treated with intrathecal chemotherapy and cranial irradiation; two toxic treatments with high morbidities. Gene-expression profiling of Notch-1-induced adhesion regulators showed that C-C chemokine receptor 7 (CCR7) was highly expressed and was associated with leukemic T-cell invasion of the CNS. Normal functions of CCR7 include lymph node homing and immune cell trafficking in response to CCR7 ligands, CC-chemokine ligand 19 (CCL19) or CCL21. In several studies CCL19 has been linked to T-cell invasion of the CNS across the blood-brain barrier (BBB). We hypothesize that antagonizing CCR7 binding to CCL19 will block migration of T-ALL into the CNS and thereby alleviate the need for toxic therapies. To test our hypothesis, we are using receptor internalization assays, calcium mobilization assays, transwell migration assays and an in vitro model of an intact human BBB. We have used flow cytometry-based CCR7 internalization assays ± CCR7 antagonist and found that we can completely block CCL19-induced receptor internalization in human CCR7-expressing CCRF-CEM, and HuT-78 T-ALL cells, and chemotaxis via β1 integrins across a fibronectin-coated transwell membrane. Our BBB employs HBEC-5i, human cerebral microvascular endothelial cells, in transwell chambers coated with gelatin and fibronectin for the adhesion of endothelial cells. HBEC-5i cells are a suitable cell line to make a 3D model of the BBB, as they form an endothelial monolayer with tight junctions (TJ), which is crucial to maintaining the integrity and inhibiting permeability of the BBB. We are establishing a transwell migration assay to assess the migration of CCRF-CEM T-ALL cells across the HBEC-5i cell monolayer. The integrity of the HBEC-5i cell monolayer is assessed by trans-epithelial electrical resistance (TEER), which measures the electrical resistance across the HBEC-5i monolayer, a measure of TJ integrity. Using TEER measurements, we observe that HBEC-5i form a monolayer that reaches an intact monolayer functioning with TJs over 72-96 hours. We are using this model system to investigate the migration of CCR7-expressing CCRF-CEM cells through the BBB ± CCL19. Subsequently, we will add the CCL19 antagonist (CCL197-77) to determine if inhibiting CCR7 is sufficient to eliminate migration of the CCRF-CEM cells through the BBB. If successful, this model will allow us to test the contribution of CCR7 signaling to integrins, the contribution of other immune cell types and the overall molecular mechanisms that mediate invasion of the CNS in a 3-D model of the BBB. Citation Format: Alondra Rodriguez, Cesar I. Cardona, Colin A. Bill, Charlotte M. Vines. Development of an in vitro blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2823.

Abstract 5402: Understanding the role of exercise in lymphatic vessel regulation in melanoma

Abstract Exercise has been demonstrated to be an effective method to remodel tumor blood vessels and to improve immune cell infiltration in multiple tumor models in mice. However, it is unclear whether aerobic exercise alters lymphatic vasculature in tumors. The lymphatic system plays an important role in tumor growth and metastasis because it regulates interstitial fluid pressure via lymph flow and immune cell trafficking, in conjunction with blood vessels. Lymphatic vessels are particularly important during metastasis because tumor cells frequently escape the primary tumor through lymphatic vasculature to establish the first site of metastasis in the lymph nodes. Here, we evaluated the effect of aerobic exercise on lymphatic vessels and lymphatic endothelial cell junctions in melanoma in male C57BL/6J mice. BP melanoma cells derived from transgenic mice with BrafV600E/+ and Pten−/− mutations were injected subcutaneously. Mice were assigned to no exercise and exercise groups when tumors reached ~30 mm3. The exercise group completed treadmill running at 12 meters/minute for 45 minutes for 5 consecutive days with 2 days of rest per week for 2 weeks before tumor harvest. Tumor lymphatic endothelial cells were identified by LYVE-1, endothelial tight junctions by ZO-1, and nuclei were identified by DAPI using immunofluorescence staining on frozen tumors. The number of total lymphatic vessels, elongated vessels( >100 µm), open lumens, and co-localization of ZO-1 with LYVE-1 was quantified on 6 random images per tumor using NIH Image J software. ANOVA and t-test were used to conduct statistical analysis (p < 0.05). Tumors from exercised mice had higher lymphatic vessel density (p=0.002) and more open lumens (p=0.001). Surprisingly, tumors from exercised mice had fewer ZO-1 positive vessels (p=0.015) and lower co-localization of ZO-1 and LYVE-1 (p = 0.0185) than tumors from non-exercised mice, suggesting fewer stable endothelial cell junctions in lymphatic vessels. The higher lymphatic vessel density and higher number of open lumens present in tumors from exercised mice may contribute to an exercise-induced improved anti-tumor immune response, which has been previously reported. However, the lower ZO-1 expression on lymphatic endothelial cells in tumors from exercised mice may indicate lymphatic vasculature dysfunction and/or hyper-permeability. Further work is needed to assess the functional changes in tumor lymphatic vasculature by exercise, and how these changes impact the anti-tumor immune response and metastasis. Citation Format: Priya Tirumala, Jonghae Lee, Keri L. Schadler. Understanding the role of exercise in lymphatic vessel regulation in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5402.

