Soluble Chemokines Research Articles

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.

Targeting immune cell recruitment in atherosclerosis.

Atherosclerosis is the primary underlying cause of myocardial infarction and stroke. Atherosclerotic cardiovascular disease is characterized by a chronic inflammatory reaction in medium-to-large-sized arteries, with its onset and perpetuation driven by leukocytes infiltrating the subendothelial space. Activation of endothelial cells triggered by hyperlipidaemia and lipoprotein retention in the arterial intima initiates the accumulation of pro-inflammatory leukocytes in the arterial wall, fostering the progression of atherosclerosis. This inflammatory response is coordinated by an array of soluble mediators, namely cytokines and chemokines, that amplify inflammation both locally and systemically and are complemented by tissue-specific molecules that regulate the homing, adhesion and transmigration of leukocytes. Despite abundant evidence from mouse models, only a few therapies targeting leukocytes in atherosclerosis have been assessed in humans. The major challenges for the clinical translation of these therapies include the lack of tissue specificity and insufficient selectivity of inhibition strategies. In this Review, we discuss the latest research on receptor-ligand pairs and interactors that regulate leukocyte influx into the inflamed artery wall, primarily focusing on studies that used pharmacological interventions. We also discuss mechanisms that promote the resolution of inflammation and highlight how major findings from these research areas hold promise as potential therapeutic strategies for atherosclerotic cardiovascular disease.

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.

Immunological signatures from irradiated cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) are abundant and influential elements of the tumor microenvironment (TME), giving support to tumor development in multiple ways. Among other mechanisms, CAFs are important regulators of immunological processes occurring in tumors. However, CAF-mediated tumor immunomodulation in the context of radiotherapy remains poorly understood. In this study, we explore effects of radiation on CAF-derived immunoregulatory signals to the TME. Primary CAF cultures were established from freshly collected human NSCLC lung tumors. CAFs were exposed to single-high or fractionated radiation regimens (1x18Gy or 3x6Gy), and the expression of different immunoregulatory cell-associated and secreted signaling molecules was analyzed 48h and 6 days after initiation of treatment. Analyses included quantitative measurements of released damage-associated molecular patterns (DAMPs), interferon (IFN) type I responses, expression of immune regulatory receptors, and secretion of soluble cytokines, chemokines, and growth factors. CAFs are able to survive ablative radiation regimens, however they enter into a stage of premature cell senescence. Our data show that CAFs avoid apoptosis and do not contribute by release of DAMPs or IFN-I secretion to radiation-mediated tumor immunoregulation. Furthermore, the secretion of relevant immunoregulatory cytokines and growth factors including TGF-β, IL-6, IL-10, TNFα, IL-1β, VEGF, CXCL12, and CXCL10 remain comparable between non-irradiated and radiation-induced senescent CAFs. Importantly, radiation exposure modifies the cell surface expression of some key immunoregulatory receptors, including upregulation of CD73 and CD276. Our data suggest that CAFs do not participate in the release of danger signals or IFN-I secretion following radiotherapy. The immune phenotype of CAFs and radiation-induced senescent CAFs is similar, however, the observed elevation of some cell surface immunological receptors on irradiated CAFs could contribute to the establishment of an enhanced immunosuppressive TME after radiotherapy.

