Respiratory complex I regulates dendritic cell maturation in explant model of human tumor immune microenvironment.

BACKGROUND: Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level. METHODS: Here we establish a patient-derived explant culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+T cell cytotoxic activity. RESULTS: We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax+metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+T cells and thus facilitating antitumor immunity. CONCLUSIONS: Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME.

Prolonged Systemic Inflammatory Response Syndrome After Cardiac Surgery.

OBJECTIVES: Cardiac surgery induces systemic inflammatory response syndrome (SIRS), leading to higher morbidity and mortality. There are no individualized predictors for worse outcomes or biomarkers for the multifactorial, excessive inflammatory response. The interest of this study was to evaluate whether a systematic use of the SIRS criteria could be used to predict postoperative outcomes beyond infection and sepsis, and if the development of an exaggerated inflammation response could be observed preoperatively. DESIGN: The study was observational, with prospectively enrolled patients. SETTING: This was a single institution study in a hospital setting combined with laboratory findings. PARTICIPANTS: The study included a cohort of 261 volunteer patients. INTERVENTIONS: Patients underwent cardiac surgery with cardiopulmonary bypass, and were followed up to 90 days. Biomarker profiling was run preoperatively. MEASUREMENTS AND MAIN RESULTS: Altogether, 17 of 261 (6.4%) patients had prolonged SIRS, defined as fulfilling at least 2 criteria on 4 consecutive postoperative days. During hospitalization, postoperative atrial fibrillation (POAF) was found in 42.2% of patients, and stroke and transient ischemic attack in 3.8% of patients. Prolonged SIRS was a significant predictor of POAF (odds ratio [OR] 4.5, 95% CI 1.2-17.3), 90-day stroke (OR 4.5, 95% CI 1.1-18.0), and mortality (OR 10.7, 95% CI 1.7-68.8). Biomarker assays showed that preoperative nerve growth factor and interleukin 5 levels were associated with prolonged SIRS (OR 5.6, 95%, CI 1.4-23.2 and OR 0.7, 95%, CI 0.4-1.0, respectively). CONCLUSIONS: Nerve growth factor and interleukin 5 can be used to predict prolonged systemic inflammatory response, which is associated with POAF, stroke, and mortality.

Bexmarilimab Activates Human Tumor-Associated Macrophages to Support Adaptive Immune Responses in Interferon-Poor Immune Microenvironments.

Immune checkpoint inhibitors (ICI) show substantially greater efficacy in inflamed tumors characterized by preexisting T-cell infiltration and IFN signaling than in noninflamed "cold" tumors, which often remain immunotherapy resistant. The cancer immunotherapy bexmarilimab, which inhibits the scavenger receptor Clever-1 to release macrophage immunosuppression and activate adaptive immunity, has shown treatment benefit in subsets of patients with advanced solid malignancies. However, the mechanisms that determine bexmarilimab therapy outcome in individual patients are unknown. Here we characterized bexmarilimab response in ovarian cancer ascites macrophages ex vivo using single-cell RNA sequencing and demonstrated increased IFN signaling and CXCL10 secretion following bexmarilimab treatment. We further showed that bexmarilimab was most efficacious in macrophages with low baseline IFN signaling, as chronic IFNγ priming abolished bexmarilimab-induced TNFα release. These results highlight an approach to target immunologically cold tumors and to increase the likelihood of their subsequent response to ICIs.

Single-cell functional genomics reveals determinants of sensitivity and resistance to natural killer cells in blood cancers.

Cancer cells can evade natural killer (NK) cell activity, thereby limiting anti-tumor immunity. To reveal genetic determinants of susceptibility to NK cell activity, we examined interacting NK cells and blood cancer cells using single-cell and genome-scale functional genomics screens. Interaction of NK and cancer cells induced distinct activation and type I interferon (IFN) states in both cell types depending on the cancer cell lineage and molecular phenotype, ranging from more sensitive myeloid to less sensitive B-lymphoid cancers. CRISPR screens in cancer cells uncovered genes regulating sensitivity and resistance to NK cell-mediated killing, including adhesion-related glycoproteins, protein fucosylation genes, and transcriptional regulators, in addition to confirming the importance of antigen presentation and death receptor signaling pathways. CRISPR screens with a single-cell transcriptomic readout provided insight into underlying mechanisms, including regulation of IFN-γ signaling in cancer cells and NK cell activation states. Our findings highlight the diversity of mechanisms influencing NK cell susceptibility across different cancers and provide a resource for NK cell-based therapies.

