Cytokine Secretion Patterns Research Articles

Type 2-like polarization and elevated CXCL4 secretion of monocyte derived macrophages upon internalization of plasma-derived exosomes from head and neck cancer patients

BackgroundExosomes are closely associated with different aspects of tumor-progression in patients with head and neck squamous cell carcinoma (HNSCC), such as angiogenesis or immune regulation. As extracellular vesicles they are involved in the intercellular communication by transferring their cargo such as proteins and nucleic acids from one cell to another. However, the influence of tumor related plasma-derived exosomes on the polarization and characteristics of monocyte derived macrophages is not fully understood.MethodsExosomes were isolated from plasma samples of healthy donors (HD) and HNSCC patients and further evaluated with regard to morphology, size and protein composition via transmission electron microscopy, nanoparticle tracking, western blot analysis and cytokine assays. Differentiation and characteristics of monocyte derived macrophages upon exosome internalization were analyzed using flow cytometry and fluorescence microscopy. Macrophage cytokine secretion patterns were analyzed by human cytokine antibody arrays and ELISA measurements.ResultsOur data revealed elevated overall plasma levels of CTLA-4, PD-L1, and TIM-3 as well as elevated exosome-associated CTLA-4, PD-L2, TIM-3, and LAG-3 levels in HNSCC patients compared to HD. Furthermore, we observed a significant type 2-like polarization and elevated CXCL4 secretion of monocyte derived macrophages upon internalization of plasma-derived exosomes from HNSCC patients, which could be visualized by fluorescence microcopy of membrane stained exosomes.ConclusionsThe study provides new insights regarding exosome driven pro-tumorigenic immune regulation in the circulation of patients with head and neck cancer and could help to better understand the individual immunologic situation.

The persistence of low CD4/CD8 ratio in chronic HIV-infection, despite ART suppression and normal CD4 levels, is associated with pre-therapy values of inflammation and thymic function

BackgroundPersistence of a low CD4/CD8 ratio is associated with an increased morbimortality in people living with HIV (PLWH) under effective antiretroviral therapy. We aimed to explore the immunological significance of a persistently low CD4/CD8 ratio, even despite normal CD4 levels, and assess whether these features vary from those associated to a low nadir-CD4, another well-established predictor of disease progression. MethodsCD4-recovered PLWH were classified by CD4/CD8 ratio after three-years of ART (viral suppression, CD4≥500; R < 0.8, n = 24 and R > 1.2, n = 28). sj/β-TRECs ratio and inflammatory-related markers were quantified. PBMCs were immunophenotyped by CyTOF and functionally characterized by ELISPOT. Subjects were also reclassified depending on nadir-CD4 (N ≤ 350/N > 350). ResultsR < 0.8 showed a differential inflammatory profile compared to R > 1.2 (increased β2-microglobulin, D-dimers and IP-10 before ART). R < 0.8 presented lower baseline thymic function, being inversely correlated with post-ART inflammation. R < 0.8 at follow-up showed most alterations in CD8 subsets (increasing frequency and exhibiting a senescent phenotype [e.g., CD57+, CD95+]) and enhanced T-cell IFNγ/IL-2 secretion. However, comparing N ≤ 350 to N > 350, the main features were altered functional markers in CD4 T-cells, despite no differences in maturational subsets, together with a restricted T-cell cytokine secretion pattern. ConclusionPersistence of low CD4/CD8 ratio in successfully-treated PLWH, with normal CD4 counts, is associated with baseline inflammation and low thymic function, and it features post-therapy alterations specific to CD8 T-cells. Differently, subjects recovered from low nadir-CD4 in this setting feature post-therapy alterations on CD4 T-cells. Hence, different mechanisms of disease progression could underlie these biomarkers, potentially requiring different clinical approaches.

Purified granulocytes in extracorporeal cell therapy: A multifaceted approach to combat sepsis-induced immunoparalysis.

