Activation Of Peripheral Blood Mononuclear Cells Research Articles

Variants of NLRP3 Protein in Haemonchus contortus Infected Sheep: Impact on Immune Cell Responsiveness to LPS In Vitro

ABSTRACTPathogen recognition is an essential component to achieve the desired outcome of host protection. Nod‐like receptor pyrin containing domain 3 (NLRP3) is a cytoplasmic pattern recognition receptor (PRR) with a wide array of agonists, such as PAMPs, DAMPs, ATP, bacterial product and viral products. Stimulation of the NLRP3 inflammasome results in proteolytic activation of IL‐1β and IL‐18, cell pyroptosis and classically, the induction of proinflammatory responses. St. Croix (STC) sheep have resistance traits exhibiting the appropriate T‐helper type 2 immune response ensuing protection during helminth parasitic infection whereas parasite‐susceptible Suffolk (SUF) sheep have an impaired response resulting in parasite establishment and adverse symptoms. The objective of these experiments was to determine if NLRP3 protein in H. contortus‐infected SUF sheep was defective using the classical activation pathway of NLRP3 inflammasome. Peripheral blood mononuclear cells (PBMCs) derived from H. contortus‐infected STC and SUF sheep were isolated from whole blood and treated (MCC950 treatment for 2 h followed by LPS treatment for 3 h, 1400 W treatment for 2 h followed by LPS treatment for 3 h, LPS treatment for 3 h or culture media for 3 h). qPCR analysis of LPS‐stimulated PBMC revealed an upregulation in inflammatory associated genes IL‐1β, TLR4, TNFα and NFκB (p < 0.0001) in STC PBMC and downregulation in IFNγ, IL‐6 and iNOS for SUF PBMC. Pharmacological inhibition of iNOS in SUF PBMC resulted in an upregulation in the expression of IFNγ. These preliminary data begin to discover a relationship between NLRP3 activation and TLR4 signalling in PBMC of STC and SUF sheep.

Blood based kinase activity profiling to predict response to immune checkpoint inhibitors in patients with advanced stage non-small cell lung cancer: The IOpener study.

e20576 Background: Treatment with immune checkpoint inhibitors (ICI) has become part of the standard of care for patients with advanced stage non-small cell lung cancer (NSCLC). Unfortunately, its success is limited to a subset of treated patients, while ICI treatment is associated with immune-related adverse events. The predictive value of kinase activity profiling of peripheral blood mononuclear cells (PBMCs) was previously shown in a discovery study (Hurkmans et al., JITC 2020). The aim of the current study was to perform a prospective and blinded validation of the predictive value of kinase activity profiles of PBMCs for response to ICI treatment in patients with NSCLC in a standardized setting. Preliminary results for this study were presented before (De Joode et al. 2022), here we present results for a significantly extended validation cohort with a follow-up of at least 1 year. Methods: PBMCs from patients with advanced stage NSCLC included in this observational multicenter study were collected prior to ICI treatment. Tyrosine kinase activity of PBMCs was profiled using a micro-array with 144 different peptide-substrates (Pamchip). Classification analysis was performed to discriminate between patients with or without progressive disease (RECIST v1.1) within 24 weeks after start of treatment. Only patients treated with first-line pembrolizumab ± chemotherapy were included. Results: A predictive model was first established based on kinase activity profiles, determined in a calibration cohort (N = 61) and subsequently applied to an independent validation cohort (N = 136). The predictive accuracy of the model in the validation cohort was 62% (CI95 = 53-70%). Interestingly, the accuracy improved when the model was combined with PD-L1 expression: for patients with a PD-L1 score < 50%, the accuracy for predicting progressive disease at 24 weeks was 68% (CI95 = 57-78%). Here, significant lower progression free survival (PFS) rates were observed for patients for whom progression was predicted compared to those for whom no progression was predicted (p = 0.0003, hazard ratio = 2.6, CI95 = 1.6-4.3).The 1-year PFS for patients with predicted progression was only 13% (CI95 = 5-31%) which implies a NPV of 87%. PFS rates were also lower for patients with predicted progression in the subgroups with PD-L1 score < 1% (p = 0.03, hazard ratio = 2.2, CI95 = 1.1-4.3) and PD-L1 score 1-49% (p = 0.002, hazard ratio = 5.0, CI95 = 1.8-14). The predictive kinase activity signature appears to be T-cell related, which will be further investigated to improve understanding of the underlying biology. Conclusions: In this prospective validation study the predictive value of kinase activity profiling of PBMCs for response to first-line ICI therapy of patients with advanced NSCLC was confirmed. The final aim is to apply the kinome analysis in clinical practice.

