Soluble Cytokine Research Articles

Soluble immune checkpoints associated with disease activity and treatment response in GD and TED.

Soluble immune checkpoints play an important role in peripheral tolerance that has seldom been investigated in Graves' disease (GD) and thyroid eye disease (TED). The objective of this work is to examine the alteration of soluble immune checkpoints in GD and TED. We performed a quantitative multiplex analysis of 17 immune checkpoint proteins in serum from 50 GD patients without TED, 28 GD patients with TED and 40 healthy controls. The association with demographical, serological, clinical features and 27 cytokines was analyzed. A follow-up was conducted in GD patients without TED. Functional outcomes of sLAG-3 and sGITR were assessed in cell cultures using rh-LAG3, rh-GITR, an antagonistic LAG-3 antibody and an antagonistic GITR antibody. GD patients with TED had distinct sICP and cytokine profiles compared with GD patients without TED. Active TED patients exhibited elevation in the levels of sBTLA, sLAG-3, sGITR, sCD80, sCD86 and sPD-L1. Further, GD patients without TED with high sBTLA, sCD27 and sCD40 levels at baseline showed a better improvement in thyrotropin receptor antibody (TRAb) titers after antithyroid drug (ATD) treatment. Adding recombinant human GITR and LAG-3 to PBMC cultures resulted in increased inflammatory cytokine secretion and decreased anti-inflammatory cytokine secretion. The present study uncovers disturbed soluble immune checkpoints and cytokines in GD patients with and without TED, and may pave the way for novel immunological screening, allowing for identification of TED patients at higher risk of developing active disease and GD patients with better treatment response after ATD treatment.

Torque teno virus: a potential marker of immune reconstitution in youths with vertically acquired HIV

Torque teno virus (TTV) viral load (VL), a component of the human virome, increases during immune suppression or dysregulation. This study aimed to explore TTV VL in youths living with vertically acquired HIV (YWVH) and its potential as an immunovirological marker. We performed an observational, retrospective study involving YWVH under antiretroviral treatment (ART) from the Spanish Cohort of HIV-infected children, adolescents, and vertically HIV-infected patients transferred to Adult Units (CoRISpe-FARO), compared to HIV-negative healthy donors (HD). Plasma TTV VL was assessed by qPCR. T-cell phenotype was analysed on cryopreserved peripheral blood mononuclear cells by flow cytometry. Correlations with baseline CD4 and CD8 and long-term virological evolution were examined. A total of 57 YWVH were compared with 23 HD. YWVH had a median CD4 T-cells of 736 cells/mm3 [IQR: 574–906], a median of 17 years [IQR: 14–20.5] since ART initiation, and 65 months [IQR: 39–116] under HIV-RNA virological control. TTV VL was higher among YWVH and in males compared with females (p < 0.05). Among YWVH, TTV VL correlated with CD4 and CD8 counts and the CD4/CD8 ratio (p = 0.002; r = − 0.39, p = 0.037; r = 0.277, p = 0.005; r = − 0.37 respectively). TTV VL correlated with activation expression markers (HLA-DR+/CD38+) on CD4 (p = 0.007, r = 0.39) and the soluble proinflammatory cytokine IL-6 (p = 0.006, r = 0.38).

The Influence of Sulfation Degree of Glycosaminoglycan-Functionalized 3D Collagen I Networks on Cytokine Profiles of In Vitro Macrophage-Fibroblast Cocultures.

Cell-cell interactions between fibroblasts and immune cells, like macrophages, are influenced by interaction with the surrounding extracellular matrix during wound healing. In vitro hydrogel models that mimic and modulate these interactions, especially of soluble mediators like cytokines, may allow for a more detailed investigation of immunomodulatory processes. In the present study, a biomimetic extracellular matrix model based on fibrillar 3D collagen I networks with a functionalization with heparin or 6-ON-desulfated heparin, as mimics of naturally occurring heparan sulfate, was developed to modulate cytokine binding effects with the hydrogel matrix. The constitution and microstructure of the collagen I network were found to be stable throughout the 7-day culture period. A coculture study of primary human fibroblasts/myofibroblasts and M-CSF-stimulated macrophages was used to show its applicability to simulate processes of progressed wound healing. The quantification of secreted cytokines (IL-8, IL-10, IL-6, FGF-2) in the cell culture supernatant demonstrated the differential impact of glycosaminoglycan functionalization of the collagen I network. Most prominently, IL-6 and FGF-2 were shown to be regulated by the cell culture condition and network constitution, indicating changes in paracrine and autocrine cell-cell communication of the fibroblast-macrophage coculture. From this perspective, we consider our newly established in vitro hydrogel model suitable for mechanistic coculture analyses of primary human cells to unravel the role of extracellular matrix factors in key events of tissue regeneration and beyond.