Abstract 4011: Locoregional GPC3 chimeric antigen receptor (CAR)-T cell therapy is associated with improved tumor control in hepatocellular carcinoma by enhancing CAR-T cell trafficking and functionality

Abstract Despite the rapid development of anti-cancer immunotherapy, the prognosis of hepatocellular carcinoma (HCC) patients is far from satisfactory. Glypican-3 (GPC3) CAR-T cell therapy has shown varied patient response. One of the potential reasons of low response rates may be due to the limited CAR-T cell infiltration. Therefore, locoregional GPC3 CAR-T therapy has been a promising option, with a hope of enhancing CAR-T cell tumor penetration. In this study, the therapeutic efficacy and underlying mechanisms of locoregional GPC3 CAR-T cell delivery were explored using HCC mouse xenografts. Orthotopic NOD-scid IL2Rgnull (NSG) HCC models were established by tumor fragment implantation into the liver. CAR-T cell injections were performed via the tail vein and portal vein to simulate systemic and locoregional administrations, respectively. Compared to the systemic treatment, locoregional therapy could better control the tumor growth, and was associated with improved liver function. In addition, the locoregional therapy group demonstrated a higher percentage of tumor-infiltrating CAR-T cells which showed an increased expression level of the cytotoxicity marker GZMB and a reduced level of the exhaustion marker LAG3. Moreover, significantly higher IFN-gamma level was found in the blood of the locoregional therapy group, indicating a better CAR-T cell functionality. In conclusion, we have demonstrated that locoregional administration of CAR-T cells is associated with increased CAR-T cell infiltration and better therapeutic efficacy. Additionally, CAR-T cells being injected locoregionally are correlated with enhanced functionality and reduced exhaustion phenotype. These findings highlight the potential of locoregional GPC3 CAR-T therapy as a promising treatment strategy for HCC patients. Citation Format: Jue Wang, Kinching Tsang, Jiale Qiu, Bo Feng. Locoregional GPC3 chimeric antigen receptor (CAR)-T cell therapy is associated with improved tumor control in hepatocellular carcinoma by enhancing CAR-T cell trafficking and functionality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4011.

Trafficking and Organs, Tissues, and Cells: An Examination of European and UK Legislation and Gaps

Abstract Substantial custodial sentences were handed down from the English Central Criminal Court for organ trafficking. Attempts to exploit vulnerable people for the purposes of organ removal, and for organ, tissue, and cells (otc) trafficking are ongoing in many jurisdictions. It is also clear from the 2019 EU evaluation of the Union legislation on blood, tissues, and cells that there is increasing commercialization in the procurement of human material for medical use, using global supply chains. Full traceability of all human material, for which there is a shortage to meet global medical needs, is required. The medical profession has drafted the Istanbul Declaration on Organ Trafficking and Transplant Tourism 2008, calling for transplant tourism to be prohibited, and full engagement with transnational law enforcement to address developing transnational organized crime in this area. This paper addresses the global, Council of Europe, EU, and UK provisions in this area.