Altered Histone Deacetylase Versus Histone Acetyltransferase Activity in Peripheral Blood Mononuclear Cells from Apparently Healthy Young Adults with Prior Exposure to Adverse Childhood Experiences

Introduction: Adverse childhood experiences (ACEs) are severe psychosocial stressors which occur in early developmental periods and promote later life cardiometabolic disease in a dose-dependent manner. ACE-related risk may be mediated in part by epigenetic alterations, particularly in myeloid lineage immune cells. Histone acetylation is a dynamic epigenetic process that regulates gene expression via modification of chromatin structure and is dictated by the opposed actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). An appropriate balance between HDAC and HAT activity is critical for maintaining cellular function, and an imbalance has been implicated in the premature development of human diseases including cardiometabolic disease. Glucocorticoids (e.g., cortisol) are well-described regulators of immune function and can influence histone acetylation through glucocorticoid receptors. Thus, we hypothesized that HDAC:HAT activity would be altered in young adults with prior ACE exposure, and that HDAC:HAT activity would be associated with salivary cortisol concentrations. Purpose: We examined HDAC:HAT activity in peripheral blood mononuclear cells (PBMCs) in apparently healthy young adults with versus without prior exposure to ACEs and explored whether circulating cortisol was associated with HDAC:HAT activity. Methods: PBMCs were isolated from whole blood collected within 1 hour of wakening from 27 young adult women who reported high (≥ 4 ACEs) ACE exposure (ACE4+; mean ± SD; age = 21 ± 3 y, BMI = 25.7 ± 4.5 kg/m2) and from 14 young adult women who reported no ACE exposure (CON; age = 21 ± 3 y, BMI = 26.1 ± 6 kg/m2). Salivary cortisol was also collected within 1 hour of wakening and 8 hours later. Nuclear proteins were isolated from PBMCs and HDAC and HAT activities were quantified by commercially available assay kits. Salivary cortisol was quantified via ELISA. Independent samples t-tests were used to examine differences in HDAC:HAT, HDAC, and HAT activity. Pearson correlation coeffcients were used to examine the relation of HDAC:HAT activity with morning and afternoon cortisol concentrations, and the diurnal cortisol slope in the complete sample. Results: HDAC:HAT activity was reduced in ACE4+ versus CON (140.4 ± 52 vs. 189.6 ± 42 arbitrary units, p = 0.004). This difference was primarily driven by reduced HDAC activity (13,706 ± 4,696 vs. 18,060 ± 3,641 ng/h/mg, p = 0.004) in ACE4+ versus CON, whereas HAT activity was not different (99.7 ± 11 vs. 96.1 ± 9 ng/h/mg, p = 0.30). HDAC:HAT activity was not associated with morning (r=0.27, p = 0.07) or afternoon (r=-0.23, p = 0.16) cortisol, but was associated with the diurnal cortisol slope (r=-0.37, p = 0.02). Discussion: Young adults with prior ACE exposure have an altered balance between HDAC and HAT activity in PBMCs that appears to be driven by lower HDAC activity, and which is negatively related to diurnal cortisol slope. These findings warrant further exploration, particularly regarding the specific HDACs implicated, their role in reduced cellular function as it relates to disease risk, and whether these can be modulated to improve function among those with ACE exposure. Further, our data support future examination of the role of glucocorticoid signaling in regulating histone acetylation in this population. This study was supported by the Center for Integrative Research on Childhood Adversity (Grant #P20GM109097), through the National Institute of General Medical Sciences (to NDMJ and TKT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PBMCs as Tool for Identification of Novel Immunotherapy Biomarkers in Lung Cancer.

Lung cancer (LC), including both non-small (NSCLC) and small (SCLC) subtypes, is currently treated with a combination of chemo- and immunotherapy. However, predictive biomarkers to identify high-risk patients are needed. Here, we explore the role of peripheral blood mononuclear cells (PBMCs) as a tool for novel biomarkers searching. We analyzed the expression of the cGAS-STING pathway, a key DNA sensor that activates during chemotherapy, in PBMCs from LC patients divided into best responders (BR), responders (R) and non-responders (NR). The PBMCs were whole exome sequenced (WES). PBMCs from BR and R patients of LC cohorts showed the highest levels of STING (p < 0.0001) and CXCL10 (p < 0.0001). From WES, each subject had at least 1 germline/somatic alteration in a DDR gene and the presence of more DDR gene mutations correlated with clinical responses, suggesting novel biomarker implications. Thus, we tested the effect of the pharmacological DDR inhibitor (DDRi) in PBMCs and in three-dimensional spheroid co-culture of PBMCs and LC cell lines; we found that DDRi strongly increased cGAS-STING expression and tumor infiltration ability of immune cells in NR and R patients. Furthermore, we performed FACS analysis of PBMCs derived from LC patients from the BR, R and NR cohorts and we found that cytotoxic T cell subpopulations displayed the highest STING expression. cGAS-STING signaling activation in PBMCs may be a novel potential predictive biomarker for the response to immunotherapy and high levels are correlated with a better response to treatment along with an overall increased antitumor immune injury.

Abstract 5556: An vascularized melanoma model for with unidirectional perfusion of circulating immune cells for advancing immune-oncology research