IMMU-48. SOLUBLE CIRCULATING IMMUNE CHECKPOINT MOLECULES IN MALIGNANT BRAIN TUMORS

Abstract Immune checkpoint therapy has been shown to be effective in several types of cancer, and tumor-directed immune activation is also a promising strategy in glioblastoma (GBM) patients. Our goal was to discover alterations in the circulating immunome of GBM patients and other cerebral tumor entities to identify soluble immune biomarkers and novel therapeutic targets. We analyzed 30 markers in plasma of 208 patients and healthy donors, using bead-based multiplex assays and flow cytometry. In a smaller cohort we further applied a high-sensitivity discovery biomarker platform with 368 markers. We included patients with 1) primary GBM (n=68), 2) recurrent GBM (n=25), 3) cerebral metastases (NSCLC, n=61; breast cancer, n=19), 4) meningioma (n=15) as well as 5) healthy donors (HD, n=20). We detected a distinct immune signature in the peripheral blood of patients with GBM, which was defined by an increased prevalence of soluble immune checkpoint markers (sCD27, sPD-L2, sGal-9), whereas soluble chemokines (CX3CL, CXCL12) were underrepresented. This signature was similar in primary and recurrent GBM, but differed markedly from other cerebral tumor entities as well as from HD. Primary GBM, but not their recurrent counterparts, additionally exhibited increased levels of neurotrophic factors (BDNF, b-NGF) and signs of immune activation with significantly increased levels of IL-18. The biomarker discovery platform further identified 76 proteins that were specifically regulated in GBM compared to HD, offering the potential to serve as biomarkers for disease activity. GBM patients with higher levels of BDNF displayed a significantly shorter survival than patients with lower levels (9.33 vs. 21.90 months; p=0.0001). Likewise patients with high concentrations of VEGF survived significantly shorter than those with low concentrations (12.97 vs. 21.90 months, p=0,0166). Taken together, we were able to delineate a specific immune profile of soluble markers in GBM patients compared to other cerebral tumor entities and to healthy individuals.

Mannose-Decorated Co-Polymer Facilitates Controlled Release of Butyrate to Accelerate Chronic Wound Healing.

Butyrate is a key bacterial metabolite that plays an important and complex role in modulation of immunity and maintenance of epithelial barriers. Its translation to clinic is limited by poor bioavailability, pungent smell, and the need for high doses, and effective delivery strategies have yet to realize clinical potential. Here, a novel polymeric delivery platform for tunable and sustainable release of butyrate consisting of a methacrylamide backbone with butyryl ester or phenyl ester side chains as well as mannosyl side chains, which is also applicable to other therapeutically relevant metabolites is reported. This platform's utility in the treatment of non-healing diabetic wounds is explored. This butyrate-containing material modulated immune cell activation in vitro and induced striking changes in the milieu of soluble cytokine and chemokine signals present within the diabetic wound microenvironment in vivo. This novel therapy shows efficacy in the treatment of non-healing wounds through the modulation of the soluble signals present within the wound, and importantly accommodates the critical temporal regulation associated with the wound healing process. Currently, the few therapies to address non-healing wounds demonstrate limited efficacy. This novel platform is positioned to address this large unmet clinical need and improve the closure of otherwise non-healing wounds.

Investigating the tumor-immune microenvironment through extracellular vesicles from frozen patient biopsies and 3D cultures.

Melanomas are highly immunogenic tumors that have been shown to activate the immune response. Nonetheless, a significant portion of melanoma cases are either unresponsive to immunotherapy or relapsed due to acquired resistance. During melanomagenesis, melanoma and immune cells undergo immunomodulatory mechanisms that aid in immune resistance and evasion. The crosstalk within melanoma microenvironment is facilitated through the secretion of soluble factors, growth factors, cytokines, and chemokines. In addition, the release and uptake of secretory vesicles known as extracellular vesicles (EVs) play a key role in shaping the tumor microenvironment (TME). Melanoma-derived EVs have been implicated in immune suppression and escape, promoting tumor progression. In the context of cancer patients, EVs are usually isolated from biofluids such as serum, urine, and saliva. Nonetheless, this approach neglects the fact that biofluid-derived EVs reflect not only the tumor, but also include contributions from different organs and cell types. For that, isolating EVs from tissue samples allows for studying different cell populations resident at the tumor site, such as tumor-infiltrating lymphocytes and their secreted EVs, which play a central anti-tumor role. Herein, we outline the first instance of a method for EV isolation from frozen tissue samples at high purity and sensitivity that can be easily reproduced without the need for complicated isolation methods. Our method of processing the tissue not only circumvents the need for hard-to-acquire freshly isolated tissue samples, but also preserves EV surface proteins which allows for multiplex surface markers profiling. Tissue-derived EVs provide insight into the physiological role of EVs enrichment at tumor sites, which can be overlooked when studying circulating EVs coming from different sources. Tissue-derived EVs could be further characterized in terms of their genomics and proteomics to identify possible mechanisms for regulating the TME. Additionally, identified markers could be correlated to overall patient survival and disease progression for prognostic purposes.

