Changes In Surface Markers Research Articles

Abstract A011: Identifying surface biomarkers from colorectal cancer-derived extracellular vesicles using a bead-based approach

Abstract Colorectal cancer (CRC) accounts for the second most common cause of cancer-related mortality globally. The search for prognostic biomarkers subserving to detected cancer at an early stage, to improve tumor classification and to stratify therapeutic patient management groups is actively ongoing. Surgical resections and chemotherapeutics remain a mainstay in the cure and control of solid tumors. Despite their usefulness in the clinical scenario, shortcomings are well recognized, leading researchers to investigate for a circulating source of tumor-derived materials in biofluids, including mostly circulating tumor DNA (ctDNA), circulating tumor cells (CTCs) and extracellular vesicles (EVs). Characterization of EVs surface markers and assessment of their molecular cargo, can lead to the identification of early metastatic colorectal cancer signatures. Cell surface markers of three CRC cell lines (HCT116, DLD1 and LOVO) were characterized using flow cytometry. EVs were isolated from 48-hour serum-deprived cell lines using the polymer pull down method (20% Polyethelene glycol (PEG)). The concentration and particle size distribution of isolated EVs was checked by the NanoSight NS300. Avidin-coated Polystyrene beads were conjugated with CD9 biotin antibodies; and were used to immunoprecipitated (IP) CD9+ EVs. Following a 2-hour incubation and a series of washing steps, the PE-labelled antibodies of interest (CD9, CD63, EpCAM, Vim, CD44, CD133 and CD29) were added and read using xMAP technology. To increase the number of surface markers that can be read in tandem, IP was multiplexed using EpCAM, CD9 and CD63. This was followed by the isolation of EVs from plasma of metastatic CRC patients (N=3) using size exclusion chromatography (Izon qEV1 columns-35nm in conjunction with the automatic fraction collector) and polymer pull down. The concentration and size were assessed by Nanosight NS300 and surface markers of tumor-derived EVs were assessed using the immunoprecipitation technique on the Luminex system. Comparison of signals between EVs isolated from cell lines and matched originator cells showed that cell surface signatures in EVs match closely with their originator cells. The mean fluorescence intensities quantified cell surface marker expression in the different exosome-bead complexes. Interestingly, one of the metastatic cancer patients with invasive colorectal adenocarcinoma and lung metastasis showed EpCAM and Vimentin positivity suggesting epithelial-mesenchymal plasticity. These results show the importance of multiplexing both at the level of immunoprecipitation and at end-measurement, given the dynamics of cell surface marker changes during disease progression. The use of the mesenchymal marker; Vimentin in a panel for cell surface markers stems from studies on biotinylated cell surface markers on various cancer cell lines and identification of Vimentin in such models. Interestingly we find Vimentin on EVs surface in this study. Citation Format: Jessica Debattista, Laura Grech, Christian Scerri, Malcolm Buhagiar, Godfrey Grech. Identifying surface biomarkers from colorectal cancer-derived extracellular vesicles using a bead-based approach [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A011.

Determining the Optimal Short-Term Storage Duration for T Cells Extracted from Peripheral Blood Prior Flow Cytometry Analysis.

Researching medical sample storage is crucial for maintaining the integrity of biological specimens and ensuring the accuracy of research investigations and diagnostic tests. Improper storage conditions can lead to sample degradation, compromising the reliability of results. Standardized storage procedures are essential for quality control, particularly in multicenter trials where samples are collected and processed at various locations. Moreover, ethical considerations dictate careful handling of patient samples to uphold privacy and rights. This study focuses on the surface phenotype of T cells, which is vital for diagnosing immunodeficiency disorders and autoimmune diseases and for monitoring disease activity and treatment efficacy. The effect of storage duration on T cell surface proteins is multifactorial, influenced by factors like protein degradation, cellular metabolism, and cytokine release. Long-term storage can lead to the gradual loss of T cell function, necessitating techniques to preserve cell activity. Changes in surface markers can affect disease diagnosis, emphasizing the importance of accurate sample processing. Findings from this study reveal time-dependent changes in T cell surface markers during storage. CD3 levels declined significantly after the fourth day, with FITC labeling proving superior to APC. CD4 levels remained consistent until the fourth day, contrasting with previous findings on foreskin tissue. HLA-DR levels declined rapidly, indicating unsuitability for storage, consistent with other studies on cryopreserved cells. CD16 and CD8 levels decreased gradually, while CD56 declined rapidly after the third day, consistent with recent research. There were detectable and significant differences after the samples were stored for an improper period, which may have affected the integrity of the results, suggesting that understanding the factors influencing T cell surface protein changes during storage is crucial for maintaining result integrity.