Abstract Understanding the interactions between the tumor the vascular systems and circulating immune cells is crucial for elucidating mechanisms of immune cell recruitment in cancer and in exploring potential immunotherapeutic approaches. However, current in vitro models often lack the fidelity to replicate these intricate dynamics observed in vivo. The OrganoPlate Graft has been developed for perfusion of peripheral blood mononuclear cells (PBMCs) through a vascular bed with a melanoma monolayer (A375) to study aspects such as immune cell adhesion, extravasation and interaction with the tumor. The OrganoPlate Graft comprises 48 chips per plate, each designed to simulate the conditions of vascular beds. The device achieves flow through passive leveling, utilizing gravity to induce a pressure drop and unidirectional flow across the vascular bed in both positive and negative inclines. This approach avoids the complications and potential artifacts introduced by external pump, reduces complexity, and increases the throughput with up to 16 plates (768 chips) served by each OrganoFlow rocker. In experimental settings, formation of the vascular bed was seen to be consistent in plates and the introduction of the chemokine CXCL-12 led to the establishment of a gradient. Subsequently, PBMCs were observed to localize in clusters as a result of extravasation towards this gradient and the A375 monolayer. This behavior is consistent with known chemotactic responses in more complex in vivo environments. A notable observation was the sustained activity of PBMCs within the system. Cells continued to perfuse through the vascular bed for up to three days after seeding. In summary, the OrganoPlate Graft provides a platform for studying immune interactions in an immunocompetent vascular bed model. Its design and functionality allow for the exploration of immune cell behaviors in proximity to tumor cells, offering potential insights for immune-oncology research. The system's ability to mimic immune cell migration and extravasation in a controlled environment, and future potential to observe tumour killing makes it a valuable tool for advancing our understanding of immune-tumor interactions and may contribute to the development of novel immune-oncology therapeutic strategies. Citation Format: Manuel Caro Torregrosa, Sander de Ruiter, Todd Burton, Karla Queiroz, Will Allen, Job Komen, Sebastiaan Trietsch, Lenie van den Broek. An vascularized melanoma model for with unidirectional perfusion of circulating immune cells for advancing immune-oncology research [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 5556.

Cytotoxicity and anti-inflammatory properties of a GLP-1 receptor agonist in a model of human peripheral blood mononuclear cell culture

&amp;lt;p&amp;gt;&amp;lt;strong&amp;gt;Introduction. &amp;lt;/strong&amp;gt;GLP-1R agonists (GLP-1RAs) are a type of anti-hyperglycemic medications used for the management of type 2 diabetes mellitus (T2DM). In addition to glucose-lowering effects, GLP-1R agonists (GLP-1RAs) also provide further advantages by promoting weight loss and controlling blood pressure. Furthermore, GLP-1RAs have been reported for their therapeutic benefits in neurological, cardiovascular, endocrine, and metabolic diseases. Emerging evidence from many clinical and experimental studies, as well as in vitro researches, suggests that GLP-1RA agonists may reduce inflamma tion and modify the immunological response, but the underlying molecu lar mechanisms remain unclear. In this research, we examined the effect of GLP-1RA on the cytotoxicity, proliferative activity, and cytokine production of human peripheral blood mononuclear cells (PBMCs) in vitro.&amp;lt;/p&amp;gt; &amp;lt;p&amp;gt;&amp;lt;strong&amp;gt; Methods. &amp;lt;/strong&amp;gt;The peripheral blood mononuclear cells (PBMCs) cultures from healthy volunteers were used. GLP-1RA (DMB), in different concentrations, was added to the cell cultures. A flow cytometry assay was used to assess cell viability, while MTT and CFSE dye dilution assays were used to quantify proliferative activity. Cytokine levels were measured using a sandwich en zyme-linked immunosorbent assay (ELISA).&amp;lt;/p&amp;gt; &amp;lt;p&amp;gt;&amp;lt;strong&amp;gt;Results.&amp;lt;/strong&amp;gt; The cytotoxicity data demonstrated that only the highest DMB concentration (1000 nM) decreased the metabolic activity (viability) of PBMCs (p&amp;amp;lt;0.05) in comparison to untreated cultures, while concentra tions of 200 nM (p&amp;amp;lt;0.05) and 1000 nM (p&amp;amp;lt;0.001) of GLP-1RA increased the proportion of late apoptotic and necrotic cells (p&amp;amp;lt;0.001). The prolif eration of PBMCs was significantly decreased in the presence of GLP-1RA at a 100 nM concentration when compared to the control (p&amp;amp;lt;0.05). The same concentration of GLP-1RA significantly reduced the production of IL-6, IL-1&amp;amp;beta;, and TNF-&amp;amp;alpha;.&amp;lt;/p&amp;gt; &amp;lt;p&amp;gt;&amp;lt;strong&amp;gt;Conclusion.&amp;lt;/strong&amp;gt; Our results suggested that GLP-1RA (DMB), at a non-toxic concentration of 100 nM, inhibited the proliferation and production of pro-inflammatory cytokines by human PBMCs in vitro. These findings open the perspective to study the immune response in detail in future experiments.&amp;lt;/p&amp;gt;

Development of a Fluorescently Labeled Ligand for Rapid Detection of DAT in Human and Mouse Peripheral Blood Monocytes.

The dopamine transporter (DAT) is one of the key regulators of dopamine (DA) signaling in the central nervous system (CNS) and in the periphery. Recent reports in a model of Parkinson's disease (PD) have shown that dopamine neuronal loss in the CNS impacts the expression of DAT in peripheral immune cells. The mechanism underlying this connection is still unclear but could be illuminated with sensitive and high-throughput detection of DAT-expressing immune cells in the circulation. Herein, we have developed fluorescently labeled ligands (FLL) that bind to surface-expressing DAT with high affinity and selectivity. The diSulfoCy5-FLL (GC04-38) was utilized to label DAT in human and mouse peripheral blood mononuclear cells (PBMCs) that were analyzed via flow cytometry. Selective labeling was validated using DAT KO mouse PBMCs. Our studies provide an efficient and highly sensitive method using this novel DAT-selective FLL to advance our fundamental understanding of DAT expression and activity in PBMCs in health and disease and as a potential peripheral biomarker.