Immune cell dysfunction plays a central role in sepsis-induced immunoparalysis. Targeted treatment using healthy donor immune cell transfusions, particularly granulocyte concentrates (GC) potentially induces tissue damage. Initial trials using GC in an extracorporeal immune cell perfusion system provided evidence for beneficial effects with fewer side effects, by separating patient and donor immune cell compartments. A multicenter clinical trial is exploring feasibility and effects of a 6-h treatment (NCT06143137). This ex vivo study examines technical feasibility and cellular effects of an extended treatment interval up to 24 h. Standard GC were purified to increase the potential storage time and subsequently implemented in the extracorporeal immune cell perfusion system. Parameters assessed included cell viability, phagocytosis activity, oxidative burst, cytokine release, and metabolic parameters of purified. GC during an extended circulation time of up to 24 h. After storage of 72 h granulocytes were viable throughout the study period and exhibited preserved functionality and metabolic activity. The findings highlight a time-dependent nature of cytokine release by neutrophils in the extracorporeal circuit, as cytokine secretion patterns showed IL-8 peaking within 6 h, while MCP-1, IL-6, IL-1β, and TNF-α increased after 24 h of circulation. Purified GC remain functional after 72 h of storage and additional 24 h in the circulating treatment model. Cytokine secretion patterns revealed a significant increase, especially between 10 and 24 h of treatment. Extending treatment time holds promise for enhancing immune response against sepsis-induced immunoparalysis. These findings provide valuable insights for optimizing immune-targeted therapeutic interventions.

Constitutive Flt3 signaling impacts conventional dendritic cell function.

The development of dendritic cells (DCs) depends on signaling via the FMS-like tyrosine kinase 3 (Flt3) receptor. How Flt3 signaling impacts terminally differentiated DC function is unknown. This is important given the increasing interest in exploiting Flt3 for vaccination and tumor immunotherapy. Here, we examined DCs in mice harboring constitutively activated Flt3 (Flt3-ITD). Flt3ITD/ITD mice possessed expanded splenic DC subsets including plasmacytoid DC, conventional DC (cDC)1, cDC2, double positive (DP) cDC1 (CD11c+ CD8+ CD11b- CD103+ CD86+), noncanonical (NC) cDC1 (CD11c+ CD8+ CD11b- CD103- CD86-) and single positive (SP) cDC1 (CD11c+ CD8+ CD11b- CD103- CD86+). Outcomes of constitutive Flt3 signaling differed depending on the cDC subset examined. In comparison with wild type (WT) DCs, all Flt3ITD/ITD cDCs displayed an altered surface phenotype with changes in costimulatory molecules, major histocompatibility complex class I (MHC I) and II (MHC II). Cytokine secretion patterns, antigen uptake, antigen proteolysis and antigen presenting function differed between WT and Flt3ITD/ITD subsets, particularly cDC2. In summary, Flt3 signaling impacts the function of terminally differentiated cDCs with important consequences for antigen presentation.

Abstract 14: Identification of tumor-reactive T cell receptors through functional single cell interaction analyses for personalized T cell therapy

Abstract Innovative immunotherapy approaches such as adoptive transfer of chimeric antigen receptor (CAR) T cells or tumor infiltrating lymphocytes (TILs) have shown great success in the treatment of solid tumors and hematological malignancies. Although treatment of multiple myeloma with CAR T cells can induce deep responses, relapses frequently occur due to antigen escape and limited CAR T cell persistence. TCR-engineered T cells may show prolonged persistence in vivo and could mediate sustained antitumor effects. A further benefit of TCR transgenic T cells is the ability to target intracellular antigens that are inaccessible to CAR T cells, expanding the range of potential targets for immunotherapy. In our project, we propose to identify T cell receptors (TCRs) specifically targeting autologous myeloma cells. Tumor-reactive T cells were identified using the Bruker Cellular Analysis Lightning® platform, allowing simultaneous functional analysis of up to 1500 individual T cell/target cell interactions on a chip. Reactive T cells were identified upon detection of secreted cytokines (IFNγ, TNFα, IL2) and measurement of 4-1BB (CD137) surface expression. Tumor-reactive T cells showing various cytokine secretion patterns and 4-1BB expression profiles were detected in each myeloma patient (on average 11.9 T cells out of 1243 cells tested per assay run). Individual tumor-reactive T cells have been isolated and their TCRs were sequenced. TCR sequences of tumor-reactive T cells were mapped to single-cell RNA sequencing data of T cells from the same patiens to reveal a gene expression signature of myeloma-reactive T cells. TCR genes of reactive T cells were cloned and overexpressed in autologous T cells for functional validation and analysis of tumor derived neoepitope specificity. In summary, we present a pipeline allowing identification of myeloma-recognizing T cells and recovery of bona fide tumor-reactive TCRs eligible for patient-individualized T cell therapy. Citation Format: Tim Robin Wagner, Niklas Kehl, Simon Steiger, Michael Kilian, Bruno Schönfelder, Tamara Boschert, Katharina Lindner, Patrick Schmidt, Karsten Rippe, Hartmut Goldschmidt, Marc-Steffen Raab, Michael Platten, Mirco Friedrich, Stefan B. Eichmüller. Identification of tumor-reactive T cell receptors through functional single cell interaction analyses for personalized T cell therapy [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 14.