Impact of urolithin A supplementation, a mitophagy activator on mitochondrial health of immune cells (MitoIMMUNE): A randomized, double-blind, placebo-controlled trial in healthy adults.

e14562 Background: Mitochondrial dysfunction and stem cell exhaustion are hallmarks of aging, driving inflammation and limiting immune function. Likewise, cancer is characterized by repression of mitochondrial dynamics in tumor-infiltrating leukocytes that fosters terminal T cell exhaustion. Yet, interventions to reliably improve immune function are still lacking. Urolithin A (UA) is a postbiotic known to improve muscle function in aged adults upon oral intake. We have recently shown that UA induces mitophagy in human CD8+ cells to induce formation of T memory stem cells, while sensitizing murine cancer to immunotherapy. Methods: In this double-blind, prospective, placebo-controlled trial (NCT05735886) we sought to characterize for the first time the effects of UA on the human immune system. Fifty healthy participants aged 45-70 years were randomized (1:1) to an intervention of single-dose (1000mg) UA or placebo per day for a study duration of 28 days. Randomization was performed based on age, gender and BMI. Exclusion criteria were prior medical conditions, BMI >35 kg/m2, smoking, and intake of prescription medication or other dietary supplements. The primary end points of the study were changes in peripheral CD3+ cells, as well as alterations in mitochondrial activity in select immune populations of the peripheral blood as assessed by flow cytometry. Results: Intake of UA was safe and well-tolerated in the intervention cohort of 25 participants. After the study period, total peripheral lymphocytes and circulating NK cells were expanded in the UA group. Performing a complete immunophenotyping via spectral flow cytometry, we found that UA elicits immune remodeling characterized by broad changes of immune surface markers and mitochondrial measurements. CD8+ cells of participants in the intervention arm displayed greater mitochondrial mass, while preferably taking on a naive phenotype and expressing more Ki67. In addition, circulating monocytes exhibited a less inflammatory signature. UA intake reduced plasma levels of several proinflammatory cytokines. Conclusions: Collectively, our study constitutes an unprecedented intervention-based assessment of immune aging that globally characterizes the effect of UA on the immune system, proposing validation in future confirmatory studies and potential combination with cancer immunotherapy. Clinical trial information: NCT05735886 .

Anti-aquaporin-4 immune complex stimulates complement-dependent Th17 cytokine release in neuromyelitis optica spectrum disorders

Proinflammatory cytokines, such as (IL: interleukin) IL-6 and IL-17A, and complement fixation are critical in the immunopathogenesis of neuromyelitis optica spectrum disorders (NMOSD). Blocking the IL-6 receptor or the C5 complement pathway reduces relapse risk. However, the role of interleukin (IL)-6 and complement in aquaporin-4 (AQP4) autoimmunity remains unclear. To investigate the role of the anti-AQP4 immunoglobulin (AQP4-IgG)/AQP4 immunocomplex on the induction and profile of ex vivo cytokine and surface marker expression in peripheral blood mononuclear cells (PBMC) culture. Isolated PBMCs obtained from 18 patients with AQP4-IgG-seropositive-NMOSD (8 treatment-naive, 10 rituximab-treated) or ten healthy controls were cultured with AQP4-immunocomplex with or without complement. Changes in PBMC surface markers and cytokine expression were profiled using flow cytometry and ELISA. PBMCs derived from treatment-naive NMOSD patients stimulated with a complex mixture of serum complement proteins produced significant elevations of IL-17A and IL-6. Rituximab-treated patients also exhibited higher IL-6 but not IL-17A release. IL-6 and IL-17A elevations are not observed without complement. Co-stimulation of PBMCs with AQP4-IgG/AQP4 immunocomplex and complement prompts a Th17-biased response consistent with the inflammatory paradigm observed in NMOSD. A possible inflammation model is proposed via antigen-specific autoreactive peripheral blood cells, including NK/NKT cells.

Quantitative Ocular Surface Changes in Patients Undergoing Immune Checkpoint Inhibitor Therapy

ABSTRACT Purpose To describe the clinical course and evaluate treatment of ocular surface changes in patients receiving immune checkpoint inhibitor (ICI) therapy. Methods Multiple markers of ocular surface dryness were evaluated in 16 patients on ICI therapy. The Wilcoxon rank-sum test was used to determine the significant change in the initial and final ocular surface indices. Results Fifty percent of the eyes demonstrated worsening Schirmer I scores; 29% showed an increase in lissamine green staining. During follow-up, 43% of patients experienced a decline in OSDI scores. Treatments included preservative-free artificial tears (88%), cyclosporine (25%), topical corticosteroids (31%), warm compresses (25%); punctal plugs (13%). Median follow-up time was 3.4 months (range:0–79 ); median ICI treatment duration was 7 months (range:1–40). Four patients died during the observation period. Conclusion A significant proportion of patients experience changes in ocular surface markers while treated with ICIs. Medical intervention can lead to stabilization of ocular surface disease.