Mononuclear cell composition and activation in blood and mucosal tissue of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic, inflammatory, antigen-driven disease of the esophagus. Tissue EoE pathology has previously been extensively characterized by novel transcriptomics and proteomic platforms, however the majority of surface marker determination and screening has been performed in blood due to mucosal tissue size limitations. While eosinophils, CD4+ T cells, mast cells and natural killer (NK) T cells were previously investigated in the context of EoE, an accurate picture of the composition of peripheral blood mononuclear cells (PBMC) and their activation is missing. In this study, we aimed to comprehensively analyze the composition of peripheral blood mononuclear cells and their activation using surface marker measurements with multicolor flow cytometry simultaneously in both blood and mucosal tissue of patients with active EoE, inactive EoE, patients with gastroesophageal reflux disease (GERD) and controls. Moreover, we set out to validate our data in co-cultures of PBMC with human primary esophageal epithelial cells and in a novel inducible mouse model of eosinophilic esophagitis, characterized by extensive IL-33 secretion in the esophagus. Our results indicate that specific PBMC populations are enriched, and that they alter their surface expression of activation markers in mucosal tissue of active EoE. In particular, we observed upregulation of the immunomodulatory molecule CD38 on CD4+ T cells and on myeloid cells in biopsies of active EoE. Moreover, we observed significant upregulation of PD-1 on CD4+ and myeloid cells, which was even more prominent after corticosteroid treatment. With co-culture experiments we could demonstrate that direct cell contact is needed for PD-1 upregulation on CD4+ T cells. Finally, we validated our findings of PD-1 and CD38 upregulation in an inducible mouse model of EoE. Herein we show significant alterations in the PBMC activation profile of patients with active EoE in comparison to inactive EoE, GERD and controls, which could have potential implications for treatment. To our knowledge, this study is the first of its kind expanding the multi-color flow cytometry approach in different patient groups using in vitro and in vivo translational models.

Reduced form of Galectin-1 Suppresses Osteoclastic Differentiation of Human Peripheral Blood Mononuclear Cells and Murine RAW264 Cells In Vitro.

Galectin-1 (Gal-1) is an evolutionarily conserved sugar-binding protein found in intra- and extracellular spaces. Extracellularly, it binds to glycoconjugates with β-galactoside(s) and functions in various biological phenomena, including immunity, cancer, and differentiation. Under extracellular oxidative conditions, Gal-1 undergoes oxidative inactivation, losing its sugar-binding ability, although it exhibits sugar-independent functions. An age-related decrease in serum Gal-1 levels correlates with decreasing bone mass, and Gal-1 knockout promotes osteoclastic bone resorption and suppresses bone formation. However, the effect of extracellular Gal-1 on osteoclast differentiation remains unclear. Herein, we investigated the effects of extracellular Gal-1 on osteoclastogenesis in human peripheral blood mononuclear cells (PBMCs) and mouse macrophage RAW264 cells. Recombinant Gal-1 suppressed the macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand-dependent osteoclast formation, actin ring formation, and bone-resorption activity of human PBMCs. Similar results were obtained for RAW264 cells. Gal-1 knockdown increased osteoclast-like cell formation, suggesting that it affected differentiation in an autocrine-like manner. Oxidized Gal-1 slightly affected differentiation, and in the presence of lactose, the differentiation inhibitory effect of galectin-1 was not observed. These findings suggest that extracellular Gal-1 inhibits osteoclast differentiation in a β-galactoside-dependent manner, and an age-related decrease in serum Gal-1 levels may contribute to reduced osteoclast activity and decreasing bone mass.

Upregulation of proBDNF/p75NTR signaling in immune cells and its correlation with inflammatory markers in patients with major depression.

ProBDNF is the precursor protein of brain-derived neurotrophic factor (BDNF) expressed in the central nervous system and peripheral tissues. Previous studies showed that the blood levels of both proBDNF and p75 neurotrophic receptors (p75NTR) in major depressive disorder (MDD) were increased, but which blood cell types express proBDNF and its receptors is not known. Furthermore, the relationship between proBDNF/p75NTR and inflammatory cytokines in peripheral blood of MDD is unclear. Peripheral blood mononuclear cells (PBMCs) and serum were obtained from depressive patients (n = 32) and normal donors (n = 20). We examined the expression of proBDNF and inflammatory markers and their correlative relationship in patients with major depression. Using flow cytometry analysis, we examined which blood cells express proBDNF and its receptors. Finally, the role of proBDNF/p75NTR signal in inflammatory immune activity of PBMCs was verified invitro experiments. Inflammatory cytokines in PBMC from MDD patients were increased and correlated with the major depression scores. The levels of IL-1β and IL-10 were also positively correlated with the major depression scores, while the levels of TNF-α and IL-6 were negatively correlated with the major depression scores. Intriguingly, the levels of sortilin were positively correlated with IL-1β. Q-PCR and Western blots showed proBDNF, p75NTR, and sortilin levels were significantly increased in PBMCs from MDD patients compared with that from the normal donors. Flow cytometry studies showed that proBDNF and p75NTR were present mainly in CD4+ and CD8+ T cells. The number of proBDNF and p75NTR positive CD4+ and CD8+ T cells from MDD patients was increased and subsequently reversed after therapeutic management. Exogenous proBDNF protein or p75ECD-Fc treatment of cultured PBMC affected the release of inflammatory cytokines invitro. ProBDNF promoted the expression of inflammatory cytokines, while p75ECD-Fc inhibited the expression of inflammatory cytokines. Given there was an inflammatory response of lymphocytes to proBDNF, it is suggested that proBDNF/p75NTR signaling may upstream inflammatory cytokines in MDD. Our data suggest that proBDNF/p75NTR signaling may not only serve as biomarkers but also may be a potential therapeutic target for MDD.