Bexmarilimab-induced macrophage activation leads to treatment benefit in solid tumors: The phase I/II first-in-human MATINS trial.

Macrophage Clever-1 contributes to impaired antigen presentation and suppression of anti-tumor immunity. This first-in-human trial investigates the safety and tolerability of Clever-1 blockade with bexmarilimab in patients with treatment-refractory solid tumors and assesses preliminary anti-tumor efficacy, pharmacodynamics, and immunologic correlates. Bexmarilimab shows no dose-limiting toxicities in part I (n = 30) and no additional safety signals in part II (n = 108). Disease control (DC) rates of 25%-40% are observed in cutaneous melanoma, gastric, hepatocellular, estrogen receptor-positive breast, and biliary tract cancers. DC associates with improved survival in a landmark analysis and correlates with high pre-treatment intratumoral Clever-1 positivity and increasing on-treatment serum interferon γ (IFNγ) levels. Spatial transcriptomics profiling of DC and non-DC tumors demonstrates bexmarilimab-induced macrophage activation and stimulation of IFNγ and T cell receptor signaling selectively in DC patients. These data suggest that bexmarilimab therapy is well tolerated and show that macrophage targeting can promote immune activation and tumor control in late-stage cancer.

Dimeric IgA specifically disables intracellular mutated oncodrivers.

A prevailing belief in the immunotherapy field has been that antibody therapy can effectively target only extracellular antigens. In this issue of Immunity, Biswas et al. demonstrate therapeutically effective targeting, neutralization, and removal of mutated oncodriver proteins from within epithelial cancer cells by treatment with pIgR-dependent, transcytosing dimeric-IgA antibodies.

A form of inherited hyperferritinemia associated with bi-allelic pathogenic variants of STAB1.

Hyperferritinemia is a frequent finding in several conditions, both genetic and acquired. We previously studied eleven healthy subjects from eight different families presenting with unexplained hyperferritinemia. Their findings suggested the existence of an autosomal-recessive disorder. We carried out whole-exome sequencing to detect the genetic cause of hyperferritinemia. Immunohistochemistry and flow cytometry assays were performed on liver biopsies and monocyte-macrophages to confirm the pathogenic role of the identified candidate variants. Through a combined approach of whole-exome sequencing and homozygosity mapping, we found bi-allelic STAB1 variants in ten subjects from seven families. STAB1 encodes the multifunctional scavenger receptor stabilin-1. Immunohistochemistry and flow cytometry analyses showed absent or markedly reduced stabilin-1 in liver samples, monocytes, and monocyte-derived macrophages. Our findings show a strong association between otherwise unexplained hyperferritinemia and bi-allelic STAB1 mutations suggesting the existence of another genetic cause of hyperferritinemia without iron overload and an unexpected function of stabilin-1 in ferritin metabolism.

The effect of acute exercise on circulating immune cells in newly diagnosed breast cancer patients.

The role of exercise in cancer prevention and control is increasingly recognized, and based on preclinical studies, it is hypothesized that mobilization of leukocytes plays an important role in the anti-tumor effect. Thus, we examined how 10-min acute exercise modulates immune cells in newly diagnosed breast cancer patients. Blood samples were taken at rest, immediately after exercise and 30 min after exercise and phenotypic characterization of major leukocyte subsets was done using 9-color flow cytometry. Total leukocyte count increased by 29%, CD8+ T cell count by 34%, CD19+ B cell count by 18%, CD56+CD16+ NK cell count by 130%, and CD14+CD16+ monocyte count by 51% immediately after acute exercise. Mobilization of CD45+, CD8+, CD19+, and CD56+CD16+ cells correlated positively with exercising systolic blood pressure, heart rate percentage of age predicted maximal heart rate, rate pressure product, and mean arterial pressure. Our findings indicate that a single bout of acute exercise of only 10 min can cause leukocytosis in breast cancer patients. Mobilization of leukocytes appear to be directly related to the intensity of exercise. It is possible that the positive effect of exercise on oncologic outcome might be partly due to immune cell mobilization as documented in the present study.