Hydrogenated Soy Phosphatidylcholine Liposomes Stimulate Differential Expression of Chemokines And Cytokines by Rat Alveolar Macrophages In Vitro

Liposomes are being developed as inhalable drug delivery systems, but concerns remain about their impact on the lungs. To better understand the impact of liposomes and their physicochemical properties on alveolar macrophages, the cytokine and chemokine expression profile of rat alveolar Nr8383 macrophages exposed to 0.1 and 1 mg/ml hydrogenated soy phosphatidylcholine (HSPC) liposomes was examined. Expression patterns varied considerably between liposomes in a concentration-dependent manner, with both anti- and pro-inflammatory chemokines/cytokines produced. Uncharged liposomes induce the greatest production of cytokines and chemokines, followed by PEGylated liposomes. The most significant increase in cytokine/chemokine expression was seen for IL-2 (up to 24-fold), IL-4 (up to 5-fold), IL-18 and VEGF (up to 10-fold), while liposome exposure significantly reduced MIP1 expression (5-fold). In summary, we demonstrate that liposome surface properties promote variable patterns of cytokine and chemokine secretion by alveolar macrophages. This suggests that the type of liposome employed may influence the type of immune response generated in the lung and by extension, dictate how inhaled liposomal nanomedicines affect the lungs response to inhaled toxicants and local infections.

1,8-Cineol Attenuates Checkpoint Molecule PDL-1 and Adhesion Molecule CX3CR1 in Circulating Monocytes in Otitis Media Patients.

Peripheral blood monocytes can be subdivided into different subsets based on the CD14/CD16 surface characteristics. Monocytes are a major source of cytokine secretion of pro-inflammatory immune responses, whereas CD16+ monocyte subsets can also contribute to persistent inflammation in the context of chronic diseases. However, the regulation and cellular characteristics of circulating monocyte subsets in patients with chronic otitis media (COM), one of the largest public health burdens, remains largely unknown. In this study, we analyzed individual distributions of circulating monocyte subsets and associated protein expression levels of adhesion protein and chemokine receptors CD11a (integrin-α L; LFA-1), CD11b (integrin-α M; Mac-1), and CD11c (integrin-α X), CX3CR1 (CX3CL1 receptor), as well as checkpoint molecule PD-L1 (programmed cell death ligand-1), in a gender-balanced cohort of 14 patients with chronic otitis media using flow cytometry, especially in view of the therapeutic impact of the natural plant-derived monoterpene oxide 1,8-Cineol. Furthermore, using the human monocyte cell line THP-1 as a model, we investigated the influence of anti-inflammatory 1,8-Cineol on monocytic cytokine secretion patterns using human cytokine arrays and ELISA measurements. The data revealed significantly elevated expression levels of all analyzed adhesion molecules in certain monocyte subsets in COM patients; CX3CR1 was especially significantly down-regulated in response to 1,8-Cineol administration. Moreover, the data revealed significantly increased monocytic PD-L1 expression levels in circulating classical and intermediate monocyte subsets from COM patients compared to healthy donors, but also a significant decrease in PD-L1 in intermediate monocytes upon 1,8-Cineol therapy compared to the pre-treatment situation. Furthermore, the increased secretion of cytokine CXCL10 by THP-1 monocytes in response to LPS was found to be strongly attenuated by 1,8-Cineol. Plasma levels of CXCL10 were also significantly increased in COM patients, but no significant differences between the pre and post 1,8-Cineol situation were observed. The present study revealed new insights into the bioactive anti-inflammatory effects of 1,8-Cineol in terms of monocyte adhesion and immune regulation. Our data suggest the potential role of cytokine CXCL10 in COM development and maintenance, which is also involved in the activity of its concomitant disease, rheumatoid arthritis.

Cathepsin D promotes polarization of tumor-associated macrophages and metastasis through TGFBI-CCL20 signaling