Dysregulation of Peripheral Blood Mononuclear Cells and Immune-Related Proteins during the Early Post-Operative Immune Response in Ovarian Cancer Patients.

Surgical treatment is a cornerstone of ovarian cancer (OC) therapy and exerts a substantial influence on the immune system. Immune responses also play a pivotal and intricate role in OC progression. The aim of this study was to investigate the dynamics of immune-related protein expression and the activity of peripheral blood mononuclear cells (PBMCs) in OC patients, both before surgery and during the early postoperative phase. The study cohort comprised 23 OC patients and 20 non-cancer controls. A comprehensive analysis of PBMCs revealed significant pre-operative downregulation in the mRNA expression of multiple immune-related proteins, including interleukins, PD-1, PD-L1, and HO-1. This was followed by further dysregulation during the first 5 post-operative days. Although most serum interleukin concentrations showed only minor changes, a distinct increase in IL-6 and HO-1 levels was observed post-operatively. Reduced metabolic and phagocytic activity and increased production of reactive oxygen species (ROS) were observed on day 1 post-surgery. These findings suggest a shift towards immune tolerance during the early post-operative phase of OC, potentially creating a window for treatment. Further research into post-operative PBMC activity could lead to the development of new or improved treatment strategies for OC.

Abstract 14475: Intrinsic T Cell Tolerance to the Autoantigen LL-37 is Mediated in Part by Platelets

Introduction: An autoimmune response to human cathelicidin antimicrobial peptide LL-37, a component of neutrophil extracellular traps (NETs), may play a role in athero-thrombosis. We have reported that acute coronary syndrome (ACS) patients have increased LL-37 auto-reactive CD8+CD69+CD137+ T cells compared to healthy controls suggesting breach of tolerance to LL-37. Platelets present antigens to CD8+ T cells via HLA Class-I but it is unknown if this pathway is involved in maintaining intrinsic tolerance to the autoantigen LL-37. In this study, we investigated the role of platelets in maintaining immune tolerance to LL-37 in healthy subjects. Methods and Results: Platelets and autologous peripheral blood mononuclear cells (PBMCs) were isolated from remnant blood of platelet donor Leukocyte Reduction System cones from the institutional blood bank. Platelet enrichment was confirmed by CD41+ flow cytometry and over 95% were HLA Class-I(+). PBMCs were co-cultured with autologous platelets that were either non-primed or primed with LL-37 peptide at a ratio of 25:1 platelet-to-PBMC. Cells were collected after 16 hours and subjected to fluorescent staining for activation markers by flow cytometry. Compared to non-primed platelets, co-culture of LL-37-primed platelets significantly reduced T cell activation in autologous PBMCs including CD8+CD69+CD137+ T cells, CD4+CD134+ T cells and CD4+CD134+CD137+ T cells (Table), supporting platelet-mediated T cell tolerance to LL-37. We then evaluate if the breach in T cell tolerance to LL-37 in ACS is related to platelet counts. CD8+CD69+CD137+ T cell response to LL-37 in PBMCs of ACS patients was negatively correlated with platelet count (N=21; Pearson r = -0.438, P=0.047). Conclusions: The results demonstrate that intrinsic immune tolerance to the autoantigen LL-37 in healthy subjects is mediated in part by platelets. Reduced platelet counts are implicated in the breach of immune tolerance to LL-37 in ACS.

A bibliometric analysis: Ca2+ fluxes and inflammatory phenotyping by flow cytometry in peripheral blood mononuclear cells.

The immune system, composed of organs, tissues, cells, and proteins, is the key to protecting the body from external biological attacks and inflammation. The latter occurs in several pathologies, such as cancers, type 1 diabetes, and human immunodeficiency virus infection. Immunophenotyping by flow cytometry is the method of choice for diagnosing these pathologies. Under inflammatory conditions, the peripheral blood mononuclear cells (PBMCs) are partially activated and generate intracellular pathways involving Ca2+-dependent signaling cascades leading to transcription factor expression. Ca2+ signaling is typically studied by microscopy in cell lines but can present some limitations to explore human PBMCs, where flow cytometry can be a good alternative. In this review, we dived into the research field of inflammation and Ca2+ signaling in PBMCs. We aimed to investigate the structure and evolution of this field in a physio-pathological context, and then we focused our review on flow cytometry analysis of Ca2+ fluxes in PBMCs. From 1984 to 2022, 3865 articles on inflammation and Ca2+ signaling in PBMCs were published, according to The Clarivate Web of Science (WOS) database used in this review. A bibliometric study was designed for this collection and consisted of a co-citation and bibliographic coupling analysis. The co-citation analysis was performed on 133 articles: 4 clusters highlighted the global context of Ca2+ homeostasis, including chemical probe development, identification of the leading players in Ca2+ signaling, and the link with chemokine production in immune cell function. Next, the bibliographic coupling analysis combined 998 articles in 8 clusters. This analysis outlined the mechanisms of PBMC activation, from signal integration to cellular response. Further explorations of the bibliographic coupling network, focusing on flow cytometry, revealed 21 articles measuring cytosolic Ca2+ in PBMCs, with only 5 since 2016. This final query showed that Ca2+ signaling analysis in human PBMCs using flow cytometry is still underdeveloped and investigates mainly the cytosolic Ca2+ compartment. Our review uncovers remaining knowledge gaps of intracellular players involved in Ca2+ signaling in PBMCs, such as reticulum and mitochondria, and presents flow cytometry as a solid option to supplement gold-standard microscopy studies.