Polymorphism in interferon alpha/beta receptor contributes to glucocorticoid response and outcome of ARDS and COVID-19.

BACKGROUND: The use of glucocorticoids has given contradictory results for treating acute respiratory distress syndrome (ARDS). The use of intravenous Interferon beta (IFN ß) for the treatment of ARDS was recently tested in a phase III ARDS trial (INTEREST), in which more than half of the patients simultaneously received glucocorticoids. Trial results showed deleterious effects of glucocorticoids when administered together with IFN ß, and therefore, we aimed at finding the reason behind this. METHODS: We first sequenced the genes encoding the IFN α/ß receptor of the patients, who participated in the INTEREST study (ClinicalTrials.gov Identifier:  NCT02622724 , November 24, 2015) in which the patients were randomized to receive an intravenous injection of IFN ß-1a (144 patients) or placebo (152 patients). Genetic background was analyzed against clinical outcome, concomitant medication, and pro-inflammatory cytokine levels. Thereafter, we tested the influence of the genetic background on IFN α/ß receptor expression in lung organ cultures and whether, it has any effect on transcription factors STAT1 and STAT2 involved in IFN signaling. RESULTS: We found a novel disease association of a SNP rs9984273, which is situated in the interferon α/ß receptor subunit 2 (IFNAR2) gene in an area corresponding to a binding motif of the glucocorticoid receptor (GR). The minor allele of SNP rs9984273 associates with higher IFNAR expression, more rapid decrease of IFN γ and interleukin-6 (IL-6) levels and better outcome in IFN ß treated patients with ARDS, while the major allele associates with a poor outcome especially under concomitant IFN ß and glucocorticoid treatment. Moreover, the minor allele of rs9984273 associates with a less severe form of coronavirus diseases (COVID-19) according to the COVID-19 Host Genetics Initiative database. CONCLUSIONS: The distribution of this SNP within clinical study arms may explain the contradictory results of multiple ARDS studies and outcomes in COVID-19 concerning type I IFN signaling and glucocorticoids.

Mapping the lymphatic system across body scales and expertise domains: A report from the 2021 National Heart, Lung, and Blood Institute workshop at the Boston Lymphatic Symposium.

Enhancing our understanding of lymphatic anatomy from the microscopic to the anatomical scale is essential to discern how the structure and function of the lymphatic system interacts with different tissues and organs within the body and contributes to health and disease. The knowledge of molecular aspects of the lymphatic network is fundamental to understand the mechanisms of disease progression and prevention. Recent advances in mapping components of the lymphatic system using state of the art single cell technologies, the identification of novel biomarkers, new clinical imaging efforts, and computational tools which attempt to identify connections between these diverse technologies hold the potential to catalyze new strategies to address lymphatic diseases such as lymphedema and lipedema. This manuscript summarizes current knowledge of the lymphatic system and identifies prevailing challenges and opportunities to advance the field of lymphatic research as discussed by the experts in the workshop.

Preclinical Evaluation of 89Zr-Desferrioxamine-Bexmarilimab, a Humanized Antibody Against Common Lymphatic Endothelial and Vascular Endothelial Receptor-1, in a Rabbit Model of Renal Fibrosis.