Ectopic CXCR2 expression cells improve the anti-tumor efficiency of CAR-T cells and remodel the immune microenvironment of pancreatic ductal adenocarcinoma

BackgroundRecent progressions in CAR-T cell therapy against pancreatic ductal adenocarcinoma (PDAC) remain disappointing, which are partially attributed to the immunosuppressive microenvironment including macrophage-mediated T cell repletion.MethodsWe first characterized the expression patterns of macrophage-relevant chemokines and identified CXCR2 as the key factor regulating T cell trafficking and tumor-specific accumulation in PDAC microenvironment. After that, we synthesized and introduced a CXCR2 expression cascade into Claudin18.2 CAR-T cells and compared the behaviors of CAR-T cells in vitro and in vivo. The therapeutic potential of CXCR2 CAR-T was evaluated in two different allogeneic models: subcutaneous allografts and metastatic PDAC models.ResultsThe results showed that CXCR2 CAR-T not only reduced the size of allografted PDAC tumors, but also completely eliminated the formation of metastases. Lastly, we investigated the tumor tissues and found that expression of ectopic CXCR2 significantly improved tumor-targeted infiltration and residence of T cells and reduced the presence of MDSCs and CXCR2 + macrophages in PDAC microenvironment.ConclusionOur studies suggested that ectopic CXCR2 played a significant and promising role in improving the efficiency of CAR-T therapy against primary and metastatic PDAC and partially reversed the immune-suppressive microenvironment.

Functional aspects of the brain lymphatic drainage system in aging and neurodegenerative diseases.

The phenomenon of an aging population is advancing at a precipitous rate. Alzheimer's disease (AD) and Parkinson's disease (PD) are two of the most common age-associated neurodegenerative diseases, both of which are primarily characterized by the accumulation of toxic proteins and the progressive demise of neuronal structures. Recent discoveries about the brain lymphatic drainage system have precipitated a growing body of investigations substantiating its novel roles, including the clearance of macromolecular waste and the trafficking of immune cells. Notably, aquaporin 4-mediated glymphatic transport, crucial for maintaining neural homeostasis, becomes disrupted during the aging process and is further compromised in the pathogenesis of AD and PD. Functional meningeal lymphatic vessels, which facilitate the drainage of cerebrospinal fluid into the deep cervical lymph nodes, are integral in bridging the central nervous system with peripheral immune responses. Dysfunction in these meningeal lymphatic vessels exacerbates pathological trajectory of the age-related neurodegenerative disease. This review explores modulatory influence of the glymphatic system and meningeal lymphatic vessels on the aging brain and its associated neurodegenerative disorders. It also encapsulates the insights of potential mechanisms and prospects of the targeted non-pharmacological interventions.

Beyond the Barrier: Unraveling the Mechanisms of Immunotherapy Resistance.

Immune checkpoint blockade (ICB) induces a remarkable and durable response in a subset of cancer patients. However, most patients exhibit either primary or acquired resistance to ICB. This resistance arises from a complex interplay of diverse dynamic mechanisms within the tumor microenvironment (TME). These mechanisms include genetic, epigenetic, and metabolic alterations that prevent T cell trafficking to the tumor site, induce immune cell dysfunction, interfere with antigen presentation, drive heightened expression of coinhibitory molecules, and promote tumor survival after immune attack. The TME worsens ICB resistance through the formation of immunosuppressive networks via immune inhibition, regulatory metabolites, and abnormal resource consumption. Finally, patient lifestyle factors, including obesity and microbiome composition, influence ICB resistance. Understanding the heterogeneity of cellular, molecular, and environmental factors contributing to ICB resistance is crucial to develop targeted therapeutic interventions that enhance the clinical response. This comprehensive overview highlights key mechanisms of ICB resistance that may be clinically translatable.

Lung lymphatic endothelial cells undergo inflammatory and prothrombotic changes in a model of chronic obstructive pulmonary disease.