Evaluating the ability of different chaperones in improving soluble expression of a triple-mutated human interferon gamma in Escherichia coli

Human interferon gamma (hIFN-γ) plays a pivotal role as a soluble cytokine with diverse functions in both innate and adaptive immunity. In a previous investigation, we pinpointed three critical amino acid residues, i.e., threonine (T) 27, phenylalanine (F) 29, and leucine (L) 30, on the IFN-γ structure, which are integral to the epitope recognized by anti-IFN-γ autoantibodies. It is crucial to impede the interaction between this epitope and autoantibodies for effective therapy in adult-onset immunodeficiency (AOID). However, the challenge arises from the diminished solubility of the T27AF29L30A mutant in Escherichia coli BL21(DE3). This study delves into a targeted strategy aimed at improving the soluble expression of IFN-γ T27AF29AL30A. This is achieved through the utilization of five chaperone plasmids: pG-KJE8, pKJE7, pGro7, pG-Tf2, and pTf16. These plasmids, encoding cytoplasmic chaperones, are co-expressed with the IFN-γ mutant in E.coli BL21(DE3), and we meticulously analyze the proteins in cell lysate and inclusion bodies using SDS-PAGE and Western blotting. Our findings reveal the remarkable efficacy of pG-KJE8, which houses cytoplasmic chaperones DnaK-DnaJ-GrpE and GroEL-GroES, in significantly enhancing the solubility of IFN-γ T27AF29AL30A. Importantly, this co-expression not only addresses solubility concerns but also preserves the functional dimerized structure, as confirmed by sandwich ELISA. This promising outcome signifies a significant step forward in developing biologic strategies for AOID.

In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts.

Pulsed electromagnetic field stimulation (PEMF) is gaining more attention as a non-invasive arthritis treatment. In our study, immortalized synovial fibroblasts (K4IM) derived from a non-arthritic donor were exposed to MAGCELL® ARTHRO, a PEMF device, with 105 mT intensity, 8 Hz frequency, and 2 × 2.5 min sessions conducted thrice with a 1 h interval, to understand the underlying mechanism in regard to the complement system. Additionally, tumor necrosis factor (TNFα, 10 ng/mL) pre-treatment prior to PEMF stimulation, as well as 3-day versus 6-day stimulation, were compared. Gene expression of C4b binding protein-alpha and -beta (C4BPα, C4BPβ), complement factor (CF)-H, CFI, CD55, CD59, Interleukin (IL-6) and TNFα was analyzed. Immunofluorescence staining of CD55, CD59, and Ki67 was conducted. Results showed the absence of C4BPα gene expression, but C4BPβ was present. One and three days of PEMF stimulation caused no significant changes. However, after six days, there was a significant increase in CD55, CFH, and CD59 gene expression, indicating cytoprotective effects. Conversely, IL-6 gene expression increased after six days of stimulation and even after a single session in TNFα pre-stimulated cells, indicating a pro-inflammatory effect. PEMF's ambivalent, i.e., enhancing complement regulatory proteins and pro-inflammatory cytokines, highlights its complexity at the molecular level.

Crosstalk between Inflammation and Atherosclerosis in Rheumatoid Arthritis and Systemic Lupus Erythematosus: Is There a Common Basis?