Bexmarilimab is a new humanized monoclonal antibody against common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) and is in clinical trials for macrophage-guided cancer immunotherapy. In addition being associated with cancer, CLEVER-1 is also associated with fibrosis. To facilitate prospective human PET studies, we preclinically evaluated 89Zr-labeled bexmarilimab in rabbits. Methods: Bexmarilimab was conjugated with desferrioxamine (DFO) and radiolabeled with 89Zr. Retained immunoreactivity was confirmed by flow cytometry. The distribution kinetics of intravenously administered 89Zr-DFO-bexmarilimab (0.1 mg/kg) were determined for up to 7 d in a rabbit model of renal fibrosis mediated by unilateral ureteric obstruction. The in vivo stability of 89Zr-DFO-bexmarilimab was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with autoradiography. Additionally, we estimated the human radiation dose from data obtained in healthy rabbits. Results: 89Zr-DFO-bexmarilimab cleared rapidly from the blood circulation and distributed to the liver and spleen. At 24 h after injection, PET/CT, ex vivo γ-counting, and autoradiography demonstrated that there was significantly higher 89Zr-DFO-bexmarilimab uptake in unilateral ureteric obstruction-operated fibrotic renal cortex, characterized by abundant CLEVER-1-positive cells, than in contralateral or healthy kidneys. The estimated effective dose for a 70-kg human was 0.70 mSv/MBq. Conclusion: The characteristics of 89Zr-DFO-bexmarilimab support future human PET studies to, for example, stratify patients for bexmarilimab treatment, evaluate the efficacy of treatment, or monitor disease progression.

Risk of fluid accumulation after cardiac surgery.

Objective: Patients undergoing heart surgery are at high risk of postoperative fluid accumulation due to long procedures and cardiopulmonary bypass. In the present study, we sought to investigate the prevalence of postoperative fluid accumulation and its relation to adverse events in patients undergoing cardiac surgery. Methods: CAREBANK is prospective, single-center cohort study focusing on the adverse events after cardiac surgery. The study population was divided into 2 groups based on 5% postoperative weight gain. All the in-hospital adverse events are registered on the database. The end points of the present study were length of hospital stay, length of intensive care unit stay, occurrence of new-onset atrial fibrillation after hospital major bleeding episodes major cardiac events, cerebrovascular events, and death. Three-month and 1-year follow-up data also include all major adverse events. Results: Altogether 1001 adult cardiac surgery patients were enrolled. The most frequent operations were coronary artery bypass grafting (56.3%). Five hundred fifty-four out of 939 (59.0%) patients had ≥5% weight gain during index hospitalization. Patients with a weight gain ≥5% were more likely to be women, have lower body mass index, had heart failure, and more often had preoperative atrial fibrillation. In-hospital period fluid accumulation was associated with reoperation due bleeding and longer total hospital stay. At 3 months' follow-up, weight gain 5% or more was associated with increased occurrence of new-onset atrial fibrillation, this was not reflected in the occurrence of strokes, transient ischemic attacks, or myocardial infarctions. Conclusions: Postoperative fluid excess is associated with adverse outcomes in cardiac surgery. Women, low-weight patients, and patients with cardiac failure or atrial fibrillation are prone to perioperative fluid accumulation.

Cancer immunotherapies transition endothelial cells into HEVs that generate TCF1+ T lymphocyte niches through a feed-forward loop.

The lack of T cell infiltrates is a major obstacle to effective immunotherapy in cancer. Conversely, the formation of tumor-associated tertiary-lymphoid-like structures (TA-TLLSs), which are the local site of humoral and cellular immune responses against cancers, is associated with good prognosis, and they have recently been detected in immune checkpoint blockade (ICB)-responding patients. However, how these lymphoid aggregates develop remains poorly understood. By employing single-cell transcriptomics, endothelial fate mapping, and functional multiplex immune profiling, we demonstrate that antiangiogenic immune-modulating therapies evoke transdifferentiation of postcapillary venules into inflamed high-endothelial venules (HEVs) via lymphotoxin/lymphotoxin beta receptor (LT/LTßR) signaling. In turn, tumor HEVs boost intratumoral lymphocyte influx and foster permissive lymphocyte niches for PD1- and PD1+TCF1+ CD8 T cell progenitors that differentiate into GrzB+PD1+ CD8 T effector cells. Tumor-HEVs require continuous CD8 and NK cell-derived signals revealing that tumor HEV maintenance is actively sculpted by the adaptive immune system through a feed-forward loop.

BRAFV600E Expression in Thyrocytes Causes Recruitment of Immunosuppressive STABILIN-1 Macrophages.