The lymphatic vasculature regulates lung homeostasis through drainage of fluid and trafficking of immune cells and plays a key role in the response to lung injury in several disease states. We have previously shown that lymphatic dysfunction occurs early in the pathogenesis of chronic obstructive pulmonary disease (COPD) caused by cigarette smoke (CS) and that this is associated with increased thrombin and fibrin clots in lung lymph. However, the direct effects of CS and thrombin on lymphatic endothelial cells (LECs) in COPD are not entirely clear. Studies of the blood vasculature have shown that COPD is associated with increased thrombin after CS exposure that causes endothelial dysfunction characterized by changes in the expression of coagulation factors and leukocyte adhesion proteins. Here, we determined whether similar changes occur in LECs. We used an in vitro cell culture system and treated human lung microvascular lymphatic endothelial cells with cigarette smoke extract (CSE) and/or thrombin. We found that CSE treatment led to decreased fibrinolytic activity in LECs, which was associated with increased expression of plasminogen activator inhibitor 1 (PAI-1). LECs treated with both CSE and thrombin together had a decreased expression of tissue factor pathway inhibitor (TFPI) and increased expression of adhesion molecules. RNA sequencing of lung LECs isolated from mice exposed to CS also showed upregulation of prothrombotic and inflammatory pathways at both acute and chronic exposure time points. Analysis of publicly available single-cell RNA sequencing of LECs as well as immunohistochemical staining of lung tissue from COPD patients supported these data and showed increased expression of inflammatory markers in LECs from COPD patients compared to those from controls. These studies suggest that in parallel with blood vessels, the lymphatic endothelium undergoes inflammatory changes associated with CS exposure and increased thrombin in COPD. Further research is needed to unravel the mechanisms by which these changes affect lymphatic function and drive tissue injury in COPD.

Hypersialylation and multiple myeloma

There is growing recognition of the importance of sialylation as a critical post translational modification in cancer. In this article we review the role of increased cell surface sialylation (hypersialylation) in Multiple Myeloma as it relates to cellular trafficking and immune evasion. Knowledge of the specific effects of sialic acid on cell trafficking machinery and modulation of immune cell interactions will identify opportunities for therapeutic interventions. The available evidence indicates that hypersialylation facilitates disease progression and negatively impacts on response to treatment and overall survival. Further research is required to fully elucidate the mechanisms through which hypersialylation influences disease biology and therapy resistance with the ultimate goal of developing new treatment approaches to improve the outcomes of patients with Multiple Myeloma.

Cutting Edge: The Tetraspanin CD53 Promotes CXCR4 Signaling and Bone Marrow Homing in B Cells.

B cell trafficking involves the coordinated activity of multiple adhesive and cytokine-receptor interactions, and the players in this process are not fully understood. In this study, we identified the tetraspanin CD53 as a critical regulator of both normal and malignant B cell trafficking. CXCL12 is a key chemokine in B cell homing to the bone marrow and secondary lymphoid organs, and both normal and malignant B cells from Cd53-/- mice have reduced migration toward CXCL12 invitro, as well as impaired marrow homing invivo. Using proximity ligation studies, we identified the CXCL12 receptor, CXCR4, as a novel, to our knowledge, CD53 binding partner. This interaction promotes receptor function, because Cd53-/- B cells display reduced signaling and internalization of CXCR4 in response to CXCL12. Together, our data suggest that CD53 interacts with CXCR4 on both normal and malignant B cells to promote CXCL12 signaling, receptor internalization, and marrow homing.

Are fetal microchimerism and circulating fetal extracellular vesicles important links between spontaneous preterm delivery and maternal cardiovascular disease risk?

Trafficking and persistence of fetal microchimeric cells (fMCs) and circulating extracellular vesicles (EVs) have been observed in animals and humans, but their consequences in the maternal body and their mechanistic contributions to maternal physiology and pathophysiology are not yet fully defined. Fetal cells and EVs may help remodel maternal organs after pregnancy-associated changes, but the cell types and EV cargos reaching the mother in preterm pregnancies after exposure to various risk factors can be distinct from term pregnancies. As preterm delivery-associated maternal complications are rising, revisiting this topic and formulating scientific questions for future research to reduce the risk of maternal morbidities are timely. Epidemiological studies report maternal cardiovascular risk as one of the major complications after preterm delivery. This paper suggests a potential link between fMCs and circulating EVs and adverse maternal cardiovascular outcomes post-pregnancies, the underlying mechanisms, consequences, and methods for and how this link might be assessed.