Curtailing virus-induced inflammation in respiratory infections: emerging strategies for therapeutic interventions.

Acute respiratory viral infections (ARVI) are the most common illnesses worldwide. In some instances, mild cases of ARVI progress to hyperinflammatory responses, which are damaging to pulmonary tissue and requiring intensive care. Here we summarize available information on preclinical and clinical effects of XC221GI (1-[2-(1-methyl imidazole-4-yl)-ethyl]perhydroazin-2,6-dione), an oral drug with a favorable safety profile that has been tested in animal models of influenza, respiratory syncytial virus, highly pathogenic coronavirus strains and other acute viral upper respiratory infections. XC221GI is capable of controlling IFN-gamma-driven inflammation as it is evident from the suppression of the production of soluble cytokines and chemokines, including IL-6, IL-8, CXCL10, CXCL9 and CXCL11 as well as a decrease in migration of neutrophils into the pulmonary tissue. An excellent safety profile of XC221GI, which is not metabolized by the liver, and its significant anti-inflammatory effects indicate utility of this compound in abating conversion of ambulatory cases of respiratory infections into the cases with aggravated presentation that require hospitalization. This drug is especially useful when rapid molecular assays determining viral species are impractical, or when direct antiviral drugs are not available. Moreover, XC221GI may be combined with direct antiviral drugs to enhance their therapeutic effects.

Pathogenesis of flares in liver inflammation in hepatitis B virus-infected women during and after pregnancy

Abstract Women with chronic hepatitis B (CHB) are at risk of increased liver inflammation during pregnancy. To investigate the pathogenesis of these hepatic flares we studied immune responses and gut microbiota in 19 women with CHB (12 without and 7 with antiviral (NUC) treatment) and 3 uninfected controls in each trimester and at two and six months postpartum. Increased serum alanine aminotransferase (ALT) activity as a liver inflammation marker was defined as a value greater than the upper limit of normal and twice the baseline value. While increased plasma levels of soluble CD163, inflammatory cytokines and chemokines generally occurred during episodes of liver inflammation, increases of IL-22 and IFN-γ levels during pregnancy preceded a postpartum ALT increase. In contrast, levels of IL-27, a cytokine produced by the trophoblast, increased also in women with normal ALT levels. The fecal microbiota of HBV-infected patients with and without NUC treatment had lower α-diversity (inverse Simpson index) than those of uninfected controls. 16srRNA amplicon analysis and PCA showed patient-specific clustering of microbiota. To identify factors predictive of a postpartum ALT increase, we compared 16srRNA DNA data from patients with a postpartum ALT increase to those with normal postpartum ALT levels (four patients per group). Combining all time points, the gut microbiota of those with postpartum ALT increase differed significantly from those with normal ALT activities (UMAP, PERMANOVA p<0.003). In conclusion, the composition of the fecal microbiota was patient-rather than time point-specific and differed between patients with and without postpartum ALT increase. Supported by NIH Bench-to-Bedside Award (B.R. and D.T.Y.L.)

Macrophages and small extracellular vesicle mediated-intracellular communication in the peritoneal microenvironment: Impact on endometriosis development.

Endometriosis is an inflammatory disease that is defined as the growth of endometrium-like tissue outside the uterus, commonly on the lining of the pelvic cavity, visceral organs and in the ovaries. It affects around 190 million women of reproductive age worldwide and is associated with chronic pelvic pain and infertility, which greatly impairs health-related life quality. The symptoms of the disease are variable, this combined with a lack of diagnostic biomarkers and necessity of surgical visualisation to confirm disease, the prognosis can take an average timespan of 6-8 years. Accurate non-invasive diagnostic tests and the identification of effective therapeutic targets are essential for disease management. To achieve this, one of the priorities is to define the underlying pathophysiological mechanisms that contribute to endometriosis. Recently, immune dysregulation in the peritoneal cavity has been linked to endometriosis progression. Macrophages account for over 50% of immune cells in the peritoneal fluid and are critical for lesion growth, angiogenesis, innervation and immune regulation. Apart from the secretion of soluble factors like cytokines and chemokines, macrophages can communicate with other cells and prime disease microenvironments, such as the tumour microenvironment, via the secretion of small extracellular vesicles (sEVs). The sEV-mediated intracellular communication pathways between macrophages and other cells within the peritoneal microenvironment in endometriosis remain unclear. Here, we give an overview of peritoneal macrophage (pMΦ) phenotypes in endometriosis and discuss the role of sEVs in the intracellular communication within disease microenvironments and the impact they may have on endometriosis progression.