Papillary thyroid carcinoma (PTC) is the most frequent histological subtype of thyroid cancers (TC), and BRAFV600E genetic alteration is found in 60% of this endocrine cancer. This oncogene is associated with poor prognosis, resistance to radioiodine therapy, and tumor progression. Histological follow-up by anatomo-pathologists revealed that two-thirds of surgically-removed thyroids do not present malignant lesions. Thus, continued fundamental research into the molecular mechanisms of TC downstream of BRAFV600E remains central to better understanding the clinical behavior of these tumors. To study PTC, we used a mouse model in which expression of BRAFV600E was specifically switched on in thyrocytes by doxycycline administration. Upon daily intraperitoneal doxycycline injection, thyroid tissue rapidly acquired histological features mimicking human PTC. Transcriptomic analysis revealed major changes in immune signaling pathways upon BRAFV600E induction. Multiplex immunofluorescence confirmed the abundant recruitment of macrophages, among which a population of LYVE-1+/CD206+/STABILIN-1+ was dramatically increased. By genetically inactivating the gene coding for the scavenger receptor STABILIN-1, we showed an increase of CD8+ T cells in this in situ BRAFV600E-dependent TC. Lastly, we demonstrated the presence of CD206+/STABILIN-1+ macrophages in human thyroid pathologies. Altogether, we revealed the recruitment of immunosuppressive STABILIN-1 macrophages in a PTC mouse model and the interest to further study this macrophage subpopulation in human thyroid tissues.

Clever-1 positive macrophages in breast cancer.

PURPOSE: Common Lymphatic Endothelial and Vascular Endothelial Receptor 1 (Clever-1) is expressed by a subset of immunosuppressive macrophages and targeting the receptor with therapeutic antibodies has been shown to activate T-cell-mediated anti-cancer immunity. The aim of this research was to study Clever-1 expression in breast cancer. Specifically, how Clever-1 + macrophages correlate with clinicopathologic factors, Tumor Infiltrating Lymphocytes (TILs) and prognosis. METHODS: Tissue microarray blocks were made from 373 primary breast cancer operation specimens. Hematoxylin and Eosin (H&E-staining) and immunohistochemical staining with Clever-1, CD3, CD4 and CD8 antibodies were performed. Differences in quantities of Clever-1 + macrophages and TILs were analyzed. Clever-1 + cell numbers were correlated with 25-year follow-up survival data and with breast cancer clinicopathologic parameters. RESULTS: Low numbers of intratumoral Clever-1 + cells were found to be an independent adverse prognostic sign. Increased numbers of Clever-1 + cells were found in high grade tumors and hormone receptor negative tumors. Tumors that had higher amounts of Clever-1 + cells also tended to have higher amounts of TILs. CONCLUSION: The association of intratumoral Clever-1 + macrophages with better prognosis might stem from the function of Clever as a scavenger receptor that modulates tumor stroma. The association of Clever-1 + macrophages with high number of TILs and better prognosis indicates that immunosuppression by M2 macrophages is not necessarily dampening adaptive immune responses but instead keeping them in control to avoid excess inflammation.

The tumor and plasma cytokine profiles of renal cell carcinoma patients.

Renal cell carcinoma (RCC) accounts for 90% of all renal cancers and is considered highly immunogenic. Although many studies have reported the circulating peripheral cytokine profiles, the signatures between the tumor tissue and matching healthy adjacent renal tissue counterparts have not been explored. We aimed to comprehensively investigate the cytokine landscape of RCC tumors and its correlation between the amount and phenotype of the tumor infiltrating lymphocytes (TILs). We analyzed the secretion of 42 cytokines from the tumor (n = 46), adjacent healthy kidney tissues (n = 23) and matching plasma samples (n = 33) with a Luminex-based assay. We further explored the differences between the tissue types, as well as correlated the findings with clinical data and detailed immunophenotyping of the TILs. Using an unsupervised clustering approach, we observed distinct differences in the cytokine profiles between the tumor and adjacent renal tissue samples. The tumor samples clustered into three distinct profiles based on the cytokine expressions: high (52.2% of the tumors), intermediate (26.1%), and low (21.7%). Most of the tumor cytokines positively correlated with each other, except for IL-8 that showed no correlation with any of the measured cytokine expressions. Furthermore, the quantity of lymphocytes in the tumor samples analyzed with flow cytometry positively correlated with the chemokine-family of cytokines, CXCL10 (IP-10) and CXCL9 (MIG). No significant correlations were found between the tumor and matching plasma cytokines, suggesting that circulating cytokines poorly mirror the tumor cytokine environment. Our study highlights distinct cytokine profiles in the RCC tumor microenvironment and provides insights to potential biomarkers for the treatment of RCC.