Cardiovascular disease is the leading cause of morbidity and mortality in patients with rheumatoid arthritis and systemic lupus erythematosus. Traditional cardiovascular risk factors, although present in lupus and rheumatoid arthritis, do not explain such a high burden of early cardiovascular disease in the context of these systemic connective tissue diseases. Over the past few years, our understanding of the pathophysiology of atherosclerosis has changed from it being a lipid-centric to an inflammation-centric process. In this review, we examine the pathogenesis of atherosclerosis in systemic lupus erythematosus and rheumatoid arthritis, the two most common systemic connective tissue diseases, and consider them as emblematic models of the effect of chronic inflammation on the human body. We explore the roles of the inflammasome, cells of the innate and acquired immune system, neutrophils, macrophages, lymphocytes, chemokines and soluble pro-inflammatory cytokines in rheumatoid arthritis and systemic lupus erythematosus, and the roles of certain autoantigens and autoantibodies, such as oxidized low-density lipoprotein and beta2-glycoprotein, which may play a pathogenetic role in atherosclerosis progression.

Extracellular vesicles in cancer: golden goose or Trojan horse.

Intercellular communication can be mediated by direct cell-to-cell contact and indirect interactions through secretion of soluble chemokines, cytokines, and growth factors. Extracellular vesicles (EVs) have emerged as important mediators of cell-to-cell and cell-to-environment communications. EVs from tumor cells, immune cells, and stromal cells can remodel the tumor microenvironment and promote cancer cell survival, proliferation, metastasis, immune evasion, and therapeutic resistance. Most importantly, EVs as natural nanoparticles can be manipulated to serve as a potent delivery system for targeted cancer therapy. EVs can be engineered or modified to improve their ability to target tumors and deliver therapeutic substances, such as chemotherapeutic drugs, nucleic acids, and proteins, for the treatment of cancer. This review provides an overview of the biogenesis and recycling of EVs, discusses their roles in cancer development, and highlights their potential as a delivery system for targeted cancer therapy.

Current trends in the role of neuroinflammation & α-synuclein in alcohol use disorder: A systematic quantitative literature review.

The neurodegenerative effects alcohol use disorder (AUD) have been well characterized and are likely due to the long-term effects of alcohol on the brain. The molecular events that underlie regional neuronal loss are a focus of current research. Chronic inflammation in the central nervous system, termed neuroinflammation, contributes to the progressive loss of neurons in the brain. Using data from genome-wide association studies and genetic and gene expression data, α-synuclein was identified as a gene of interest for AUD almost 10 years ago. Despite this and the well-recognized role of α-synuclein in mediating neuroinflammation in other neurodegenerative diseases, its role in alcohol-induced brain damage and AUD is yet to be elucidated. This systematic literature review quantifies and analyzes relationships between AUD, α-synuclein, and neuroinflammation. The review identified fewer studies focused on the role in AUD of α-synuclein (30) than on neuroinflammation (177), with published studies heavily centered on the myeloid differentiation primary response 88 (MyD88)-dependent toll-like receptor 4 (TLR4) pathway. The systematic review revealed that no original literature investigates the roles of α-synuclein and neuroinflammation in AUD and that there are significantly fewer published articles on the role of α-synuclein in AUD than in other neuroinflammatory conditions. Studies of the role of neuroinflammation in AUD are largely centered on the TLR4 signaling cascade, followed by TLR2 and TLR3, and soluble cytokines such as IL-10, IL-1β, and TNF-α. Key research themes identified in other neurodegenerative disorders provide new insights for further investigation in AUD.

Specimen type validation and establishment of normal cytokine reference intervals in cerebrospinal fluid