Staphylococcus aureus Infection Induces the Production of the Neutrophil Chemoattractants CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 by Murine Osteoblasts.

Staphylococcus aureus is the principal causative agent of osteomyelitis, a serious bacterial infection of bone that is associated with progressive inflammatory damage. Bone-forming osteoblasts have increasingly been recognized to play an important role in the initiation and progression of detrimental inflammation at sites of infection and have been demonstrated to release an array of inflammatory mediators and factors that promote osteoclastogenesis and leukocyte recruitment following bacterial challenge. In the present study, we describe elevated bone tissue levels of the potent neutrophil-attracting chemokines CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 in a murine model of posttraumatic staphylococcal osteomyelitis. RNA sequencing (RNA-Seq) gene ontology analysis of isolated primary murine osteoblasts showed enrichment in differentially expressed genes involved in cell migration and chemokine receptor binding and chemokine activity following S. aureus infection, and a rapid increase in the expression of mRNA encoding CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7, in these cells. Importantly, we have confirmed that such upregulated gene expression results in protein production with the demonstration that S. aureus challenge elicits the rapid and robust release of these chemokines by osteoblasts and does so in a bacterial dose-dependent manner. Furthermore, we have confirmed the ability of soluble osteoblast-derived chemokines to elicit the migration of a neutrophil-like cell line. As such, these studies demonstrate the robust production of CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 by osteoblasts in response to S. aureus infection, and the release of such neutrophil-attracting chemokines provides an additional mechanism by which osteoblasts could drive the inflammatory bone loss associated with staphylococcal osteomyelitis.

Different cytokine and chemokine profiles in hospitalized patients with COVID-19 during the first and second outbreaks from Argentina show no association with clinical comorbidities.

COVID-19 severity has been linked to an increased production of inflammatory mediators called "cytokine storm". Available data is mainly restricted to the first international outbreak and reports highly variable results. This study compares demographic and clinical features of patients with COVID-19 from Córdoba, Argentina, during the first two waves of the pandemic and analyzes association between comorbidities and disease outcome with the "cytokine storm", offering added value to the field. We investigated serum concentration of thirteen soluble mediators, including cytokines and chemokines, in hospitalized patients with moderate and severe COVID-19, without previous rheumatic and autoimmune diseases, from the central region of Argentina during the first and second infection waves. Samples from healthy controls were also assayed. Clinical and biochemical parameters were collected. Comparison between the two first COVID-19 waves in Argentina highlighted that patients recruited during the second wave were younger and showed less concurrent comorbidities than those from the first outbreak. We also recognized particularities in the signatures of systemic cytokines and chemokines in patients from both infection waves. We determined that concurrent pre-existing comorbidities did not have contribution to serum concentration of systemic cytokines and chemokines in COVID-19 patients. We also identified immunological and biochemical parameters associated to inflammation which can be used as prognostic markers. Thus, IL-6 concentration, C reactive protein level and platelet count allowed to discriminate between death and discharge in patients hospitalized with severe COVID-19 only during the first but not the second wave. Our data provide information that deepens our understanding of COVID-19 pathogenesis linking demographic features of a COVID-19 cohort with cytokines and chemokines systemic concentration, presence of comorbidities and different disease outcomes. Altogether, our findings provide information not only at local level by delineating inflammatory/anti-inflammatory response of patients but also at international level addressing the impact of comorbidities and the infection wave in the variability of cytokine and chemokine production upon SARS-CoV-2 infection.