A16 ALTERATIONS TO BOTH INTESTINAL BARRIER INTEGRITY AND THE MICROBIOTA CONSPIRE DURING AGING TO PROMOTE DISEASE PROGRESSION IN A MOUSE MODEL OF MULTIPLE SCLEROSIS

Abstract Background Multiple sclerosis (MS) is a chronic, inflammatory condition impacted by both genetic and environmental factors. Aged individuals have an increased risk of progressive multiple sclerosis (pMS), as well as altered microbiota community composition and elevated levels of circulating gut microbiota-derived metabolites due to compromised gut barrier function. Fecal microbiota transplantation (FMT) studies confirm that an MS-associated microbiome can potentiate disease onset and enhance neuroinflammation in experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Aims 1) Assess whether an aged microbiota alone is sufficient to promote age-associated progressive-like disease in our mouse model of EAE 2) Determine whether altered intestinal barrier integrity results in central nervous system-specific immune changes and/or disease course during EAE Methods To assess the impact of an aged microbiota on EAE outcomes we performed age-disparate human-derived FMT from young vs aged healthy co-habiting, first degree relatives into recipient mice. Then, to further interrogate the role of the gut-brain axis in the association of age and pMS we isolated the compromised intestinal barrier seen in aged individuals as a variable via two methods – Muc2-/- mice, which exhibit a defective mucus barrier, and DSS treated mice. Results Following adoptive transfer of young EAE-primed T cells, ‘young’ FMT recipients exhibited disease resolution while an ‘aged’ FMT lead to severe, non-remitting EAE relative to both controls and ‘young’ FMT recipients. This difference in outcomes was not seen in all donor pairs, suggesting that worsened EAE is not a universal feature of the aged microbiota. We collected cecal material, blood, and central nervous system (CNS) tissue from an age-differential FMT mouse pair and performed metabolomic analysis. We found robust differences in the metabolomic profile of young vs aged FMT recipients before and after EAE induction. Further, in both models of altered barrier integrity, we observed altered myeloid cell trafficking to and immune activation in the CNS, suggesting that the intestinal barrier itself plays a critical role in immune activation during EAE. Conclusions These findings suggest specific age-related microbiota-derived metabolites, which have increased access to the host system due to a leaky intestinal barrier, may be associated with increased risk of progressive disease and may represent promising therapeutic targets for MS. Funding Agencies CIHRMS Canada

Plasma S1P Orchestrates the Reverse Transendothelial Migration of Aortic Intimal Myeloid Cells in Mice.

Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs. Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

Persistent immune abnormalities discriminate post-COVID syndrome from convalescence

BackgroundInnate lymphoid cells (ILCs) are key organizers of tissue immune responses and regulate tissue development, repair, and pathology. Persistent clinical sequelae beyond 12 weeks following acute COVID-19 disease, named post-COVID syndrome (PCS), are increasingly recognized in convalescent individuals. ILCs have been associated with the severity of COVID-19 symptoms but their role in the development of PCS remains poorly defined.Methods and resultsHere, we used multiparametric immune phenotyping, finding expanded circulating ILC precursors (ILCPs) and concurrent decreased group 2 innate lymphoid cells (ILC2s) in PCS patients compared to well-matched convalescent control groups at > 3 months after infection or healthy controls. Patients with PCS showed elevated expression of chemokines and cytokines associated with trafficking of immune cells (CCL19/MIP-3b, FLT3-ligand), endothelial inflammation and repair (CXCL1, EGF, RANTES, IL-1RA, PDGF-AA).ConclusionThese results define immunological parameters associated with PCS and might help find biomarkers and disease-relevant therapeutic strategies.

Abstract B028: Investigating immune activity caused by a novel anti-cancer cell penetrating peptide

Abstract Activating the immune system against cancerous cells represents a promising therapy against multiple cancer types, particularly breast cancer. The epidermal growth factor receptor (EGFR) represents an emerging target in immunotherapy. A novel anti-cancer cell penetrating peptide was recently discovered to affect immune recruitment and potentially function in a murine model of breast cancer. This peptide, termed cSNX1.3, is a targeted therapy to block the nuclear localization of the epidermal growth factor receptor (EGFR), a phenomenon thought to occur primarily in cancerous cells. This suggests that nuclear localized EGFR may play a role in suppressing an anti-cancer immune response. To gain a better understanding of this in vivo data, we used in vitro immune cell co-culture techniques, migration assays, and other techniques to investigate immune recruitment and function after cancer cells are treated with this novel peptide therapy. In addition, we are investigating the mechanism of cSNX1.3 mediated cell death to determine if it can induce immunogenic cell death. To determine if nuclear EGFR plays a role in immune cell suppression, we are using a CRISPR/Cas9 strategy to mutate the nuclear localization sequence. This will provide a valuable tool to better understand the seemingly cancer-specific trafficking phenomenon. Together, this work will expand our understanding of therapy induced immune cell recruitment and trafficking. Citation Format: Ryan Hecksel, Angelica Escoto, Joyce Schroeder. Investigating immune activity caused by a novel anti-cancer cell penetrating peptide [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B028.