Cerebrospinal fluid (CSF) is a critical body fluid to examine in attempts to discover potential biomarkers for neuroinflammatory and other disorders of the central nervous system (CNS). Serum and/or plasma cytokine levels have been associated with a variety of inflammatory conditions, and some have been shown to be actionable therapeutic targets. Less is known, however, about cytokine levels in CSF. Serum and plasma cytokine testing is widely available in clinical and research laboratories, but cytokine testing in CSF is extremely limited and if performed, accompanied by a disclaimer that it is an unvalidated specimen type. In this study, we validate CSF as a suitable specimen type and determine normal reference intervals for multiple cytokines as well as a soluble cytokine receptor. CSF was validated as a specimen type for testing using a laboratory developed multiplexed cytokine assay previously validated to measure 13 cytokines/markers in serum and plasma. Performance parameters including specimen dilution, specimen interference, linearity and precision were examined. Reference intervals were established using 197 normal and control CSF specimens by non-parametric quantile-based methods. CSF cytokine analysis demonstrated within and between run precision of <10% and < 20% CV, respectively and linearity of ±15% for all analytes throughout the analytical measurement range of the assay. Reference intervals for the 13 cytokines/markers were established from 197 normal and control CSF specimens (78 Male; mean 44.8 y ± 21.7 SD, 119 Female; mean 42.8 y ± 20.3 SD). Cytokine concentrations in CSF from normal donors and controls were less than the lower limit of quantitation of our assay for 6 of the 13 measured cytokines/markers. The chemokine IL8 demonstrated the highest concentration of all analytes measured. CSF demonstrated acceptable performance as a specimen type in our multiplexed cytokine assay. By validating CSF as a specimen type and establishing normal reference intervals for cytokine concentrations in CSF, their potential as biomarkers for infectious, autoimmune and other inflammatory CNS disorders can be more appropriately investigated.

Prognostic and diagnostic effects of high serum midkine levels in patients with hepatocellular carcinoma.

Midkine (MK) is a soluble cytokine, and its serum levels strongly correspond to protein expression levels in tumors. The present study aimed to clarify the clinicopathological and prognostic significance of serum MK (s-MK) in patients with hepatocellular carcinoma (HCC). Serum samples were obtained before surgery from 123 patients with HCC who had undergone surgery between January 2012 and December 2020. The receiver operating characteristic curve revealed that the best cut-off value for s-MK in differentiating HCC from healthy cases was 426 pg/ml. The clinicopathological variables and overall survival of patients were compared between the s-MK-positive group and s-MK-negative group. The sensitivity, specificity and accuracy of s-MK were 82.1, 97.4 and 88.0%, respectively. An s-MK-positive status was significantly associated with the number of tumors (≥2). The positivity rate of s-MK was significantly higher compared with that of α-fetoprotein and protein-induced by vitamin K absence-II. In total, only 28% of the patients were positive for s-MK. The s-MK-positive group showed significantly worse overall survival compared with the s-MK-negative group. Moreover, multivariate analysis revealed that an s-MK-positive status was independently associated with poor prognosis. s-MK was useful in detecting early HCC. The findings of this study indicated that the s-MK-positive status is associated with the number of tumors and can act as an independent prognostic risk factor.

Peptoid-Cross-Linked Hydrogel Stiffness Modulates Human Mesenchymal Stromal Cell Immunoregulatory Potential in the Presence of Interferon-Gamma.

Human mesenchymal stromal cell (hMSC) manufacturing requires the production of large numbers of therapeutically potent cells. Licensing with soluble cytokines improves hMSC therapeutic potency by enhancing secretion of immunoactive factors but typically decreases proliferative ability. Soft hydrogels, however, have shown promise for boosting immunomodulatory potential, which may compensate for decreased proliferation. Here, hydrogels are cross-linked with peptoids of different secondary structures to generate substrates of various bulk stiffnesses but fixed network connectivity. Secretions of interleukin 6, monocyte chemoattractive protein-1, macrophage colony-stimulating factor, and vascular endothelial growth factor are shown to depend on hydrogel stiffness in the presence of interferon gamma (IFN-γ) supplementation, with soft substrates further improving secretion. The immunological function of these secreted cytokines is then investigated via coculture of hMSCs seeded on hydrogels with primary peripheral blood mononuclear cells (PBMCs) in the presence and absence of IFN-γ. Cocultures with hMSCs seeded on softer hydrogels show decreased PBMC proliferation with IFN-γ. To probe possible signaling pathways, immunofluorescent studies probe the nuclear factor kappa B pathway and demonstrate that IFN-γ supplementation and softer hydrogel mechanics lead to higher activation of this pathway. Overall, these studies may allow for production of more efficacious therapeutic hMSCs in the presence of IFN-γ.

Stem Cell Secretions as a Potential Therapeutic Agent for Autism Spectrum Disorder: A Narrative Review.