Abstract 5851: Cancer associated fibroblast crosstalk through VEGF increases tumor cell proliferation in human pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a classically heterogeneous tumor for which tumor cells comprise less than 40% of the total tumor mass. To better understand mechanisms of intercellular interactions driving tumor response or resistance to chemotherapy, it is crucial to account for the complex tumor microenvironment (TME). Cancer associated fibroblasts (CAFs) have been implicated as key drivers supporting mechanisms that both promote and restrain tumor growth. Using our model system that investigates interactions between CAFs and cancer cells, we show a novel intercellular interaction through VEGF signaling that drives proliferation. Methods: We interrogate intracellular crosstalk in the PDAC TME using a novel 3-dimensional (3D), patient-matched coculture system of patient-derived organoids (PDO) and CAFs, obtained from patients undergoing surgical resection. Molecular characterization is performed by profiling with a 1200 analyte multiplex ELISA screen, multiparameter flow cytometry, bulk RNA sequencing of CAF and PDO cocultures, imaging mass cytometry (IMC) on patient samples, and qPCR. Results: Secretome profiling of 7 patient-derived CAF lines demonstrated patient-specific heterogeneity in proteins such as: FGF-12, FGF3, and CD79a. Common CAF-derived signaling proteins with the potential to regulate intercellular crosstalk that were seen across all samples include CRP, IL-6R, R-Spondin and others. To better understand how factors change with intercellular interactions, we set up direct CAF-PDO cocultures over 4 days to identify global transcriptional changes using bulk RNAseq. Phenotypically, coculture drives gene expression changes in both PDO subtype (basal vs classical) and CAF subtype (prevalence of iCAFs vs myCAFs). We used these transcriptome data to identify pathways modulated by CAF-PDO crosstalk. Profiling coculture supernatant, we identified upregulation in VEGF secretion. In tandem, when we investigate cellular surface marker changes, we see upregulation of VEGFR2 on the surface of CAFs by flow cytometry. This interaction is accompanied by an increase in PDO proliferation seen in the coculture conditions. Ongoing experiments aim to investigate the impact of this relationship spatially using matched patient tissue. Conclusions: Interactions between cancer cells and CAFs compound the complexity of the biology of the TME and contribute to poor patient outcomes; therefore, we have put forth a model that better represents these interactions through patient matched PDO - CAF cocultures. Using this model, we demonstrate a novel interaction through VEGF that enhances tumor cell proliferation. We introduce a targeted approach to investigating the complex biology of the TME to inform the mechanisms driving cancer biology in individual patients that can also be used to develop novel therapeutic targets. Citation Format: Samantha Guinn, Joseph Tandurella, Jignasha Patel, Richard Burkhart, Jacquelyn Zimmerman, Elizabeth Jaffee. Cancer associated fibroblast crosstalk through VEGF increases tumor cell proliferation in human pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5851.

Evaluation of Changes in Leukocyte Surface Markers in the Early Diagnosis of Late-Onset Neonatal Sepsis

Abstract Objective This study aimed to evaluate the cluster of differentiation (CD)64, CD16, CD11b, CD63 human leukocyte antigen-DR (HLA-DR), and CD62L leukocyte surface marker abnormalities using flow cytometry in the early diagnosis of late-onset neonatal sepsis. Methods Forty-four neonates were included in this study. Of them, 22 neonates with clinical late-onset neonatal sepsis were included in the study group, and the remaining 22 neonates without sepsis were considered the control group. Complete sepsis screening was performed. Additionally, monocyte and neutrophil surfaces marker were examined using flow cytometry. Results The expression of the leukocyte surface markers CD16 and CD64 on monocytes and neutrophils was significantly higher in the study group than in the control group (p < 0.05), while the CD63, CD62L, CD11b, and HLA-DR levels were similar to those in the control group (p > 0.05). Furthermore, receiver operating characteristic curve analysis indicated that neutrophil CD64 (nCD64) is a diagnostic marker for neonatal sepsis, with an area under the curve of 0.901. The CD64 and CD16, which are the respective leukocyte surface markers on neutrophils and monocytes, are useful tests in the early diagnosis of late-onset neonatal sepsis. Conclusion In addition to acute phase proteins, cell surface antigens such as CD16 and more specifically CD64 should be used in routine investigations for the early diagnosis of late-onset neonatal sepsis. Such use in combination with acute phase reactants can improve diagnostic accuracy.

Characterization of Intestinal Mesenchymal Stromal Cells From Patients With Inflammatory Bowel Disease for Autologous Cell Therapy.

Therapy with mesenchymal stromal cells (MSCs) has shown promise in inflammatory bowel disease-leveraging their immunosuppressive and regenerative properties. However, the potential immunogenic complications of allogenic MSCs sourced from different tissues raise concern. Thus, we assessed the fitness and functionality of autologous intestinal MSCs as a potential platform for cellular therapy. Mucosal biopsy-derived MSCs from Crohn's disease (n = 11), ulcerative colitis (n = 12), and controls (n = 14) were analyzed by microscopy and flow cytometry for doubling-time, morphology, differentiation potential, and immunophenotype. Gene expression, cell-subtype composition, along with surface marker and secretome changes after IFN-γ priming were measured by bulk and single-cell RNA sequencing coupled with a 30-plex Luminex panel. MSCs expanded ex vivo demonstrate canonical MSC markers, similar growth kinetics, and tripotency regardless of the patient phenotype. Global transcription patterns were similar at baseline though inflammatory bowel disease (IBD) rectal MSCs showed changes in select immunomodulatory genes. IFN-γ priming resulted in upregulation of shared immunoregulatory genes (particularly in PD-1 signaling) and overrode the transcriptional differences observed at baseline. Furthermore, MSCs secrete key immunomodulatory molecules at baseline and in response to IFN-γ including CXCL10, CXCL9, and MCP-1. Overall, MSCs from IBD patients have normal transcriptional and immunomodulatory properties with therapeutic potential and can be sufficiently expanded.