FRI126 Autoreactive T Cells To Angiotensin II Type 1 Receptor In Salt-dependent Hypertension Due To Primary Aldosteronism

Abstract Disclosure: R. Scarpa: None. M. Piazza: None. B. Caroccia: None. S. Carraro: None. F. Vianello: None. F. Cinetto: None. C. Agostini: None. T.M. Seccia: None. G. Rossi: None. Compelling evidence suggested a role of acquired immunity in hypertension and hypertension-mediated organ damage (HMOD), but the pathogenic role of this acquired immunity is still unknown. Primary Aldosteronism, the paradigm of salt-dependent hypertension, is characterised by a more prominent HMOD than primary (‘essential’) hypertension and by high titer of autoantibodies against the type 1 angiotensin receptor (AT1R). As T cells are broadly recognised as HMOD-mediators, we aim to investigate: 1) the presence of AT1R-autoreactive T cells in PA; 2) aldosterone rapid and chronic effect on AT1R-autoreactive T lymphocytes; 3) the presence and functional role of know aldosterone receptors on T lymphocytes. The presence of AT1R-autoreactive T cells in 11 PA patients was investigated by flow cytometry after stimulation with pool of peptides spanning the full length of AT1R. The expression of mineralocorticoid receptor (MR) and G Protein-Coupled Estrogen Receptor (GPER) on T cells was tested by measuring mRNA copy number by droplet digital PCR and immunoblotting from healthy donors (HD) subjects and PA patients. Peripheral blood mononuclear cells (PBMCs) from PA patients were exposed to aldosterone 10-10 M with or without MR antagonist (canrenone) and GPER agonist (G1) and antagonist (G36). We evaluated the effect of aldosterone on CD8+ T cells by measuring IFNɣ release with flow cytometry. PA patients were found to have CD8+ AT1R-autoreactive T cells after activation of PBMC with AT1R peptides. MR and GPER were detected both at mRNA and protein level in CD4+ and CD8+ T cells with the following rank of expression GR&amp;gt;MR&amp;gt;&amp;gt;GPER. Aldosterone significantly increased IFNɣ release in CD8+ T-cell both under chronic and acute stimulation. Chronic exposure to aldosterone increased IFN-γ production by CD8+ T cells by acting via the MR, as it was prevented (p &amp;lt; 0.004) by canrenone, while the rapid aldosterone action was MR and GPER mediated, as it was reduced by canrenone (p &amp;lt; 0.003), but also by G36 (p &amp;lt; 0.02), and mimicked by G1. These results showed the presence of AT1R-autoreactive T cells in patients with PA. Moreover, upon of MR and GPER receptor stimulation with aldosterone, AT1-R specific CD8+ T cells undergo profound changes that mediated a strong cytotoxic response towards AT1-R-expressing organs. Presentation: Friday, June 16, 2023

Short-term assays for mesenchymal stromal cell immunosuppression of T-lymphocytes.

Trauma patients are susceptible to coagulopathy and dysfunctional immune responses. Mesenchymal stromal cells (MSCs) are at the forefront of the cellular therapy revolution with profound immunomodulatory, regenerative, and therapeutic potential. Routine assays to assess immunomodulation activity examine MSC effects on proliferation of peripheral blood mononuclear cells (PBMCs) and take 3-7 days. Assays that could be done in a shorter period of time would be beneficial to allow more rapid comparison of different MSC donors. The studies presented here focused on assays for MSC suppression of mitogen-stimulated PBMC activation in time frames of 24 h or less. Three potential assays were examined-assays of apoptosis focusing on caspase activation, assays of phosphatidyl serine externalization (PS+) on PBMCs, and measurement of tumor necrosis factor alpha (TNFα) levels using rapid ELISA methods. All assays used the same initial experimental conditions: cryopreserved PBMCs from 8 to 10 pooled donors, co-culture with and without MSCs in 96-well plates, and PBMC stimulation with mitogen for 2-72 h. Suppression of caspase activity in activated PBMCs by incubation with MSCs was not robust and was only significant at times after 24 h. Monitoring PS+ of live CD3+ or live CD4+/CD3+ mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, 2 h, although no increase in the percentage of PS+ cells was seen with time. The ability of MSC in co-culture to suppress PBMC PS+ externalization compared favorably to two concomitant assays for MSC co-culture suppression of PBMC proliferation, at 72 h by ATP assay, or at 96 h by fluorescently labeled protein signal dilution. TNFα release by mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, with accumulating signal over time. However, suppression levels with MSC co-culture was reliably seen only after 24 h. Takeaways from these studies are as follows: (1) while early measures of PBMC activation is evident at 2-6 h, immunosuppression was only reliably detected at 24 h; (2) PS externalization at 24 h is a surrogate assay for MSC immunomodulation; and (3) rapid ELISA assay detection of TNFα release by PBMCs is a robust and sensitive assay for MSC immunomodulation at 24 h.