Hepatic stellate cell-released CXCL1 aggravates HCC malignant behaviors through the MIR4435-2HG/miR-506-3p/TGFB1 axis.

Hepatic stellate cell (HSC) activation is a critical event in the development of hepatic fibrosis and hepatocellular carcinoma (HCC). By the release of soluble cytokines, chemokines, and chemotaxis, HSCs affect HCC cell phenotypes through a complex tumor microenvironment. In this study, weighted gene co-expression network analysis (WGCNA) was used to identify the TGF-β signaling pathway as a key signaling pathway in Hep3B cells cultured in HSC conditioned medium. MIR4435-2HG is a hub lncRNA associated with the TGF-β signaling pathway and HSC activation. HSC-condition medium (CM) culture induced HCC cell malignant behaviors, which were partially reversed by MIR4435-2HG silencing. miR-506-3p directly bound to MIR4435-2HG and the 3'UTR of TGFB1. Similarly, overexpression of miR-506-3p also attenuated HSC-CM-induced malignant behavior of HCC cells. In HSC-CM cultured HCC cells, the effects of MIR4435-2HG knockdown on TGFB1 expression and HCC cell phenotypes were partially reversed by miR-506-3p inhibition. HSCs affected HCC cell phenotypes by releasing CXCL1. In an orthotopic xenotransplanted tumor model of HCC cells plus HSCs in mice, CXCR2 knockdown in HCC cells significantly inhibited tumorigenesis, which was partially reversed by MIR4435-2HG overexpression in HCC cells. In HCC tissue samples, the levels of CXCL1, TGF-β1, and MIR4435-2HG were upregulated, while miR-506-3p expression was downregulated. In conclusion, HSC-released CXCL1 aggravated HCC cell malignant behaviors through the MIR4435-2HG/miR-506-3p/TGFB1 axis. In addition to CXCL1, the MIR4435-2HG/miR-506-3p/TGFB1 axis might also be the underlying target for HCC therapy.

Circulating immune markers and risks of non-Hodgkin lymphoma subtypes: A pooled analysis.

Although prediagnostic circulating concentrations of the immune activation markers soluble CD27 (sCD27), sCD30 and chemokine ligand-13 (CXCL13) have been associated with non-Hodgkin lymphoma (NHL) risk, studies have been limited by sample size in associations with NHL subtypes. We pooled data from eight nested case-control studies to investigate subtype-specific relationships for these analytes. Using polytomous regression, we calculated odds ratios (ORs) with 95% confidence intervals (CIs) relating study-specific analyte tertiles to selected subtypes vs controls (n=3310): chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; n=623), diffuse large B cell lymphoma (DLBCL; n=621), follicular lymphoma (FL; n=398), marginal zone lymphoma (MZL; n=138), mantle cell lymphoma (MCL; n=82) and T cell lymphoma (TCL; n=92). We observed associations with DLBCL for elevated sCD27 [OR for third vs first tertile (ORT3 )=2.2, 95% CI=1.6-3.1], sCD30 (ORT3 = 2.0, 95% CI=1.6-2.5) and CXCL13 (ORT3 = 2.3, 95% CI=1.8-3.0). We also observed associations with sCD27 for CLL/SLL (ORT3 = 3.3, 95% CI=2.4-4.6), MZL (ORT3 = 7.7, 95% CI=3.0-20.1) and TCL (ORT3 = 3.4, 95% CI=1.5-7.7), and between sCD30 and FL (ORT3 = 2.7, 95% CI=2.0-3.5). In analyses stratified by time from phlebotomy to case diagnosis, the sCD27-TCL and all three DLBCL associations were equivalent across both follow-up periods (<7.5, ≥7.5 years). For other analyte-subtype comparisons, associations were stronger for the follow-up period closer to phlebotomy, particularly for indolent subtypes. In conclusion, we found robust evidence of an association between these immune markers and DLBCL, consistent with hypotheses that mechanisms related to immune activation are important in its pathogenesis. Our other findings, particularly for the rarer subtypes MZL and TCL, require further investigation.