Multiomics Analysis Confirms Effective Target Engagement for RVU120 - a First-in-Class CDK8/19 Kinase Inhibitor in AML and MR-MDS Patients and Reveals the Mechanism of Action

RVU120 is a first-in-class CDK8/19 kinase inhibitor. The primary objective of an ongoing Phase Ib study of RVU120 in AML and HR-MDS patients is to determine the preliminary safety profile and the recommended phase 2 dose of RVU120 as a single agent. Characterization of pharmacodynamic (PD) effects is a secondary objective of the trial. PD analysis supports the determination of the recommended phase 2 dose, further clinical development and ultimately positioning of this novel class of targeted agents in the treatment strategies for AML and MDS patients. A battery of tests has been used to establish the relationship between pharmacokinetics (PK) and PD activity, confirm the on-target activity of RVU120, and reveal a mechanism of action of CDK8/19 inhibitors in preclinical models. Specific protein binding of RVU120 in leukemic and normal blood cells has been verified by capturing CDK8/19 kinases after a competition with ATP acyl-phosphate biotin-labeled probes. Changes in CDK8- dependent phosphorylation of STAT5 were used as proximal biomarkers and were measured by flow cytometry. Transcriptome changes were used as downstream biomarkers and were determined by the integrated analysis of bulk RNAseq, ChIPseq and ATACseq. The molecular activity of RVU120 was correlated with anti-leukemic activity and surface marker changes. In patients, PD activity of RVU120 was established by measuring changes in pSTAT5 levels, directly in blast cells and ex vivo in established AML cell lines, exposed to plasma collected from patients treated with RVU120. Downstream transcriptomic effects in patients were assessed by RNAseq and phenotypic changes were tracked by flow cytometry surface marker panels. Preclinical profiling indicated effective CDK8/19 target engagement and inhibition of pSTAT5 levels at low nanomolar concentrations of RVU120, in both established cell lines and primary leukemic cells. The antileukemic activity was tightly correlated with levels of biomarker inhibition, however, in a group of super-responders enriched for NPM1 mutants and leukemic stem cells, efficacy was achieved at partial biomarker inhibition levels. Downstream profiling revealed inhibition of homeobox genes and induction of STAT5-dependent genes as two major transcriptomic programs regulated by RVU120. Homeobox genes inhibited by RVU120 are essential for the viability and self-renewal of leukemic cells, whereas many STAT5 -dependent genes are known as master regulators of erythropoiesis. Consistently, treatment of healthy and transformed CD34+ cells with RVU120 results in the commitment to erythropoiesis. Profiling of patient samples largely confirmed preclinical findings. Inhibition of pSTAT5 in CD34+ blasts from AML and MDS patients tightly correlated with achieved exposures after the first dose and at a steady state. At the time of submission pSTAT5 inhibition has already reached >50%, a threshold that based on preclinical predictions is sufficient for robust efficacy in selected groups of super-responder patients. Further increase at higher doses, is expected to result in deeper, more durable responses in broader populations of patients. Broad transcriptomic changes were observed in patients after treatment with RVU120. Notably, potent inhibition of homeobox genes was observed in NPM1 mutant blasts and induction of erythroid master regulator genes was observed in SF3B1 mutant cells with erythroid arrest. Clear hallmarks of erythroid differentiation were further identified in several patients by specific changes in surface markers. These patients achieved meaningful clinical responses such as reduction of blasts and transfusion independence, respectively.

Effects of LSD1 Inhibition with Img-7289 on the Leukemia Initiating Cell Population with Induction of Differentiation and Cell Death in Pediatric Relapsed/Refractory AML