Nonclinical Characterization of Bexmarilimab, a Clever-1-Targeting Antibody for Supporting Immune Defense Against Cancers.

Common lymphatic endothelial and vascular endothelial receptor-1 (Clever-1) is a multifunctional type-1 transmembrane protein that plays an important role in immunosuppression against tumors. Clever-1 is highly expressed in a subset of human tumor-associated macrophages and associated with poor survival. In mice, Clever-1 supports tumor growth and metastasis formation, and its deficiency or blockage induces T-cell-dependent killing of cancer cells. Therefore, targeting Clever-1 could lead to T-cell activation and restoration of immune response also in patients with cancer. This is studied in an on-going clinical trial [Macrophage Antibody To INhibit immune Suppression (MATINS); NCT03733990] in patients with advanced solid tumors where bexmarilimab, a humanized IgG4 antibody against human Clever-1, shows promising safety and efficacy. Here, we report the humanization and nonclinical characterization of physicochemical properties, biological potency, and safety profile of bexmarilimab. Bexmarilimab showed high affinity to Clever-1 on KG-1 cells and bound to Clever-1 on the surface of classical and intermediate monocytes derived from healthy human blood. Bexmarilimab inhibited the internalization of its natural ligand acetylated low-density lipoprotein into KG-1 cells and increased TNFα secretion from macrophages but did not impair phagocytic clearance. Bexmarilimab did not induce significant cytokine release in human whole-blood cultures, did not contain nonsafe immunogenic glycans, or show any significant binding to human Fcγ receptors or complement pathway component C1q. In vivo, bexmarilimab showed dose-dependent duration of monocyte Clever-1 receptor occupancy in cynomolgus monkeys but did not induce a cytokine storm up to a dose of 100 mg/kg. In conclusion, these data support the clinical development of bexmarilimab for the restoration of immune response in cancers.

A distinct M2 macrophage infiltrate and transcriptomic profile decisively influence adipocyte differentiation in lipedema.

Lipedema is a chronic and progressive adipose tissue disorder, characterized by the painful and disproportionate increase of the subcutaneous fat in the lower and/or upper extremities. While distinct immune cell infiltration is a known hallmark of the disease, its role in the onset and development of lipedema remains unclear. To analyze the macrophage composition and involved signaling pathways, anatomically matched lipedema and control tissue samples were collected intra-operatively from gender- and BMI-matched patients, and the Stromal Vascular Fraction (SVF) was used for Cytometry by Time-of-Flight (CyTOF) and RNA sequencing. The phenotypic characterization of the immune component of lipedema versus control SVF using CyTOF revealed significantly increased numbers of CD163 macrophages. To gain further insight into this macrophage composition and molecular pathways, RNA sequencing of isolated CD11b+ cells was performed. The analysis suggested a significant modification of distinct gene ontology clusters in lipedema, including cytokine-mediated signaling activity, interleukin-1 receptor activity, extracellular matrix organization, and regulation of androgen receptor signaling. As distinct macrophage populations are known to affect adipose tissue differentiation and metabolism, we evaluated the effect of M2 to M1 macrophage polarization in lipedema using the selective PI3Kγ inhibitor IPI-549. Surprisingly, the differentiation of adipose tissue-derived stem cells with conditioned medium from IPI-549 treated SVF resulted in a significant decreased accumulation of lipids in lipedema versus control SVF. In conclusion, our results indicate that CD163+ macrophages are a critical component in lipedema and re-polarization of lipedema macrophages can normalize the differentiation of adipose-derived stem cells in vitro evaluated by the cellular lipid accumulation. These data open a new chapter in understanding lipedema pathophysiology and may indicate potential treatment options.