Enhanced inflammasome-mediated inflammation and impaired autophagy in peripheral blood mononuclear cells is associated with non-alcoholic fatty liver disease severity

AimsIdentification of the progress of non-alcoholic fatty liver disease (NAFLD) is crucial for their effective treatment. Circulating peripheral blood mononuclear cells (PBMC) could be a surrogate monitor instead of complicated and expensive biopsies. Changes in immuno-metabolic status in NAFLD patients may be reflected by an expression of different PBMC-specific molecular markers. It was hypothesized that impaired autophagy with enhanced inflammasome activation is a critical molecular event in PBMC that could contribute to systemic inflammation associated with NAFLD progression. Main methodsA cross-sectional study with a sample size of 50 subjects were undertaken from a governmental facility in Kolkata, India. Major anthropometric, biochemical, and dietary parameters were recorded. Cellular and serum samples of NAFLD patients were analyzed for oxidative stress, inflammation, inflammasome activation, and autophagic flux by western blot, flow cytometry, immunocytochemistry. Key findingsBaseline anthropometric and clinical parameters were found associated with NAFLD severity. Elevated systemic inflammation was reflected by higher proinflammatory markers like iNOS, Cox-2, IL-6, TNF-α, IL-1β, hsCRP in the serum of NAFLD subjects (p < 0.05). ROS-induced NLRP3 inflammasomes marker proteins were upregulated (p < 0.05) in PBMC along with NAFLD severity. Expression of autophagic markers such as LC3B, Beclin-1 and its regulator pAMPKα were found diminished (p < 0.05) with a concomitant rise of p62. Colocalization of NLRP3 with LC3B proteins in PBMC was found diminished along NAFLD severity. SignificancePresent data provide mechanistic evidence of impaired autophagy and intracellular ROS triggered inflammasome activation in PBMC, which could potentially exacerbate NAFLD severity.

Mitochondrial reprogramming in peripheral blood mononuclear cells of patients with glycogen storage disease type Ia

BackgroundGlycogen storage disease type Ia (GSDIa) is an inborn metabolic disorder caused by the deficiency of glucose-6-phospatase-α (G6Pase-α) leading to mitochondrial dysfunction. It remains unclear whether mitochondrial dysfunction is present in patients’ peripheral blood mononuclear cells (PBMC) and whether dietary treatment can play a role. The aim of this study was to investigate mitochondrial function in PBMC of GSDIa patients.MethodsTen GSDIa patients and 10 age-, sex- and fasting-time matched controls were enrolled. Expression of genes involved in mitochondrial function and activity of key fatty acid oxidation (FAO) and Krebs cycle proteins were assessed in PBMC. Targeted metabolomics and assessment of metabolic control markers were also performed.ResultsAdult GSDIa patients showed increased CPT1A, SDHB, TFAM, mTOR expression (p < 0.05) and increased VLCAD, CPT2 and citrate synthase activity in PBMC (p < 0.05). VLCAD activity directly correlated with WC (p < 0.01), BMI (p < 0.05), serum malonycarnitine levels (p < 0.05). CPT2 activity directly correlated with BMI (p < 0.05).ConclusionMitochondrial reprogramming is detectable in PBMC of GSDIa patients. This feature may develop as an adaptation to the liver enzyme defect and may be triggered by dietary (over)treatment in the frame of G6Pase-α deficiency. PBMC can represent an adequate mean to assess (diet-induced) metabolic disturbances in GSDIa.

Epigenomic landscape exhibits interferon signaling suppression in the patient of myocarditis after BNT162b2 vaccination

After the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, a novel mRNA vaccine (BNT162b2) was developed at an unprecedented speed. Although most countries have achieved widespread immunity from vaccines and infections, yet people, even who have recovered from SARS-CoV-2 infection, are recommended to receive vaccination due to their effectiveness in lowering the risk of recurrent infection. However, the BNT162b2 vaccine has been reported to increase the risk of myocarditis. To our knowledge, for the first time in this study, we tracked changes in the chromatin dynamics of peripheral blood mononuclear cells (PBMCs) in the patient who underwent myocarditis after BNT162b2 vaccination. A longitudinal study of chromatin accessibility using concurrent analysis of single-cell assays for transposase-accessible chromatin with sequencing and single-cell RNA sequencing showed downregulation of interferon signaling and upregulated RUNX2/3 activity in PBMCs. Considering BNT162b2 vaccination increases the level of interferon-α/γ in serum, our data highlight the immune responses different from the conventional responses to the vaccination, which is possibly the key to understanding the side effects of BNT162b2 vaccination.