M2-like tumor-associated macrophages (TAMs) are risk factors for cancer progression and metastasis. However, the mechanisms underlying their polarization are still not fully understood. Although cathepsin D (Cat D) has been reported as a procarcinogenic factor, little is known about the functional role of Cat D in the tumor microenvironment (TME). This study aimed to explore the effect and molecular mechanisms of Cat D in the TME. Cat D knockout (KO) altered the cytokine secretion pattern and induced TAM reprogramming from the M2 to M1 subtype, thereby preventing epithelial-mesenchymal transition and tumor metastasis. Mechanistically, we identified transforming growth factor beta-induced protein (TGFBI) as a Cat D target protein that is specifically associated with TAM polarization. Elevated TGFBI expression in Cat D KO cancer cells resulted in a decline in M2-like TAM polarization. Our RNA-sequencing results indicated that the cancer cell-secreted chemokine CCL20 is a major secretory chemokine for Cat D-TGFBI-mediated TAM polarization. In contrast, Cat D overexpression accelerated TAM polarization into M2-like cells by suppressing TGFBI expression. In addition, the double Cat D and TGFBI KO rescued the inhibitory effects of Cat D KO on tumor metastasis by controlling TAM and T-cell activation. These findings indicated that Cat D contributes to cancer metastasis through TGFBI-mediated TAM reprogramming. Cat D deletion inhibits M2-like TAM polarization through TGFBI-mediated CCL20 expression, reprogramming the immunosuppressive TME. Our results open a potential new avenue for therapy focused on eliminating tumor metastasis.

Distinguishing the impact of distinct obstructive sleep apnea syndrome (OSAS) and obesity related factors on human monocyte subsets

Obstructive sleep apnea syndrome (OSAS) and obesity go hand in hand in the majority of patients and both are associated with a systemic inflammation, immune disturbance and comorbidities such as cardiovascular disease. However, the unambiguous impact of OSAS and obesity on the individual inflammatory microenvironment and the immunological consequences of human monocytes has not been distinguished yet. Therefore, aim of this study was to investigate the impact of OSAS and obesity related factors on the inflammatory microenvironment by performing flow cytometric whole blood measurements of CD14/CD16 monocyte subsets in normal weight OSAS patients, patients with obesity but without OSAS, and patients with OSAS and obesity, compared to healthy donors. Moreover, explicitly OSAS and obesity related plasma levels of inflammatory mediators adiponectin, leptin, lipocalin and metalloproteinase-9 were determined and the influence of different OSAS and obesity related factors on cytokine secretion and expression of different adhesion molecules by THP-1 monocytes was analysed. Our data revealed a significant redistribution of circulating classical and intermediate monocytes in all three patient cohorts, but differential effects in terms of monocytic adhesion molecules CD11a, CD11b, CD11c, CX3CR1, CD29, CD49d, and plasma cytokine levels. These data were reflected by differential effects of OSAS and obesity related factors leptin, TNFα and hypoxia on THP-1 cytokine secretion patterns and expression of adhesion molecules CD11b and CD49d. In summary, our data revealed differential effects of OSAS and obesity, which underlines the need for a customized therapeutic regimen with respect to the individual weighting of these overlapping diseases.

Alteration of Immunoregulatory Patterns and Survival Advantage of Key Cell Types in Food Allergic Children.

All allergic responses to food indicate the failure of immunological tolerance, but it is unclear why cow's milk and egg (CME) allergies resolve more readily than reactivity to peanuts (PN). We sought to identify differences between PN and CME allergies through constitutive immune status and responses to cognate and non-cognate food antigens. Children with confirmed allergy to CME (n = 6) and PN (n = 18) and non-allergic (NA) (n = 8) controls were studied. Constitutive secretion of cytokines was tested in plasma and unstimulated mononuclear cell (PBMNC) cultures. Blood dendritic cell (DC) subsets were analyzed alongside changes in phenotypes and soluble molecules in allergen-stimulated MNC cultures with or without cytokine neutralization. We observed that in allergic children, constitutively high plasma levels IL-1β, IL-2, IL-4, IL-5 and IL-10 but less IL-12p70 than in non-allergic children was accompanied by the spontaneous secretion of sCD23, IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12p70, IFN-γ and TNF-α in MNC cultures. Furthermore, blood DC subset counts differed in food allergy. Antigen-presenting cell phenotypic abnormalities were accompanied by higher B and T cell percentages with more Bcl-2 within CD69+ subsets. Cells were generally refractory to antigenic stimulation in vitro, but IL-4 neutralization led to CD152 downregulation by CD4+ T cells from PN allergic children responding to PN allergens. Canonical discriminant analyses segregated non-allergic and allergic children by their cytokine secretion patterns, revealing differences and areas of overlap between PN and CME allergies. Despite an absence of recent allergen exposure, indication of in vivo activation, in vitro responses independent of challenging antigen and the presence of unusual costimulatory molecules suggest dysregulated immunity in food allergy. Most importantly, higher Bcl-2 content within key effector cells implies survival advantage with the potential to mount abnormal responses that may give rise to the manifestations of allergy. Here, we put forward the hypothesis that the lack of apoptosis of key immune cell types might be central to the development of food allergic reactions.