Background: Outcomes for pediatric patients with relapsed or refractory acute myelogenous leukemia (AML) are poor, with <30% overall survival in recurrent settings. Additionally, patients harboring high-risk molecular features, including KMT2A/MLL-rearrangements or GLIS2 fusion genes, experience poor outcomes. LSD1 inhibition has been evaluated as a potential therapeutic approach in adult patients with AML, but its role in pediatric AML remains limited. In this study, we characterize the anti-leukemic effect of an LSD1 inhibitor, IMG-7289, across a panel of pediatric AML models in vitro and in vivo and evaluate its effect on leukemia initiating cells (LICs). Methods: Dose-response effect of IMG-7289 on cell viability was assessed across 11 pediatric leukemia cell lines. Induction of apoptosis and cell cycle arrest was assessed by flow cytometric analysis of Annexin V and 7-AAD respectively. Flow cytometry was also used to analyze a panel of cell surface markers to assess for differentiation effect. Pediatric AML patient-derived xenograft (PDX) mouse models, including an acute megakaryoblastic leukemia (AMKL) model and a KMT2A-rearranged (KMT2A-r) AML model, established by implanting AML cells into NSG-S mice, were used for in vivo validation studies and limiting dilution secondary transplantation studies. PDX models were treated with IMG-7289 (25 mg/kg PO) or Vehicle daily for 21 days (n=6/arm). Engraftment was determined using flow cytometry to assess expression of human CD45 (hCD45) in the bone marrow. Leukemia initiating cell (LIC) frequency was evaluated by transplanting serial dilutions (2x105, 105, 104, 103, and 102 cells) of bone marrow cells derived from Vehicle- or IMG-7289-treated PDX models into untreated donor mice. The LIC frequency was estimated using the Extreme Limiting Dilution Analysis (ELDA) software (Walter and Eliza Hall Bioinformatics Institute of Medical Research), and log-rank analysis was used to compare event-free survival (EFS), defined as time from secondary transplant to engraftment. Results: Seven pediatric AML cell lines demonstrated sensitivity to IMG-7289, including 2 AMKL cell lines, M07e (IC50: 0.03 μM ± 0.01) and CMK (IC50: 0.065 μM ± 0.003), and 2 KMT2A-r AML cell lines, MV4;11 (IC50: 0.007 μM ± 0.001) and MOLM14. Treatment with IMG-7289 leads to induction of apoptosis (DMSO 8.8% vs IMG-7289 30.3%, P=0.0005) and cell-cycle arrest (G0/G1 phase populations: DMSO 65.5% vs IMG-7289 74.7%, P<0.0001) in MV4;11 cells. To investigate the effect of IMG-7289 on induction of cell differentiation, cell surface marker changes were evaluated over time in IMG-7289-treated AML. MV4;11 and M07e cells treated over 25 days showed increased expression of the monocytic cell surface markers CD14 and CD86 compared to DMSO control. Additionally, decreased expression of megakaryocytic markers CD41 and CD42b were observed in IMG-7289-treated M07e cells. To evaluate the in vivo activity of IMG-7289, a KMT2A-r AML PDX model was treated with IMG-7289 for 21 days. We observed a significant reduction in hCD45+ cells in IMG-7289-treated mice (27.5% hCD45+) vs Vehicle-treated mice (76% hCD45+, P=0.0012). To evaluate effects of IMG-7289 on the LIC population, serial transplantation of cells derived from Vehicle-treated or IMG-7289-treated AMKL PDX model was performed. Assessment of leukemia engraftment (flow cytometric analysis for hCD45+ cells in mouse bone marrow) at 20 weeks post-secondary transplant demonstrated an LIC frequency of 1 in 4.9x104 in IMG-7289-treated mice representing a ~500-fold reduction in LIC number compared to Vehicle control. Furthermore, a significant improvement in EFS was observed in IMG-7289-treated mice (median survival = 33 days) compared to Vehicle control (median survival = 76 days, P=0.0009, log-rank). Conclusions: In conclusion, treatment with IMG-7289 results in decreased viability, as demonstrated by induction of apoptosis and cell cycle arrest, as well as differentiation induction in pediatric AML cell lines. The in vitro and in vivo activity observed in pediatric AML is also observed in models with high-risk phenotypes (KMT2A-r AML and AMKL). Activity of LSD1 inhibition on the LIC population may represent a promising strategy to mitigate relapse or refractory disease.

A multifaceted high-throughput assay for probing antigen-specific antibody-mediated primary monocyte phagocytosis and downstream functions

Monocytes are highly versatile innate immune cells responsible for pathogen clearance, innate immune coordination, and induction of adaptive immunity. Monocytes can directly and indirectly integrate pathogen-destructive instructions and contribute to disease control via pathogen uptake, presentation, or the release of cytokines. Indirect pathogen-specific instructions are conferred via Fc-receptor signaling and triggered by antibody opsonized material. Given the tremendous variation in polyclonal humoral immunity, defining the specific antibody-responses able to arm monocytes most effectively remains incompletely understood. While monocyte cell line-based assays have been used previously, cell lines may not faithfully recapitulate the full biology of monocytes. Thus, here we describe a multifaceted antigen-specific method for probing antibody-dependent primary monocyte phagocytosis (ADMP) and secondary responses. The assay not only reliably captures phagocytic uptake of immune complexes, but also detects unique changes in surface markers and cytokine secretions profiles, poorly detected by monocytic cell lines. The assay captures divergent polyclonal-monocyte recruiting activity across subjects with varying SARS-CoV-2 disease severity and also revealed biological nuances in Fc-mutant monoclonal antibody activity related to differences in Fc-receptor binding. Thus, the ADMP assay is a flexible assay able to provide key insights into the role of humoral immunity in driving monocyte phenotypic transitions and downstream functions across many diseases.