CCR10-mediated Enhancement of T Cell Trafficking for Improved Tumor Immunotherapy.

The effectiveness of adoptive T cell therapy for solid tumors remains suboptimal, partly attributed to insufficient T cell infiltration into the tumor site. A promising strategy involves directing T cells towards the tumor utilizing tumor-specific chemokine receptors. We analyzed chemokine receptor expression in activated T cells and chemokine expression in breast and lung cancer using The Cancer Genome Atlas (TCGA) data. Subsequently, we generated 1G4 T cell receptor-engineered T (TCR-T) cells with CCR10 and performed in vitro and in vivo efficacy tests. CCR10 exhibited insufficient expression in various human T cells. Analysis of TCGA RNA sequencing data revealed elevated expression of the chemokine CCL28, the corresponding chemokine for CCR10, in breast and lung cancer. Consequently, we generated CCR10-1G4 TCR-T cells. CCR10-1G4 dual expressing TCR-T cells exhibited comparable cellular cytotoxicity but increased mobility compared to 1G4 TCR-T cells in vitro. Furthermore, injecting CCR10-1G4 dual expressing TCR-T cells into a xenograft tumor model demonstrated enhanced in vivo trafficking and a greater reduction of tumor burden. This study highlights the potential of CCR10 for developing efficient adoptive T-cell treatments targeting solid tumors.

The Role of Chemokines in Orchestrating the Immune Response to Pancreatic Ductal Adenocarcinoma

Chemokines are small molecules that function as chemotactic factors which regulate the migration, infiltration, and accumulation of immune cells. Here, we comprehensively assess the structural and functional role of chemokines, examine the effects of chemokines that are present in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME), specifically those produced by cancer cells and stromal components, and evaluate their impact on immune cell trafficking, both in promoting and suppressing anti-tumor responses. We further explore the impact of chemokines on patient outcomes in PDAC and their role in the context of immunotherapy treatments, and review clinical trials that have targeted chemokine receptors and ligands in the treatment of PDAC. Lastly, we highlight potential strategies that can be utilized to harness chemokines in order to increase cytotoxic immune cell infiltration and the anti-tumor effects of immunotherapy.

P041 Assessment of Serum Biomarkers associated with Vedolizumab Therapy Response in Inflammatory Bowel Disease

Abstract Background Vedolizumab (VDZ) is an anti α4β7-integrin inflammatory bowel disease (IBD) therapy postulated to reduce immune cell trafficking to the intestine. A significant proportion of patients fail to respond to VDZ. We aimed to assess the effect of VDZ on a panel serum inflammatory proteins (IPs) during induction therapy for IBD with the aim of identifying a serum biomarker of VDZ therapy response. We also aimed to gain further insights in VDZ mechanism of action by evaluating changes in serum IP concentrations during induction. Methods Patients commencing VDZ for IBD were prospectively recruited. All received standard VDZ induction. Baseline and week 6 serum concentrations of 54 IPs were measured by ELISA (Meso Scale Discovery, USA). Clinical outcomes were evaluated at weeks 14 and 30 of VDZ therapy. Corticosteroid-free remission (SFR) was defined as persistence of VDZ therapy, absence of corticosteroid therapy, a partial Mayo score ≤1 Ulcerative Colitis (UC) or HBI <5 Crohn’s Disease (CD). The associations between baseline serum IP concentrations and VDZ therapy outcome at weeks 14 and 30 were evaluated. Changes in IP concentrations from week 0 to week 6 of VDZ therapy were assessed. P values <0.05 following multiple test correction were considered significant in all analyses. Results Thirty-nine patients were included: 51% male, 51% UC, median [range]: age 52 [18.2-75.8] years; disease duration 13.4 [0.4–40.8] years; clinical Mayo subscore 4 [0–9]; and HBI 7 [1–20]. 28(72%) had received prior anti-TNF therapy. In univariate analysis, at higher baseline serum TNF-β concentration and lower IL-22, VEGF-C, and IL-7 concentrations were associated with week 14 SFR, p values p=0.003, 0.034, 0.042, and 0.045 respectively. Higher baseline serum IL-4, MDC and MCP-4 concentrations and lower baseline IL-10 concentration were associated with week 30 SFR, p=0.011, 0.026, 0.028 and 0.03 respectively. Significance was not maintained after correction for multiple testing. From baseline to week 6 post VDZ initiation, significant increases were observed in the concentration of six IPs: bFGF, eotaxin, eotaxin-3, MIP-1β, TARC and TNF-β, p=0.034, 0.0012, 0.0013, 0.043, 0.015 and 0.0077 respectively; and a significant decrease in one IP, IL-15, p=0.0004. The significant changes observed in IL-15, eotaxin, and eotaxin-3 concentrations from baseline to week 6 following VDZ initiation remained significant after multiple test correction. Conclusion No serum inflammatory protein was identified as a biomarker of SFR in patients receiving VDZ therapy for IBD. VDZ was observed to significantly affect serum IPs known to be involved in chemotaxis. Further study is required to identify biomarkers of VDZ therapy response.