Modulation of NLRP3 Inflammasome in Monocytes By HT-6184 in a Single-Cell Proteomic Study

Background The NLRP3 inflammasome is associated with the activation of monocytes and plays a crucial role in the innate immune response. The NLRP3 inflammasome is a multi-protein complex expressed primarily in monocytes and other immune cells. When monocytes encounter danger signals, such as pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), the NLRP3 inflammasome is assembled, leading to the maturation and secretion of potent pro-inflammatory cytokines, interleukin-1β (IL-1β), and interleukin-18 (IL-18). These cytokines, in turn, induce a robust inflammatory response to clear infections, promote tissue repair, and coordinate adaptive immunity. However, dysregulated NLRP3 inflammasome activation has been associated with various inflammatory disorders, such as autoimmune diseases, metabolic diseases, and neurodegenerative conditions. Purpose This study investigated the effects of a potent, allosteric NLRP3 inflammasome modulator, HT-6184, on cytokine release response from the activation of NLRP3 in human monocytes. Methods Human peripheral blood mononuclear cells (PBMCs) were utilized, and monocytes were isolated using a Miltenyi pan monocyte isolation kit. Subsequently, these isolated monocytes were exposed to different concentrations of HT-6184 (300 nM and 100 nM) before being stimulated with lipopolysaccharide (LPS) at a concentration of 10 ng/mL, a potent activator of the NLRP3 inflammasome. After 24 hours, the secreted cytokines were analyzed at the single-cell level using the Isospark, with data analysis performed using the Isospeak software. Results The results demonstrated that monocytes treated with LPS alone exhibited high levels of pro-inflammatory cytokines, including effector and stimulatory cytokines, and chemoattractive chemokines such as IL-8, MIF, MIP-1b, and TNF-alpha. In contrast, monocytes treated with HT-6184 and LPS showed reduced cytokine secretion, approaching levels observed in the control group. Notably, the HT- 6184-treated group displayed a lower incidence of polyfunctionality (2%) than the LPS-only group (11%), indicating fewer cells capable of simultaneously secreting multiple cytokines. The 3D-TSNE graph analysis revealed that the HT-6184 and LPS-treated groups clustered with the control group, signifying similar cytokine secretion profiles. In contrast, the LPS-only group exhibited a distinct pattern of cytokine secretion. Conclusions These findings indicate that HT-6184 effectively inhibits the release of pro-inflammatory cytokines upon NLRP3 activation in monocytes. Further research should explore the characteristics of HT-6184 and its ability to modulate the inflammasome in diverse in vitro and in vivo models. The modulation of the NLRP3 inflammasome by HT-6184 holds potential therapeutic implications for managing inflammatory disorders associated with dysregulated NLRP3 activation.

Assessing Radiosensitivity: Effects of Acute Ionizing Radiation on Inflammation and Apoptosis in Macrophage Cell Line (RAW 264.7).

Introduction: The responses of biological systems to various types of radiation have multifaceted dimensions. In the field of ionizing radiation, in vitro external gamma radiation therapy has primarily been studied as a model to elucidate the challenges that biological systems face from radiation effects. Exposure of cells/organisms to gamma radiation results in a cascade of ionization events that can cause severe and irreversible biological damage. However, the biological responses and oxidative stress-related mechanisms under acute radiation conditions remain poorly understood in inflammatory systems. The present study aimed to provide a model of the effect of ionizing radiation on macrophages, which play a pivotal role in the mechanisms of inflammation, to assess the impact of radiotherapy as an approach to treating inflammatory diseases. Methods: A macrophage cell line (RAW 264.7) was cultured and exposed to different doses of gamma radiation (4, 6, 8, 10 Gy). Cell viability, apoptosis, cell cycle, migration, nitric oxide (NO) and prostaglandin E2 (PGE2) production, expression of pro-inflammatory and apoptotic genes, and cytokine secretion of macrophages were also evaluated. Results: The results showed that gamma radiation at 4 Gy had a low effect on macrophage characteristics and cytokine secretion patterns. In contrast, higher doses (8 and 10 Gy) increased DNA damage, expression of apoptotic genes, and secretion of NO and PGE2 cytokines. 6 Gy radiation, the maximum radiation dose, showed moderate non-destructive effects and inflammation process modulation. In this study, doses higher than 6 Gy of Gamma radiation caused cell mortality. Conclusion: It appears that 6 Gy of gamma radiation modulates the inflammatory cascade caused by macrophage cells.

Umbilical Cord Blood-Derived Monocytes as A Reliable Source of Functional Macrophages for Biomedical Research.