Enhancing arginase 2 expression using target site blockers as a strategy to modulate macrophage phenotype

Macrophages are plastic cells playing a crucial role in innate immunity. While fundamental in responding to infections, when persistently maintained in a pro-inflammatory state they can initiate and sustain inflammatory diseases. Therefore, a strategy that reprograms pro-inflammatory macrophages toward an anti-inflammatory phenotype could hold therapeutic potential in that context. We have recently shown that arginase 2 (Arg2), a mitochondrial enzyme involved in arginine metabolism, promotes the resolution of inflammation in macrophages and it is targeted by miR-155. Here, we designed and tested a target site blocker (TSB) that specifically interferes and blocks the interaction between miR-155 and Arg2 mRNA, leading to Arg2 increased expression and activity. In bone marrow-derived macrophages transfected with Arg2 TSB (in the presence or absence of the pro-inflammatory stimulus LPS), we observed an overall shift of the polarization status of macrophages toward an anti-inflammatory phenotype, as shown by significant changes in surface markers (CD80 and CD71), metabolic parameters (mitochondrial oxidative phosphorylation) and cytokines secretion (IL-1β, IL-6, and TNF). Moreover, in an in vivo model of LPS-induced acute inflammation, intraperitoneal administration of Arg2 TSB led to an overall decrease in systemic levels of pro-inflammatory cytokines. Overall, this proof-of-concept strategy represent a promising approach to modulating macrophage phenotype.

The cytokines interleukin-6 and interferon-\u03b1 induce distinct microglia phenotypes

BackgroundElevated production of the cytokines interleukin (IL)-6 or interferon (IFN)-α in the central nervous system (CNS) is implicated in the pathogenesis of neurological diseases such as neuromyelitis optica spectrum disorders or cerebral interferonopathies, respectively. Transgenic mice with CNS-targeted chronic production of IL-6 (GFAP-IL6) or IFN-α (GFAP-IFN) recapitulate important clinical and pathological features of these human diseases. The activation of microglia is a prominent manifestation found both in the human diseases and in the transgenic mice, yet little is known about how this contributes to disease pathology.MethodsHere, we used a combination of ex vivo and in situ techniques to characterize the molecular, cellular and transcriptomic phenotypes of microglia in GFAP-IL6 versus GFAP-IFN mice. In addition, a transcriptomic meta-analysis was performed to compare the microglia response from GFAP-IL6 and GFAP-IFN mice to the response of microglia in a range of neurodegenerative and neuroinflammatory disorders.ResultsWe demonstrated that microglia show stimulus-specific responses to IL-6 versus IFN-α in the brain resulting in unique and extensive molecular and cellular adaptations. In GFAP-IL6 mice, microglia proliferated, had shortened, less branched processes and elicited transcriptomic and molecular changes associated with phagocytosis and lipid processing. In comparison, microglia in the brain of GFAP-IFN mice exhibited increased proliferation and apoptosis, had larger, hyper-ramified processes and showed transcriptomic and surface marker changes associated with antigen presentation and antiviral response. Further, a transcriptomic meta-analysis revealed that IL-6 and IFN-α both contribute to the formation of a core microglia response in animal models of neurodegenerative and neuroinflammatory disorders, such as Alzheimer’s disease, tauopathy, multiple sclerosis and lipopolysaccharide-induced endotoxemia.ConclusionsOur findings demonstrate that microglia responses to IL-6 and IFN-α are highly stimulus-specific, wide-ranging and give rise to divergent phenotypes that modulate microglia responses in neuroinflammatory and neurodegenerative diseases.

Lack of Atorvastatin Effect on Monocyte Gene Expression and Inflammatory Markers in HIV-1-infected ART-suppressed Individuals at Risk of non-AIDS Comorbidities.

Background:Many people living with HIV have persistent monocyte activation despite viral suppression by antiretroviral therapy (ART), which contributes to non-AIDS complications including neurocognitive and other disorders. Statins have immunomodulatory properties that might be beneficial by reducing monocyte activation.Methods:We previously characterized monocyte gene expression and inflammatory markers in 11 HIV-positive individuals on long-term ART (HIV/ART) at risk for non-AIDS complications because of low nadir CD4+ counts (median 129 cells/uL) and elevated hsCRP. Here, these individuals participated in a double-blind, randomized, placebo-controlled crossover study of 12 weeks of atorvastatin treatment. Monocyte surface markers were assessed by flow cytometry, plasma mediators by ELISA and Luminex, and monocyte gene expression by microarray analysis.Results:Among primary outcome measures, 12 weeks of atorvastatin treatment led to an unexpected increase in CCR2+ monocytes (P=0.04), but did not affect CD16+ or CD163+ monocytes, nor levels in plasma of CCL2/MCP-1 or sCD14. Among secondary outcomes, atorvastatin treatment was associated with decreased plasma hsCRP (P=0.035) and IL-2R (P=0.012). Treatment was also associated with increased total CD14+ monocytes (P=0.015), and increased plasma CXCL9 (P=0.003) and IL-12 (P<0.001). Comparable results were seen in a subgroup that had inflammatory marker elevations at baseline. Atorvastatin treatment did not significantly alter monocyte gene expression or normalize aberrant baseline transcriptional patterns.Conclusions:In this study of aviremic HIV+ individuals at high risk of non-AIDS events, 12 weeks of atorvastatin did not normalize monocyte gene expression patterns nor lead to significant changes in monocyte surface markers or plasma mediators linked to non-AIDS comorbidities.