Abstract 4858: Effect of mTOR activity in peripheral blood mononuclear cells with metformin in high-risk prostate cancer patients receiving external beam radiotherapy and androgen deprivation therapy

Abstract Introduction: There is considerable interest in the antineoplastic properties of metformin for prostate cancer treatment, however, the underlying mechanism of action of metformin in this setting is poorly understood. It is suspected that metformin’s antineoplastic activity is related to inhibition of mTOR (mammalian target of rapamycin), which ultimately inhibits cell proliferation. In this pilot study, we assessed the mTOR activity of peripheral blood mononuclear cells (PBMCs) from high-risk Prostate cancer (PCa) patients treated with androgen deprivation therapy (ADT) and external beam radiotherapy (EBRT) plus metformin versus placebo. We hypothesized decreased mTOR expression in PBMCs from patients receiving metformin versus placebo. Methods: Normoglycemic adult males with high-risk PCa receiving ADT &amp; EBRT were randomized to metformin 500 mg 3 times daily or placebo. Peripheral blood samples were collected from participants ≥ 6 months after starting study drug, buffy coats were extracted, and PBMCs were isolated ≤2 hours after specimen collection using ficoll-hypaque density gradient method. The mTOR expression was assessed by phosphorylation of serine 2448 (pmTOR; S2448). Activity of mTOR was measured in both unstimulated or stimulated (using CD3/CD28 antibodies) CD4+ and CD8+ T cells. Results: Samples from 15 patients were analyzed (11 placebo &amp; 4 metformin). We observed lower expression of pmTOR in PBMCs in patients receiving metformin compared to placebo indicating metformin demonstrated inhibition of mTOR activity in PBMCs. A general trend of mTOR inhibition was observed in the metformin group across the cell population and conditions (unstimulated vs. stimulated T cell populations). However, the most pronounced difference was observed in unstimulated CD4+ cells, which is of interest since they represent the closest physiological state to the study participants. Conclusion: In this pilot study, we observed mTOR inhibition in the PBMCs of PCa patients treated with metformin. Further assessment of mTOR activity amongst PCa patients receiving metformin is warranted in order to confirm these findings. Citation Format: Febin Antony, Anuraag Shrivastav, Arbind Dubey, Nawaid Usmani, Aldrich Ong, Rashmi Koul, Harvey Quon, Gokulan Sivananthan, William Hunter, Don Yee, Lindsay Rowe, Brita Danielson, Andrew Plesniarski, Ruey-Chyi Su, Julian O. Kim. Effect of mTOR activity in peripheral blood mononuclear cells with metformin in high-risk prostate cancer patients receiving external beam radiotherapy and androgen deprivation therapy. [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 4858.

Abstract 6791: Inovative in vivo (in ovo) CAM model to predict efficacy and mode of action of a new antitumor vaccine stc-1010 on human colorectal adenocarcinoma

Abstract BACKGROUND: Colorectal cancer (CRC) is the second-most deadly cancer. Therapeutic resistance to immuno-oncology drives the need for new treatments. Stimulated tumor cell (STC) vaccine (Brenus Pharma) is composed of selected tumor cell lines, stimulated to overexpress tumor-associated or tumor-specific antigens and neoantigens including resistance factors. Haptenization of these proteins forms an immunogenic complex which stimulate the immune system to recognize and target the patient’s tumor cells expressing same resistance factors. We report in vivo results of STC-1010 vaccine, on human CRC adenocarcinoma from HT29 cell using chorioallantoic membrane (CAM) assay developed by Inovotion in immune reactive model. METHODS: This study was carried out in 2 steps: Firstly, three batches of naïve chicken embryos were stimulated by injections of STC1010 at Embryo Development Day (EDD) 11 and EDD13. At EDD18 of each batch, chicken peripheral blood mononuclear cells (PBMCs) were collected and used as anti-tumor reagent to treat respectively, at EDD 11, EDD 13 and EDD16 chicken embryos xenografted with HT29 cells. Activation of PBMCs was evaluated by IL2 and IL12 secretion quantified by ELISA. At EDD18, i.e., 9 days post-graft, in ovo anti-tumor efficacy was evaluated by tumor weight, metastatic invasion (qPCR analysis of human Alu sequence in lower CAM) and quantification of tumor-infiltrating by CD8, CD4, IFN-gamma, Perforin and TNF-alpha. RESULTS: Compared to negative control, STC-1010 vaccine induced: 1) significant increase of IL-12 and IL-2 secretion in peripheric blood during the generation of all three batches of PBMCs, confirming previous results (IL-12: +52%, p=0.0003 ; IL-2: +482%, p=0.0033); 2) a significant expression of IFN-gamma in tumor (+130,83%, p=0.0185); 3) a tendency to increase infiltrating cells: CD4+: +79,2%, CD8+: +29,4% , Perforin: +105,5%, TNFα : +78,63% confirmed by immunohistochemistry and translated into 4) a significant increase of tumor necrosis (p = 0.0267); and 5) a tendency of metastasis regression (-49%); with 6) no embryonic toxicity/mortality (daily evaluation of embryonic viability) induced by STC-1010. CONCLUSION: This in ovo study confirms efficacy of the STC-1010 observed in previous CRC syngeneic models and gives more insight about STC mechanism of action with the activation and maturation of dendritic cells, induction of CD8+ and LTh1 against tumor as the main driver of the response, all without toxicity. Inovotion’s CAM model could be used for indication screening and as a pre-proof-of-concept before syngeneic model study. Citation Format: Yan Wang, Arnaud Peyronnier, Benoit Pinteur, Lionel Chalus, Corinne Tortorelli, Paul Bravetti, Jean Viallet, François Ghiringhelli, Philippe Fornies. Inovative in vivo (in ovo) CAM model to predict efficacy and mode of action of a new antitumor vaccine stc-1010 on human colorectal 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 6791.