Macrophages are multifunctional immune cells widely used in immunological research. While autologous macrophages have been widely used in several biomedical applications, allogeneic macrophages have also demonstrated similar or even superior therapeutic potential. The umbilical cord blood (UCB) is a well-described source of abundant allogenic monocytes and macrophages that is easy to collect and can be processed without invasive methods. Current monocyte isolation procedures frequently result in heterogenous cell products, with limited yields, activated cells, and high cost. This study outlines a simple isolation method that results in high yields and pure monocytes with the potential to differentiate into functional macrophages. In the experimental study, we describe a simple and efficient protocol to isolate highpurity monocytes. After collection of human UCB samples, we used a gradient-based procedure composed of three consecutive gradient steps: i. Hydroxyethyl starch-based erythrocytes sedimentation, followed by ii. Mononuclear cells (MNCs) isolation by Ficoll-Hypaque gradient, and iii. Separation of monocytes from lymphocytes by a slight hyperosmolar Percoll gradient (0.573 g/ml). Then the differentiation potential of isolated monocytes to pro- and antiinflammatory macrophages were evaluated in the presence of granulocyte colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF), respectively. The macrophages were functionally characterized as well. A high yield of monocytes after isolation (25 to 50 million) with a high purity (>95%) could be obtained from every 100-150 ml UCB. Isolated monocytes were defined based on their phenotype and surface markers expression pattern. Moreover, they possess the ability to differentiate into pro- or anti-inflammatory macrophages with specific phenotypes, gene/surface protein markers, cytokine secretion patterns, T-cell interactions, and phagocytosis activity. Here we describe a simple and reproducible procedure for isolation of pure monocytes from UCB, which could be utilized to provide functional macrophages as a reliable and feasible source of allogenic macrophages for biomedical research.

Nasal IL-13 production identifies patients with late-phase allergic responses

There is limited knowledge on how local cytokine secretion patterns after nasal allergen challenge correlate with clinical symptoms especially with regard to the "late allergic response," which occurs in approximately 40% to 50% of patients with allergy. We sought to characterize the immunologic and clinical nasal responses to birch pollen allergen challenge with a special focus on the late allergic response. In this randomized, double-blind, placebo-controlled trial, birch pollen-allergic participants were challenged with birch pollen extract (n= 20) or placebo (n= 10) on 3 consecutive days. On days 1 and 3, nasal secretions were collected at selected time points over a 24-hour time course for the measurement of 33 inflammatory mediators. Clinical responses were determined through subjective symptom scores and objective nasal airflow measurements. Provoked participants had significantly greater clinical responses and showed significant increases in tryptase and the soluble IL-33 receptor serum stimulation 2 (sST2) in nasal secretions within minutes compared with the placebo group. Eight of 20 provoked participants displayed high IL-13 levels 2 to 8 hours after allergen provocation. This group also showed significant changes in clinical parameters, with a secondary drop in nasal airflow measured by peak nasal inspiratory flow and increased symptoms of nasal obstruction, which significantly differed from IL-13 nonresponders after 6 hours. IL-13 response status correlates with clinical responses and type 2 cytokine responses in the late phase after allergen provocation.

Immune and inflammatory responses of human macrophages, dendritic cells, and T-cells in presence of micro- and nanoplastic of different types and sizes

Environmental pollution by microplastics (MPs) is a growing concern regarding their impact on aquatic and terrestrial systems and human health. Typical exposure routes of MPs are dermal contact, digestion, and inhalation. Recent in vitro and in vivo studies observed alterations in immunity after MPs exposure, but systemic studies using primary human immune cells are scarce. In our investigation, we addressed the effect of polystyrene (PS) and poly methyl methacrylate (PMMA) in three different sizes (50–1100 nm) as well as amino-modified PS (PS-NH2; 50 nm) on cells of the adaptive and innate immune system. T-cells isolated from human peripheral blood mononuclear cells (PBMCs) were least affected regarding the cytotoxicity but displayed increased activation marker expression after 72 h, and strongly modulated cytokine secretion patterns. Conversely, phagocytic dendritic cells and macrophages derived from isolated monocytes were highly sensitive to pristine MPs. Their marker expression suggested a downregulation of the inflammatory phenotypes indicative of M2 macrophage induction after MPs exposure for 24 h. Our results showed that even pristine MPs affected immune cell function and inflammatory phenotype dependent on MPs polymers, size, and immune cell type.

Boost of innate immunity cytokines as biomarkers of response to extracorporeal photopheresis in patients with leukaemic cutaneous T-cell lymphoma.