ECCO Grant Dissecting the role of S1PR1 modulator in the mucosal regeneration of Ulcerative Colitis

Abstract Background and Aims Ozanimod, a potent sphingosine 1-phosphate (S1P) 1/5 receptor agonist, first approved for the treatment of adults with moderately to severely active UC in the USA and in the EU, has showed promising results in terms of both tolerability and efficacy reducing inflammatory processes and inducing disease remission. The selectivity of Ozanimod for S1PR1 is 27 times that for S1PR5. Therefore, is plausible that the beneficial effects of Ozanimod are mediated by S1PR1, which is constitutively expressed not only by immune cells, but also by epithelial, endothelial, and stromal cells. However, its function on non-immune cells has been neglected. Based on preliminary data demonstrating a faster recovery of mucosal damage in mice with a conditional deletion of S1PR1 in the epithelia and endothelia compartments, our hypothesis is that the inactivation of S1PR1 not only inhibits immune cell trafficking, but also promotes tissue repair. Based on this background, the main objectives will be to exploit the Ozanimod effects during the process of mucosa healing, and to investigate the molecular pathways activated by Ozanimod on epithelial and endothelial cells. Methods Pre-clinical validation of Ozanimod, in vivo model of UC in association with conditional knockout mice for S1PR1 on epithelial and endothelial cells, stimulation of primary endothelial cells from UC patients and human induced pluripotent stem cell-derived intestinal organoids (iHOs) will be used to address our aims. Transcriptomics, functional assay, immune characterization, WB validation will be performed. Anticipated Impact In short and medium term, we will improve the knowledge on the role of S1PR1 in the processes of mucosal healing and identify the molecular pathways involved in the response to the mucosal damage mediated by the ozanimod in non-immune cells. The results of this study may improve the treatment options for UC patients.

The Role of CCL24 in Primary Sclerosing Cholangitis: Bridging Patient Serum Proteomics to Preclinical Data.

Primary sclerosing cholangitis (PSC) is an inflammatory and fibrotic biliary disease lacking approved treatment. We studied CCL24, a chemokine shown to be overexpressed in damaged bile ducts, and its involvement in key disease-related mechanisms. Serum proteomics of PSC patients and healthy controls (HC) were analyzed using the Olink® proximity extension assay and compared based on disease presence, fibrosis severity, and CCL24 levels. Disease-related canonical pathways, upstream regulators, and toxicity functions were elevated in PSC patients compared to HC and further elevated in patients with high CCL24 levels. In vitro, a protein signature in CCL24-treated hepatic stellate cells (HSCs) differentiated patients by disease severity. In mice, CCL24 intraperitoneal injection selectively recruited neutrophils and monocytes. Treatment with CM-101, a CCL24-neutralizing antibody, in an α-naphthylisothiocyanate (ANIT)-induced cholestasis mouse model effectively inhibited accumulation of peribiliary neutrophils and macrophages while reducing biliary hyperplasia and fibrosis. Furthermore, in PSC patients, CCL24 levels were correlated with upregulation of monocyte and neutrophil chemotaxis pathways. Collectively, these findings highlight the distinct role of CCL24 in PSC, influencing disease-related mechanisms, affecting immune cells trafficking and HSC activation. Its blockade with CM-101 reduces inflammation and fibrosis and positions CCL24 as a promising therapeutic target in PSC.