Comparison of early changes in ocular surface markers and tear inflammatory mediators after femtosecond lenticule extraction and FS-LASIK.

To compare the short-term impacts of femtosecond lenticule extraction (FLEx) and femtosecond laser-assisted laser in situ keratomileusis (FS-LASIK) on ocular surface measures and tear inflammatory mediators. This prospective comparative nonrandomized clinical study comprised 75 eyes (75 patients). Totally 20 male and 15 female patients (age 21.62±3.25y) with 35 eyes underwent FLEx, and 26 male and 14 female patients (age 20.18±3.59y) with 40 eyes underwent FS-LASIK. Central corneal sensitivity, noninvasive tear breakup time, corneal fluorescein staining, Schirmer I test, tear meniscus height, and ocular surface disease index were evaluated in all patients. Tear concentrations of nerve growth factor (NGF), interleukin-1α (IL-1α), transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and matrix metalloproteinase-9 (MMP-9) were assessed by multiplex antibody microarray. All measurements were performed preoperatively, and 1d, 1wk, and 1mo postoperatively. Patients who underwent FLEx exhibited a more moderate reduction in central corneal sensation and less corneal fluorescein staining than those in the FS-LASIK group 1wk after the procedure (P<0.01). NGF was significantly higher 1d and 1wk after surgery in the FS-LASIK group than in the FLEx group (P<0.01). By contrast, compared to those in the FLEx group, higher postoperative values and slower recovery of tear TGF-β1, IL-1α, and TNF-α concentrations were observed in the FS-LASIK group (P<0.01). Tear concentrations of NGF, TGF-β1, TNF-α, and IL-1α were correlated with ocular surface changes after FLEx or FS-LASIK surgery. There is less early ocular surface disruption and a reduced inflammatory response after FLEx than after FS-LASIK. NGF, TGF-β1, TNF-α, and IL-1α may contribute to the process of ocular surface recovery.

Anti-neutrophil cytoplasmic antibodies (ANCA): Antigen interactions and downstream effects.

Neutrophils are the most abundant leukocytes in circulation and are key "first responders" in the immune response to infectious and non-infectious stimuli. Unlike other immune cells, neutrophils can mount a robust response (including a change in surface markers and the production of extracellular traps and reactive oxygen species) just minutes after sensing a disturbance. It has been speculated that, in some individuals, the activation of neutrophils inadvertently leads to the generation of anti-neutrophil cytoplasmic autoantibodies (ANCA) against particular neutrophil proteins (antigens) such as myeloperoxidase (MPO) and proteinase 3 (PR3). In these individuals, continuous ANCA-antigen interactions are thought to drive persistent activation of neutrophils, chronic immune activation, and disease, most notably, small vessel vasculitis. There are significant gaps however in our understanding of the underlying mechanisms and even the pathogenicity of ANCA given that vasculitis can develop in the absence of ANCA, and that ANCA have been found in circulation in other conditions with no apparent contribution to disease. These gaps are particularly evident in the context of human studies. Herein, we review knowledge on neutrophil-derived ANCA antigens PR3 and MPO, ANCA generation, and ANCA-antigen interaction(s) that may promote immune activation and disease.

LTA1 and dmLT enterotoxin-based proteins activate antigen-presenting cells independent of PKA and despite distinct cell entry mechanisms.

Enterotoxin-based proteins are powerful manipulators of mucosal immunity. The A1 domain of heat-labile enterotoxin from E. coli, or LTA1, is a newer adjuvant from this family under investigation for intranasal vaccines. Although LTA1 has been examined in mouse vaccination studies, its ability to directly stimulate immune cells compared to related adjuvant proteins has not been well explored. Here, we perform the first rigorous examination of LTA1’s effect on antigen presenting cells (APC) using a human monocyte cell line THP-1. To better understand LTA1’s stimulatory effects, we compared it to dmLT, or LT-R192G/L211A, a related AB5 adjuvant in clinical trials for oral or parenteral vaccines. LTA1 and dmLT both activated APCs to upregulate MHC-II (HLA-DR), CD86, cytokine secretion (e.g., IL-1β) and inflammasome activation. The effect of LTA1 on surface marker changes (e.g., MHC-II) was highly dose-dependent whereas dmLT exhibited high MHC-II expression regardless of dose. In contrast, cytokine secretion profiles were similar and dose-dependent after both LTA1 and dmLT treatment. Cellular activation by LTA1 was independent of ganglioside binding, as pre-treatment with purified GM1 blocked the effect of dmLT but not LTA1. Unexpectedly, while activation of the inflammasome and cytokine secretion by LTA1 or dmLT was blocked by the protein kinase A inhibitor H89 (similar to previous reports), these responses were not inhibited by a more specific PKA peptide inhibitor or antagonist; thus Indicating that a novel and unknown mechanism is responsible for inflammasome activation and cytokine secretion by LT proteins. Lastly, LTA1 stimulated a similar cytokine profile in primary human monocytes as it did in THP1 cells, including IL-1β, IL-6, IL-8, MIP-1α, MIP-1β, and TNFα. Thus, we report that LTA1 protein programs a dendritic cell-like phenotype in APCs similar to dmLT in a mechanism that is independent of PKA activation and GM1 binding and entry.