Role of Innate and Adaptive Cytokines in the Survival of COVID-19 Patients

SARS-CoV-2 is a new coronavirus characterized by a high infection and transmission capacity. A significant number of patients develop inadequate immune responses that produce massive releases of cytokines that compromise their survival. Soluble factors are clinically and pathologically relevant in COVID-19 survival but remain only partially characterized. The objective of this work was to simultaneously study 62 circulating soluble factors, including innate and adaptive cytokines and their soluble receptors, chemokines and growth and wound-healing/repair factors, in severe COVID-19 patients who survived compared to those with fatal outcomes. Serum samples were obtained from 286 COVID-19 patients and 40 healthy controls. The 62 circulating soluble factors were quantified using a Luminex Milliplex assay. Results. The patients who survived had decreased levels of the following 30 soluble factors of the 62 studied compared to those with fatal outcomes, therefore, these decreases were observed for cytokines and receptors predominantly produced by the innate immune system—IL-1α, IL-1α, IL-18, IL-15, IL-12p40, IL-6, IL-27, IL-1Ra, IL-1RI, IL-1RII, TNFα, TGFα, IL-10, sRAGE, sTNF-RI and sTNF-RII—for the chemokines IL-8, IP-10, MCP-1, MCP-3, MIG and fractalkine; for the growth factors M-CSF and the soluble receptor sIL2Ra; for the cytokines involved in the adaptive immune system IFNγ, IL-17 and sIL-4R; and for the wound-repair factor FGF2. On the other hand, the patients who survived had elevated levels of the soluble factors TNFβ, sCD40L, MDC, RANTES, G-CSF, GM-CSF, EGF, PDGFAA and PDGFABBB compared to those who died. Conclusions. Increases in the circulating levels of the sCD40L cytokine; MDC and RANTES chemokines; the G-CSF and GM-CSF growth factors, EGF, PDGFAA and PDGFABBB; and tissue-repair factors are strongly associated with survival. By contrast, large increases in IL-15, IL-6, IL-18, IL-27 and IL-10; the sIL-1RI, sIL1RII and sTNF-RII receptors; the MCP3, IL-8, MIG and IP-10 chemokines; the M-CSF and sIL-2Ra growth factors; and the wound-healing factor FGF2 favor fatal outcomes of the disease.

A Stable Chemokine Gradient Controls Directional Persistence of Migrating Dendritic Cells.

Navigation of dendritic cells (DCs) from the site of infection to lymphoid organs is guided by concentration gradients of CCR7 ligands. How cells interpret chemokine gradients and how they couple directional sensing to polarization and persistent chemotaxis has remained largely elusive. Previous experimental systems were limited in the ability to control fast de novo formation of the final gradient slope, long-lasting stability of the gradient and to expose cells to dynamic stimulation. Here, we used a combination of microfluidics and quantitative in vitro live cell imaging to elucidate the chemotactic sensing strategy of DCs. The microfluidic approach allows us to generate soluble gradients with high spatio-temporal precision and to analyze actin dynamics, cell polarization, and persistent directional migration in both static and dynamic environments. We demonstrate that directional persistence of DC migration requires steady-state characteristics of the soluble gradient instead of temporally rising CCL19 concentration, implying that spatial sensing mechanisms control chemotaxis of DCs. Kymograph analysis of actin dynamics revealed that the presence of the CCL19 gradient is essential to stabilize leading edge protrusions in DCs and to determine directionality, since both cytoskeletal polarization and persistent chemotaxis are abrogated in the range of seconds when steady-state gradients are perturbed. In contrast to Dictyostelium amoeba, DCs are unable to decode oscillatory stimulation of soluble chemokine traveling waves into a directional response toward the wave source. These findings are consistent with the notion that DCs do not employ adaptive temporal sensing strategies that discriminate temporally increasing and decreasing chemoattractant concentrations in our setting. Taken together, in our experimental system DCs do not depend on increasing absolute chemokine concentration over time to induce persistent migration and do not integrate oscillatory stimulation. The observed capability of DCs to migrate with high directional persistence in stable gradients but not when subjected to periodic temporal cues, identifies spatial sensing as a key requirement for persistent chemotaxis of DCs.