Extracorporeal photopheresis (ECP) has emerged as a systemic first-line immunomodulatory therapy in leukaemic cutaneous T-cell lymphoma (L-CTCL) and is now beginning to be utilized in other T-cell-mediated diseases. Although ECP has been used for nearly 30 years, its mechanisms of action are not sufficiently understood, and biomarkers for response are scarce. We aimed to investigate the immunomodulatory effects of ECP on cytokine secretion patterns in patients with L-CTCL, to help elucidate its mechanism of action. A total of 25 patients with L-CTCL and 15 healthy donors (HDs) were enrolled in this retrospective cohort study. Concentrations of 22 cytokines were simultaneously quantified by using multiplex bead-based immunoassays. Neoplastic cells in patients' blood were evaluated by flow cytometry. Firstly, we observed a distinct cytokine profile pattern difference between L-CTCLs and HDs. There was a significant loss of tumour necrosis factor (TNF)-α, and significant increase of interleukins (IL)-9, IL-12 and IL-13 in the sera of patients with L-CTCL compared with HDs. Secondly, patients with L-CTCL who received ECP were classified as treatment responders and nonresponders according to the quantitative reduction of malignant burden in their blood. We evaluated cytokine levels in culture supernatants from patients' peripheral blood mononuclear cells (PBMCs) at baseline and 27 weeks after ECP initiation. Strikingly, PBMCs purified from ECP responders released statistically higher concentrations of innate immune cytokines IL-1α, IL-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF) and TNF-α in comparison with ECP nonresponders. In parallel, responders showed clearance of erythema, reduction of malignant clonal T cells in the blood, and a potent boost of relevant innate immune cytokines in individual patients with L-CTCL. Taken together, our results demonstrate that ECP stimulates the innate immune network, and facilitates redirection of the tumour-biased immunosuppressive microenvironment towards proactive antitumour immune responses. The alterations of IL-1α, IL-1β, GM-CSF and TNF-α can be used as biomarkers of response to ECP in patients with L-CTCL.

Maintenance of chronicity signatures in fibroblasts isolated from recessive dystrophic epidermolysis bullosa chronic wound dressings under culture conditions

BackgroundRecessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare inherited skin disease caused by variants in the COL7A1 gene, coding for type VII collagen (C7), an important component of anchoring fibrils in the basement membrane of the epidermis. RDEB patients suffer from skin fragility starting with blister formation and evolving into chronic wounds, inflammation and skin fibrosis, with a high risk of developing aggressive skin carcinomas. Restricted therapeutic options are limited by the lack of in vitro models of defective wound healing in RDEB patients.ResultsIn order to explore a more efficient, non-invasive in vitro model for RDEB studies, we obtained patient fibroblasts derived from discarded dressings) and examined their phenotypic features compared with fibroblasts derived from non-injured skin of RDEB and healthy-donor skin biopsies. Our results demonstrate that fibroblasts derived from RDEB chronic wounds (RDEB-CW) displayed characteristics of senescent cells, increased myofibroblast differentiation, and augmented levels of TGF-β1 signaling components compared to fibroblasts derived from RDEB acute wounds and unaffected RDEB skin as well as skin from healthy-donors. Furthermore, RDEB-CW fibroblasts exhibited an increased pattern of inflammatory cytokine secretion (IL-1β and IL-6) when compared with RDEB and control fibroblasts. Interestingly, these aberrant patterns were found specifically in RDEB-CW fibroblasts independent of the culturing method, since fibroblasts obtained from dressing of acute wounds displayed a phenotype more similar to fibroblasts obtained from RDEB normal skin biopsies.ConclusionsOur results show that in vitro cultured RDEB-CW fibroblasts maintain distinctive cellular and molecular characteristics resembling the inflammatory and fibrotic microenvironment observed in RDEB patients’ chronic wounds. This work describes a novel, non-invasive and painless strategy to obtain human fibroblasts chronically subjected to an inflammatory and fibrotic environment, supporting their use as an accessible model for in vitro studies of RDEB wound healing pathogenesis. As such, this approach is well suited to testing new therapeutic strategies under controlled laboratory conditions.

Secondary infection of Fasciola gigantica in buffaloes shows a similar pattern of serum cytokine secretion as in primary infection.