Autism spectrum disorder (ASD) is a neurodevelopmental illness characterized by impaired social interaction and restricted repetitive behaviors or interests. The rising prevalence of ASD diagnosis has triggered a surge in research into investigating the underlying neuropathological processes and finding new therapeutic approaches. ASD is characterized by neuroinflammation and dysregulation of neuro-immune cross-talk, which suggests that stem cell treatment might be a potential therapeutic approach. The beneficial and restorative effects of stem cells are mainly due to their paracrine activity, in which stem cells generate and release extracellular vesicles such as exosomes and distinct secreted non-vesicle soluble proteins, including, growth factors, chemokines, cytokines, and immunomodulatory molecules referred to as the Secretome. In this paper, we reviewed the existing research exploring the therapeutic potential of stem cell secretome focusing on their role in addressing ASD pathology. Furthermore, we proposed a comprehensive mechanism of action for stem cell secretions, encompassing the broader secretome as well as the specific contribution of exosomes, in alleviating ASD neuropathology. Across the reviewed studies, exosomes and secreted soluble factors of the transplanted stem cell demonstrate a potential efficacy in ameliorating autistic-like behaviors. The proposed mechanism of action involves the modulation of signaling pathways implicated in neuroinflammation, angiogenesis, cellular apoptosis, and immunomodulation.

Abstract P46: Small Extracellular Vesicles from Therapy-induced Senescent TNBC Elicit Paracrine Anti-tumour Effects

Abstract Triple-negative breast cancers (TNBC), associated with poor prognosis and high tumour recurrence, are often treated with taxanes. However, cells may bypass treatment-induced cell death via mitotic slippage, resulting in multinucleated polyploid cells and therapy-induced senescence (TIS). TIS cancer cells represent a population of residual disease and may modulate therapy resistance and recurrence through secretion of the senescence-associated secretory phenotype (SASP). While SASP have been heavily linked with pro-tumorigenic behaviour mediated through soluble cytokines, small extracellular vesicles (sEVs) derived from TIS cancer cells represent an underappreciated aspect of the senescent tumour secretome, as its mechanistic role in mediating paracrine effects remains poorly understood. In this study, we uncovered an unexpected role for TIS-derived sEVs within the secretome, which conferred paracrine anti-tumour activity in recipient TNBC cancer cells, in contrast to SASP that support pro-tumorigenic behaviour. SEVs profiling through mass-spectrometry revealed that DKK1, a negative regulator of WNT signalling, is enriched in TIS-derived sEVs. Further investigation into the tumour microenvironment with animal models and neoadjuvant chemo-treated patient samples revealed STING-dependent immune response. Hence, our study suggests that sEVs should be considered as distinct SASP entities within the senescent tumour secretome and provide an additional basis for combinatorial use of senomorphics with taxanes to potentially modulate senescence-associated sEVs while suppressing pro-tumorigenic SASP factors. Citation Format: Matius Robert, Rekha Jakhar, Bijin Veonice Au, Gracie Wee Ling Eng, Alvin Kunyao Gao, You Heng Chuah, Karishma Sachaphibulkij, Lina Hsiu Kim Lim, Derrick Sek Tong Ong, Elaine Hsuen Lim, Yoko Itahana, Koji Itahana, Jit Kong Cheong, John Edward Connolly, Karen Carmelina Crasta. Small Extracellular Vesicles from Therapy-induced Senescent TNBC Elicit Paracrine Anti-tumour Effects [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P46.

Calcium phosphate ceramic-induced osteoimmunomodulation: Submicron-surface-treated macrophage-derived exosomes driving osteogenesis

The development of osteoimmunology has highlighted the crucial role of biomaterial surface topography in modulating macrophage responses, subsequently affecting bone formation. However, the underlying mechanism remains largely unexplored. This study introduced two biphasic calcium phosphate (BCP) ceramics with different surface architecture, finding that compared to micron-scale counterparts (BCP2, ∼3.07 μm), BCP with submicron-scale structure (BCP1, ∼0.66 μm) enhanced ectopic bone formation and facilitated M2 macrophage polarization in vivo. Previous studies have focused predominantly on soluble cytokines secreted by macrophages, the significance of exosomes in intercellular communication has been overlooked. This study isolated exosomes from macrophages treated with either submicron- or micron-scale surfaces, designated as B1-Exos and B2-Exos, respectively. Notably, B1-Exos could significantly improve osteogenic differentiation of mesenchymal stem cells (MSCs), potentially attributable to miRNA cargos within these exosomes. SmallRNA sequencing, western blotting, qRT-PCR, and bioinformatics analyses indicated that submicron-sized BCP1 ceramic markedly elevated miR-142a-5p levels in macrophage-derived exosomes, activating the PTEN/AKT signaling pathway, and consequently guiding the differentiation of MSCs towards osteoblast lineage. These findings not only deepen our understanding of the biomaterial-mediated osteoinduction mechanism, but also inform the design of bone repair materials with specialized surface design for proper immunomodulation to initiate osteogenesis.