Abstract 685: Circulating immune markers and risks of non-Hodgkin lymphoma subtypes: A pooled analysis

Abstract Background: Peripheral blood levels of soluble CD27 (sCD27), sCD30 and chemokine ligand-13 (CXCL13) are proposed markers of immune activation, the effects of which may influence the development of non-Hodgkin lymphoma (NHL). Pre-diagnostic circulating levels of sCD27, sCD30 and CXCL13 have been associated with NHL, although individual studies have typically been underpowered to assess associations for individual NHL subtypes. We pooled data from eight case-control studies nested within general-population cohorts to investigate subtype-specific relationships with these immune markers. Methods: After pooling, immune marker data for 2,455 cases diagnosed &amp;gt;2 years after blood collection and 3,310 controls were available for analysis. Using polytomous regression models, we calculated odds ratios (ORs) with 95% confidence intervals (CIs) relating study-specific tertiles of each immune marker to the following subtypes: chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; n=623), diffuse large B-cell lymphoma (DLBCL; n=621), follicular lymphoma (FL; n=398), marginal zone lymphoma (MZL; n=138), mantle cell lymphoma (MCL; n=82) and T-cell lymphoma (TCL; n=92). Results: We observed associations with DLBCL for elevated levels of sCD27 [OR for 3rd vs. 1st tertile (ORT3) = 2.2, 95% CI = 1.6-3.1; Ptrend = 9.3x10-6), sCD30 (ORT3 = 2.0, 95% CI 1.6-2.5; Ptrend = 6.5 x10-10) and CXCL-13 (ORT3 = 2.3, 95% CI 1.8-3.0; Ptrend = 3.9 x10-12). These associations remained in a model simultaneously adjusting for all three markers. We also observed associations with sCD27 for CLL/SLL (ORT3 = 3.3, 95% CI = 2.4-4.6; Ptrend = 1.6x10-13), MZL (ORT3 =7.7, 95% CI 3.0-20.1; Ptrend = 2.3x10-6) and TCL (ORT3 = 3.4, 95% CI 1.5-7.7; Ptrend = 0.003), and between sCD30 and FL (ORT3 = 2.7, 95% CI 2.0-3.5; Ptrend = 1.7x10-12), all of which remained after adjustment for the other immune markers. In analyses stratified by follow-up time from blood collection to case diagnosis, the sCD27-TCL association and all three DLBCL associations were equivalent across both follow-up periods (&amp;gt;2-&amp;lt;7.5, ≥7.5 years). The sCD27-CLL/SLL, sCD27-MZL and sCD30-FL associations were weaker for cases diagnosed ≥7.5 years post-phlebotomy compared to the earlier follow-up period but remained statistically significant. Conclusions: In this pooled analysis, to our knowledge the first of its kind, we found robust evidence of an association between the three immune markers and DLBCL, consistent with hypotheses that mechanisms related to immune activation are important in the etiology of this malignancy. Other findings are notable, particularly the strong associations with sCD27 for the rare subtypes MZL and TCL, but require further investigation before causal inferences can be drawn. Citation Format: Jongeun Rhee, Brenda M. Birmann, Anneclaire J. De Roos, Mara M. Epstein, Otoniel Martinez-Maza, Elizabeth C. Breen, Lynn I. Levin, Kala Visvanathan, H Dean Hosgood, Thomas Rohan, Lihong Qi, Qing Lan, Nathaniel Rothman, Mark P. Purdue. Circulating immune markers and risks of non-Hodgkin lymphoma subtypes: A pooled analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 685.

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell–bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.

Timeline Kinetics of Systemic and Airway Immune Mediator Storm for Comprehensive Analysis of Disease Outcome in Critically Ill COVID-19 Patients.