As a natural host of Fasciola gigantica, buffalo is widely infected by F. gigantica. Its impact on buffalo production has caused great losses to the husbandry sector, and repeat infection is non-negligible. In buffaloes experimentally infected with F. gigantica, primary and secondary infection have yielded the same rate of fluke recovery, indicating a high susceptibility of buffalo to F. gigantica, which contributes to the high infection rate. Determining the immunological mechanism of susceptibility will deepen the understanding of the interaction between F. gigantica and buffalo. Here, we explored the immune response of buffaloes against primary and secondary F. gigantica infection, with a focus on cytokines' dynamics explored through serum cytokine detection. Buffaloes were assigned to three groups: group A (noninfected, n = 4), group B (primary infection, n = 3), and group C (secondary infection, n = 3). Group B was infected via oral gavage with 250 viable F. gigantica metacercariae, and group C was infected twice with 250 metacercariae at an interval of 4 weeks. The second infection of group C was performed simultaneously with that of group B. Whole blood samples were collected pre-infection (0 weeks) and at 1-6, 10, and 12 weeks after that. The serum levels of seven cytokines (IFN-γ, IL-4, IL-5, IL-10, IL-13, TGF-β, and IL-17) were simultaneously determined using ELISA and further analyzed. In the present study, no significant changes in Th1-type cytokines production were detected in early infection, both in primary and secondary infections, while the Th2-type response was strongly induced. A comparison of primary and secondary infection showed no significant difference in the cytokine secretion, which may indicate that the re-infection at 4 weeks after primary infection could not induce a robust adaptive immune response. The full extent of interaction between buffalo and F. gigantica in re-infection requires further study.

Thelper1 and T helper2 cytokine production patterns in stressed beta2-adrenergic receptor-deficient mice during chlamydia genital infection.

Murine CD4+ T cells have two distinct cytokine secretion patterns to play different functions. We have demonstrated that cold-induced stress (CIS) suppresses the immune system leading to increased intensity of Chlamydia muridaum in mice. We have reported that beta2-adrenergic receptor (b2-AR) knockout (KO) mice resist chlamydia genital infection. However, the cytokine profile of CD4+ T cells is not well explored. This study aimed to determine the cytokine production of Th1 and Th2 types in stressed and non-stressed b2-AR KO mice. We investigated the cytokine production levels of stressed and non-stressed mice during C. muridarum genital infection. Significantly increased production of cytokines was observed in plates pre-coated with anti-CD3/CD28 or in the presence of Con A or LPS. A decrease in the production of IFN-g and IL-2, whereas an increase in the secretion of IL-10, IL-13, and IL-23 in CD4+ T cells of stressed wild-type mice was obtained; however, the secretion of IFN-g and IL-2 was restored in T cells of b2-AR KO mice. Moreover, in vitro proliferation of CD4 T cells in the presence of b2-AR antagonists, ICI 118, 551 stimulated the production of Th1 cytokines, whereas b2-AR agonist, Fenoterol, decreased the production of Th1-type cytokines. Overall, Th1 cytokine responses are reduced in stressed mice suggesting that the cytokine status was polarized toward a Th2 immune response that can be restored by removing b2-AR from immune cells.

Abstract 4071: Identification of myeloma-specific T cell receptors by functional single cell interaction analyses

Abstract Innovative immunotherapy approaches such as adoptive transfer of chimeric antigen receptor (CAR) T cells or tumor infiltrating lymphocytes (TILs) have shown great success in the treatment of solid tumors and hematological malignancies. Although treatment of multiple myeloma with CAR T cells can induce deep responses, relapses frequently occur due to antigen escape and limited CAR T cell persistence. TCR-engineered T cells may mediate sustained antitumor effects upon recognition of intracellular targets, thereby significantly increasing the range of relevant target antigens. In our project, we propose to identify T cell receptors (TCRs) specifically targeting autologous myeloma cells. Tumor-reactive T cells were identified using the Berkeley Lights Lightning platform, allowing simultaneous functional analysis of up to 1500 individual T cell/target cell interactions per run. Reactive T cells have been identified upon detection of secreted chemokines (IFNγ, TNFα, IL-2) and by measurement of CD137 surface expression. Tumor-reactive T cells showing various cytokine secretion patterns and CD137 expression profiles could be detected in each myeloma patient (7 to 26 of approx.1200 cells tested per patient). Individual tumor reactive T cells have been isolated for TCR sequencing. Recovered TCR genes will be cloned and overexpressed in autologous T cells for functional validation and analysis of tumor derived neoepitope specificity. In summary, we present a pipeline allowing identification of myeloma-recognizing T cells and recovery of bona fide tumor-reactive TCRs eligible for patient-individualized T cell therapy. Citation Format: Tim R. Wagner, Eren Boğa, Patrick Schmidt, Wolfram Osen, Michael Platten, Hartmut Goldschmidt, Marc S. Raab, Mirco J. Friedrich, Stefan B. Eichmüller. Identification of myeloma-specific T cell receptors by functional single cell interaction analyses. [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 4071.