Abstract 5229: Enhancing solid tumor treatment with engineered blood-derived Vδ1 platform: Safe and effective targeting of mesothelin-expressing tumors with Vδ1 γδT cells

Abstract Mesothelin (MSLN) is a solid tumor-associated antigen that has been thoroughly studied and is expressed in several tumor indications that have significant unmet therapeutic needs. Vδ1-enriched γδT cells differentiate themselves from other MSLN-targeted modalities by mitigating the challenges of targeting tumors with heterogeneous MSLN expression through their inherent capability to detect malignant cells while sparing normal cells. The ability to effectively combat cancer cells that express mesothelin is significantly increased by the expression of a chimeric antigen receptor (CAR). Novel armoring is being evaluated to improve engineered Vδ1 persistence in the solid tumor microenvironment. Cytokine armoring, through novel engineering, enhances sensitivity to cytokines and recovers cell fitness that is impaired by 'traditional' cytokine armoring options. Extensive in vitro and in vivo safety and efficacy studies were carried out to validate the product's potency. These studies aimed to determine cytotoxicity, expansion, and persistence. MSLN CAR cytokine-armored Vδ1 cells exhibited high post-thaw viability and survival. Furthermore, cytokine-armored Vδ1 cells displayed elevated innate receptor expression and gene-engineered cells maintained their baseline innate phenotype, indicating that they can maintain their ‘innate’ effectiveness over time. In vitro studies demonstrated broad anti-tumor activity against multiple tumor cell lines expressing various levels of mesothelin. Additionally, in long-term cytotoxicity assays, cytokine-armored cells demonstrated improved tumor-clearance capabilities. The effector cells also exhibited an enhanced innate cytotoxic phenotype that equips the cells with the capability to persist and proliferate in response to soluble cytokines and upon cytokine trans-presentation by adjacent cells. In vitro studies further revealed that there is a distinction in the killing activity between tumor and normal cells, which is important for ensuring that the product is safe for use in patients. An orthotopic lung tumor-bearing mouse model was developed to confirm their anti-tumor effectiveness in vivo. The model's outcomes demonstrated that MSLN CAR cytokine-armored Vδ1 cells exhibit higher in vivo efficacy in comparison to their unarmored counterparts at low effector-to-target ratios. With no significant healthy tissue damage found, the histopathology report confirmed an excellent safety profile. Terminal biodistribution studies demonstrated that armored drug candidates preferentially home to and persist in tumor-bearing tissues. Overall, these findings imply that the product might be highly effective in treating tumors that express mesothelin. This strategy could greatly advance our knowledge of how Vδ1-enriched γδT cells function as a platform against solid tumors. Citation Format: Nandhu Mohan Sobhana, Andre Simoes, Sarah Edwards, Sweta Mishra, Gary Gu, George Papadopoulos, Stefan Kiesgen, Rebecca Alade, njing Li, Andrea Venuso, Jyothi Kumaran, Kristy Waddington, Tabassum Huseni, Rajeev Karattil, Lilly Reynolds, Maria Sharif, David Wotherspoon, Corina Tudor, Istvan Kovacs, Mei Rosa Ng, Yana Wang, Lan Cao. Enhancing solid tumor treatment with engineered blood-derived Vδ1 platform: Safe and effective targeting of mesothelin-expressing tumors with Vδ1 γδT cells [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 5229.