Flow Cytometric Allergy Diagnosis: Basophil Activation Techniques.

The basis of flow cytometric allergy diagnosis is the quantification of changes in the expression of basophilic surface membrane markers (Ebo et al., Clin Exp Allergy 34: 332-339, 2004). Upon encountering specific allergens recognized by surface receptor FcεRI-bound IgE, basophils not only secrete and generate quantifiable bioactive mediators but also upregulate the expression of different markers (e.g., CD63, CD203c) which can be detected by multicolor flow cytometry using specific monoclonal antibodies (Ebo et al., Cytometry B Clin Cytom 74: 201-210, 2008). Here, we describe two flow cytometry-based protocols which allow the detection of surface marker activation (Method 1) and changes in intragranular histamine (Method 2), both reflecting different facets of basophil activation.

Inhibition of Cell Cycle Progression, Induction of Apoptosis, and Changes in Surface Markers of MEG-01 Megakaryoblastic Cells Exposed to a Random Positioning Machine

One cause of thrombocytopenia in astronauts after spaceflight is decreased platelet production. To increase our understanding of thrombopoiesis in humans while in space, we investigated the effects of simulated microgravity, achieved using a random positioning machine (RPM), on megakaryoblastic cells (MEG-01 cell line). Exposure of MEG-01 cells to simulated microgravity for up to one week significantly increased cellular apoptosis compared to the static group (1 g-control). Flow cytometry analysis of the cell cycle revealed a significant increase in the percentage of cells in the G0/G1 phase after one week of RPM-exposure compared to the static group. Additionally, after one week, a difference in morphology was detected between the cells of the static group and the cells exposed to microgravity conditions. The expression of the CD33 surface marker was significantly decreased after a one week of microgravity exposure compared to the 1 g-control. We, therefore, concluded that in MEG-01 cells, simulated microgravity induces apoptosis, inhibits cell cycle progression of cells from G0/G1 into S phase, decreases cell proliferation, and decreases the expression of surface markers. We believe that, with insufficient physiological compensation, these changes under microgravity conditions may lead to disorders of megakaryocytes differentiation and/or decreased platelet production. It should also be noted that the human cell line MEG-01 could be a useful model for studying the effects of simulated microgravity on platelet production because of their ability to generate platelet-like particles.

Abstract 1559: TLR7/8 immune-stimulating antibody conjugates elicit robust myeloid activation leading to enhanced effector function and anti-tumor immunity in pre-clinical models

Abstract Deficient anti-tumor immunity often results from an immunosuppressive tumor microenvironment (TME) that renders antigen presenting cells (APCs) unable to effectively stimulate T cells. Recent studies indicate that local delivery of immunostimulatory adjuvants can activate tumor resident APCs, driving uptake, processing and presentation of tumor neoantigens to T cells that mediate anti-tumor immunity. To overcome challenges associated with intratumoral delivery of such adjuvants, we developed a novel class of TLR immune-stimulating antibody conjugates (TAC) that comprise a TLR7/8 agonist conjugated to tumor-targeting monoclonal antibodies. In vitro co-cultures with human cancer cell lines and leukocytes revealed that TACs potently activate primary APCs, leading to increased co-stimulatory molecule expression (e.g. CD40, CD86) and secretion of pro-inflammatory cytokines (e.g. TNFα). The TACs also enhanced antibody-mediated effector functions such as ADCC and ADCP. Surprisingly, these constructs also induced dendritic cell (DC) differentiation from monocytes, as measured by changes in cellular morphology and DC surface markers (e.g. CD14 downregulation). CyTOF-based analysis of intracellular signaling in human leukocytes revealed a unique signaling signature of the conjugate compared to a mixture of its components, suggesting a novel biological mechanism by which the conjugate stimulates APCs. Finally, we demonstrated in vivo efficacy in syngeneic tumor models in which TAC treatment led to tumor clearance and development of immunologic memory. These results provide a strong rationale for this technology as a platform for cancer immunotherapy. Citation Format: Shelley E. Ackerman, Joseph C. Gonzalez, Josh D. Gregorio, Jason C. Paik, Felix J. Hartmann, Justin A. Kenkel, Arthur Lee, Angela Luo, Cecelia I. Pearson, Murray L. Nguyen, Benjamin Ackerman, Lauren Y. Sheu, Richard P. Laura, Steven J. Chapin, Brian S. Safina, Sean C. Bendall, David Dornan, Edgar G. Engleman, Michael N. Alonso. TLR7/8 immune-stimulating antibody conjugates elicit robust myeloid activation leading to enhanced effector function and anti-tumor immunity in pre-clinical models [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 1559.