In the present study, the levels of serum and airway soluble chemokines, pro-inflammatory/regulatory cytokines, and growth factors were quantified in critically ill COVID-19 patients (total n=286) at distinct time points (D0, D2-6, D7, D8-13 and D>14-36) upon Intensive Care Unit (ICU) admission. Augmented levels of soluble mediators were observed in serum from COVID-19 patients who progress to death. An opposite profile was observed in tracheal aspirate samples, indicating that systemic and airway microenvironment diverge in their inflammatory milieu. While a bimodal distribution was observed in the serum samples, a unimodal peak around D7 was found for most soluble mediators in tracheal aspirate samples. Systems biology tools further demonstrated that COVID-19 display distinct eccentric soluble mediator networks as compared to controls, with opposite profiles in serum and tracheal aspirates. Regardless the systemic-compartmentalized microenvironment, networks from patients progressing to death were linked to a pro-inflammatory/growth factor-rich, highly integrated center. Conversely, patients evolving to discharge exhibited networks of weak central architecture, with lower number of neighborhood connections and clusters of pro-inflammatory and regulatory cytokines. All in all, this investigation with robust sample size landed a comprehensive snapshot of the systemic and local divergencies composed of distinct immune responses driven by SARS-CoV-2 early on severe COVID-19.

TransCon IL-2 β/γ, a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2 analog, demonstrates improved pharmacokinetics and profound expansion of cytotoxic immune cells in non-human primates.

2563 Background: Recombinant interleukin-2 (IL-2, aldesleukin) is an approved cancer immunotherapy but causes severe side effects including cytokine release syndrome (CRS) and vascular leak syndrome (VLS). This is believed to be due to activation of IL-2Rα+ endothelial cells and inflammatory eosinophils as well as high Cmax due to the short half-life requiring frequent high dose administrations. Also, potent activation of immunosuppressive IL-2Rα+ regulatory T cells (Tregs) may limit IL-2’s efficacy. TransCon IL-2 β/γ is a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2 analog designed to optimally address these shortcomings. Methods: Naïve male and female cynomolgus monkeys received 2-3 doses of TransCon IL-2 β/γ intravenously Q2W. Blood samples were taken to assess plasma concentrations of IL-2 β/γ (active drug), serum cytokines, hematology and immunophenotyping. Histopathology was also performed. Results: Administration of TransCon IL-2 β/γ induced robust increases in absolute lymphocyte counts (ALC), with no/minor increases in eosinophil counts. The increase in ALC was primarily driven by potent activation and expansion of CD8+ T cells, NK cells and γδ T cells, relative to CD4+ T cells and Tregs. At doses with maximum pharmacodynamic (PD) responses, TransCon IL-2 β/γ induced on average &gt; 20-fold ALC increases, &gt; 19-fold CD8+ T cell increases, &gt; 50-fold Effector Memory CD8+ T cell increases, &gt; 18-fold NK cell increases and &gt; 400-fold γδ T cell increases compared to pre-dose levels. These changes corresponded with a low Cmax and a long effective half-life ( &gt; 30 hours) for IL-2 β/γ. A clear induction of soluble CD25 and chemokine MCP-1 was seen after dosing, while levels of IL-5, IL-6, IFN-γ and TNF-α remained low. Further, histopathology showed no signs of vascular damage, tissue necrosis, pulmonary edema or eosinophilia. Conclusions: In cynomolgus monkeys, TransCon IL-2 β/γ demonstrated remarkable PD responses in CD8+ T cells, NK cells and γδ T cells over Tregs and eosinophils. No signs of VLS or CRS were observed. These results are likely due to the selective IL-2Rβ/γ bias along with the slow and sustained release of IL-2 β/γ resulting in low Cmax and long circulatory half-life enabled by the TransCon prodrug technology. The massive expansion of γδ T cells, which are known to possess considerable anti-tumor activity, is to our knowledge a unique finding for TransCon IL-2 β/γ among IL-2 variants in clinical development. Altogether, the responses seen in monkeys are suggestive of a potentially substantial improvement in the therapeutic index beyond what has been achieved by aldesleukin or new IL-2 variants to date. TransCon IL-2 β/γ is currently being evaluated in a clinical Phase 1/2 trial as monotherapy and in combination with pembrolizumab (NCT05081609).