Small extracellular vesicles in plasma carry luminal cytokines that remain undetectable by antibody-based assays in cancer patients and healthy donors

BackgroundSmall (30–150 nm) extracellular vesicles (sEV), also known as exosomes, play a key role in cell-to-cell signaling. They are produced by all cells, circulate freely and are present in all body fluids. Evidence indicates that cytokines are present on the surface and/or in the lumen of sEV. The contribution of intravesicular cytokines to cytokine levels in plasma are unknown.MethodssEV were isolated by ultrafiltration/size exclusion chromatography from pre-cleared plasma obtained from patients with head and neck squamous cell carcinoma (HNSCC) and healthy donors (HDs). Multiplex immunoassays were used to measure cytokine levels in paired untreated and detergent-treated (0.5% Triton X-100) plasma and plasma-derived detergent-treated sEV. Non-parametric tests were used to assess differences in cytokine levels.ResultsThe presence of cytokines in sEV isolated from patients’ and HDs’ plasma was confirmed by immunoblots and on-bead flow cytometry. sEV-associated cytokines were functional in various in vitro assays. Levels of cytokines in sEV varied among the HNSCC patients and were generally significantly higher than the levels observed in sEV from HDs. Compared to untreated plasma, levels for the majority (40/51) of the evaluated proteins were significantly higher in detergent-treated plasma (P < 0.0001–0.03). In addition, levels of 24/51 proteins in sEV, including IL6, TNFRII, IL-17a, IFNa and IFNg, were significantly positively correlated with the difference between levels detected in detergent-treated plasma and untreated plasma.DiscussionThe data indicate that sEV-associated cytokines account for the differences in cytokine levels measured in detergent-treated versus untreated plasma. Ab-based assays using untreated plasma detect only soluble cytokines and miss cytokines carried in the lumen of sEV. Permeabilization of sEV with a mild detergent allows for Ab-based detection of sEV-associated and soluble cytokines in plasma. The failure to detect cytokines carried in the sEV lumen leads to inaccurate estimates of cytokine levels in body fluids.A schema illustrating the presence of cytokines on the surface or in the lumen of small extracellular vesicles (sEV).

Preclinical Characterization of the Anti-Leukemia Activity of the CD33/CD16a/NKG2D Immune-Modulating TriNKET® CC-96191.

Increasing efforts are focusing on natural killer (NK) cell immunotherapies for AML. Here, we characterized CC-96191, a novel CD33/CD16a/NKG2D immune-modulating TriNKET®. CC-96191 simultaneously binds CD33, NKG2D, and CD16a, with NKG2D and CD16a co-engagement increasing the avidity for, and activation of, NK cells. CC-96191 was broadly active against human leukemia cells in a strictly CD33-dependent manner, with maximal efficacy requiring the co-engagement of CD16a and NKG2D. A frequent CD33 single nucleotide polymorphism, R69G, reduced CC-96191 potency but not maximal activity, likely because of reduced CD33 binding. Similarly, the potency, but not the maximal activity, of CC-96191 was reduced by high concentrations of soluble CD33; in contrast, the soluble form of the NKG2D ligand MICA did not impact activity. In the presence of CD33+ AML cells, CC-96191 activated NK cells but not T cells; while maximum anti-AML efficacy was similar, soluble cytokine levels were 10- to >100-fold lower than with a CD33/CD3 bispecific antibody. While CC-96191-mediated cytolysis was not affected by ABC transporter proteins, it was reduced by anti-apoptotic BCL-2 family proteins. Finally, in patient marrow specimens, CC-96191 eliminated AML cells but not normal monocytes, suggesting selectivity of TriNKET-induced cytotoxicity toward neoplastic cells. Together, these findings support the clinical exploration of CC-96191 as in NCT04789655.

Soluble receptors in cancer: mechanisms, clinical significance, and therapeutic strategies.

Soluble receptors are soluble forms of receptors found in the extracellular space. They have emerged as pivotal regulators of cellular signaling and disease pathogenesis. This review emphasizes their significance in cancer as diagnostic/prognostic markers and potential therapeutic targets. We provide an overview of the mechanisms by which soluble receptors are generated along with their functions. By exploring their involvement in cancer progression, metastasis, and immune evasion, we highlight the importance of soluble receptors, particularly soluble cytokine receptors and immune checkpoints, in the tumor microenvironment. Although current research has illustrated the emerging clinical relevance of soluble receptors, their therapeutic applications remain underexplored. As the landscape of cancer treatment evolves, understanding and targeting soluble receptors might pave the way for novel strategies for cancer diagnosis, prognosis, and therapy.

Aspergillus fumigatus antigen-reactive Th17 cells are enriched in bronchoalveolar lavage fluid in severe equine asthma.

Equine asthma (EA) is a common disease of adult horses with chronic respiratory pathology and common neutrophilic airway inflammation. It presents with hyperreactivity to hay dust components such as molds, and underlying dysregulated T cell responses have been suggested. Thus far, T cells have been analysed in EA with conflicting results and the antigen reactivity of T cells has not been demonstrated. Serological and epidemiological data point to the relevance of Aspergillus fumigatus as an antigen source in EA. Here, we aimed to identify and characterise Aspergillus antigen-reactive T cells in EA. Cryopreserved bronchoalveolar lavage cells (BALC) and peripheral blood mononuclear cells (PBMC) from healthy horses (HE, n=9) and those with mild-moderate (MEA, n=3) or severe asthma (SEA, n=8) were stimulated in vitro with the recombinant A. fumigatus antigens Asp f 1, or Asp f 7 combined with Asp f 8, to assess antigen reactivity, and with phorbol-12-myristat-13-acetate and ionomycin (P/i) to assess overall T cell reactivity. Stimulated cells were analysed by flow cytometry for CD4, CD8, IL-17, IL-4, and IFN-γ. Cytokine expression in all lymphocytes, and in CD4+ or CD8+ T cells, was quantified and compared between the groups. In BAL fluid (BALF), soluble cytokines and chemokines were quantified by bead-based assays. Antigen restimulation of BALC with Asp f 1 or Asp f 7/8 provoked higher frequencies of IL-17+ lymphocytes, CD4+IL-17+ Th17 cells, and CD4+IL-4+ Th2 cells in SEA than in HE, whereas MEA and HE were similar. Antigen stimulation of PBMC did not result in group differences. P/i stimulation of BALC resulted in increased IL-17+ lymphocyte and CD4+IL-17+ Th17 cell frequencies in MEA compared with HE but the limited number of horses with MEA must be considered. P/i-stimulated PBMC from MEA or SEA contained more IL-17+ lymphocytes compared with HE. Cytokines were hardly detected in BALF and similar between the groups but CCL2 and CCL5 concentrations were increased in BALF from SEA or MEA, respectively, compared with HE. Horses with SEA have increased Aspergillus antigen-reactive Th17 cells in their airways, emphasising local T cell responses to this mold, which were quantified in EA for the first time here.

Suppression of inflammation by Baccharis punctulata and Baccharis trimera through modulation of innate and adaptive immune responses

Inflammatory processes, involving both innate and adaptive responses, are essential for controlling pathogens and maintaining homeostasis. However, excessive inflammation can cause chronic conditions including autoimmune diseases, atherosclerosis, and cancer. Alternative treatment options for inflammation are crucial, and exploring plants with anti-inflammatory properties holds significance. The genus Baccharis (Asteraceae family) is widespread across the Americas and is traditionally used to treat various disorders, including inflammatory diseases. Several biological activities of Baccharis species have been described; however, their immunomodulatory effects have not been widely evaluated. In this study, we analyzed the immunomodulatory activity of two Baccharis species, Baccharis punctulata, and Baccharis trimera, using an in vitro inflammation model involving monocytic cells and splenocytes to evaluate both innate and adaptive immune responses. Both B. punctulata and B. trimera reduced lipopolysaccharide (LPS)-induced inflammatory mediators, including soluble CD14 and pro-inflammatory cytokines, in THP-1 cells. Furthermore, they suppressed nitric oxide (NO) production in splenocytes, demonstrating a dampened innate immune response. In addition, both species attenuated concanavalin A (ConA)-induced splenocyte proliferation, suggesting an anti-inflammatory effect on the adaptive immune response. In summary, the extracts demonstrated significant anti-inflammatory activity, affecting both innate and adaptive immune responses, and underscoring their potential as treatments for inflammatory diseases and sources of anti-inflammatory molecules.