TREM2 macrophages drive NK cell paucity and dysfunction in lung cancer.

Natural killer (NK) cells are commonly reduced in human tumors, enabling many to evade surveillance. Here, we sought to identify cues that alter NK cell activity in tumors. We found that, in human lung cancer, the presence of NK cells inversely correlated with that of monocyte-derived macrophages (mo-macs). In a murine model of lung adenocarcinoma, we show that engulfment of tumor debris by mo-macs triggers a pro-tumorigenic program governed by triggering receptor expressed on myeloid cells 2 (TREM2). Genetic deletion of Trem2 rescued NK cell accumulation and enabled an NK cell-mediated regression of lung tumors. TREM2+ mo-macs reduced NK cell activity by modulating interleukin (IL)-18/IL-18BP decoy interactions and IL-15 production. Notably, TREM2 blockade synergized with an NK cell-activating agent to further inhibit tumor growth. Altogether, our findings identify a new axis, in which TREM2+ mo-macs suppress NK cell accumulation and cytolytic activity. Dual targeting of macrophages and NK cells represents a new strategy to boost antitumor immunity.

Escaping Negative Selection: ILC You in the Gut.

How commensal-specific T cells are controlled in the periphery is poorly understood. In a recent issue of Science, Hepworth et al. (2015) show that ILC3s induce apoptosis of microbiota-specific CD4 T cells in a form of extrathymic negative selection.

Neutrophils and neutrophil extracellular traps enhance venous thrombosis in mice bearing human pancreatic tumors.

Pancreatic cancer is associated with a high incidence of venous thromboembolism. Neutrophils have been shown to contribute to thrombosis in part by releasing neutrophil extracellular traps (NET). A recent study showed that increased plasma levels of the NET biomarker, citrullinated histone H3 (H3Cit), are associated with venous thromboembolism in patients with pancreatic and lung cancer but not in those with other types of cancer, including breast cancer. In this study, we examined the contribution of neutrophils and NET to venous thrombosis in nude mice bearing human pancreatic tumors. We found that tumor-bearing mice had increased circulating neutrophil counts and levels of granulocyte-colony stimulating factor, neutrophil elastase, H3Cit and cell-free DNA compared with controls. In addition, thrombi from tumor-bearing mice contained increased levels of the neutrophil marker Ly6G, as well as higher levels of H3Cit and cell-free DNA. Thrombi from tumor-bearing mice also had denser fibrin with thinner fibers consistent with increased thrombin generation. Importantly, either neutrophil depletion or administration of DNase I reduced the thrombus size in tumor-bearing but not in control mice. Our results, together with clinical data, suggest that neutrophils and NET contribute to venous thrombosis in patients with pancreatic cancer.

Segmental arterial mediolysis: a vasculitis mimicker. A single centre experience.

OBJECTIVES: Segmental arterial mediolysis (SAM) is a rare vasculopathy of unknown aetiology. It is non-atherosclerotic, non-inflammatory, non-hereditary, non-infectious, large to medium-sized arteriopathy. SAM is a condition which in some circumstances behaves as a vasculitis mimicker and should be recognised in order to provide appropriate treatment and avoid unnecessary immune-suppressive therapy. METHODS: We report a single-centre experience of 6 consecutive SAM cases (3 males and 3 females). A literature search of cases reported with SAM was performed and data summarised. RESULTS: Abdominal or flank pain was the presenting symptom in 5 of the 6 patients. CT angiography (CTA) was the method of diagnosis in all 6 patients. 3 patients underwent therapeutic angiography; 2 with angiographic embolisation because of bleeding, and one patient needed a stent insertion because of left renal infarction. 2 patients underwent FDG-PET to rule out vasculitis. Serological tests were negative in all case, but C-reactive protein was elevated in 4 of them. 2 patients were treated with angiographic embolisation due to bleeding, 2 treated with anti-platelet therapy, one with stent insertion, and one with antihypertensive treatment. A medical literature review of 160 additional cases shows that abdominal or flank pain was the chief complaint in the vast majority of the cases. Renal and abdominal medium-sized arteries were the most commonly involved. CTA was the preferred method of diagnosis. CONCLUSIONS: SAM should be suspected in cases presenting with abdominal or flank pain. Angiographic features should be carefully studied by experienced radiologists to rule out vasculitis.

Oral and Topical Sirolimus for Vascular Anomalies: A Multicentre Study and Review.

Vascular anomalies (VAs) may be associated with significant morbidity and mortality. The aim of this study was to evaluate the efficacy and safety of sirolimus (rapamycin) in the treatment of children and young adults with complicated VAs. A retrospective chart was created that included 19 patients treated with sirolimus for complicated VAs. Concurrently, a search of the PubMed database for VA cases treated with sirolimus was conducted. Descriptive analysis was performed and the efficiency rate of sirolimus was calculated. This retrospective study included 19 patients, 17 of whom were treated with oral sirolimus and 2 with topical sirolimus. Clinical improvement occurred in 15 patients (79%). One patient experienced near-complete resolution. Only 2 patients showed poor response and discontinued treatment. The literature review analysed 150 cases of VA treated with sirolimus. Sirolimus was efficient in 85% of cases, including 5 cases of complete resolution. Sirolimus appears to be an effective and safe treatment for children and young adults with complicated VAs.

Immune phenotype of peripheral blood mononuclear cells in patients with high-risk non-muscle invasive bladder cancer.

PURPOSE: To explore the immune phenotype of peripheral blood mononuclear cells (PBMC) in patients with high-risk non-muscle invasive bladder cancer (NMIBC). METHODS: We prospectively collected blood samples from patients with high-risk NMIBC treated at our institution. PBMC were analyzed by flow cytometry to determine the frequency of T cells and NK cells and the expression of immunoregulatory molecules (Tim-3, TIGIT and PD-1). PBMC from healthy donors (HD) were included for comparison, and associations with response to BCG were investigated. RESULTS: A total of 38 patients were included, 19 BCG responders and 19 BCG refractory. Compared to 16 PBMC from HD, the frequency of total NK cells was significantly higher in patients with NMIBC [15.2% (IQR: 11.4, 22.2) vs. 5.72% (IQR: 4.84, 9.79); p = 0.05], whereas the frequency of T cells was not statistically different. Both Tim-3 and TIGIT expressions were significantly higher in NMIBC compared to HD, particularly in NK cells [13.8% (11.0; 22.4) vs. 5.56% (4.20; 10.2) and 34.9% (18.9; 53.5) vs. 1.82% (0.63; 5.16), respectively; p < 0.001]. Overall, the expression of PD-1 in all cell types was low in both NMIBC patients and HD. The immune phenotype was not significantly different before and after initiation of BCG. However, the proportion of CD8+ T cells before BCG was significantly higher in responders. CONCLUSION: The immune phenotype of PBMC from patients with high-risk NMIBC was significantly different from HD, regardless of the presence of disease or the initiation of BCG. Peripheral CD8+ T cells could play a role in response to BCG.

Novel mechanisms underlying the immediate and transient global tolerization of splenic dendritic cells after vaccination with a self-antigen.

Dendritic cells (DCs) are important orchestrators of the immune response, ensuring that immunity against pathogens is generated, whereas immunity against healthy tissues is prevented. Using the tumor Ag MUC1, we previously showed that i.v. immunization of MUC1 transgenic mice, but not wild-type, with a MUC1 peptide resulted in transient tolerization of all splenic DCs. These DCs did not upregulate costimulatory molecules and induced regulatory T cells rather than effector T cells. They were characterized by suppressed expression of a cohort of pancreatic enzymes not previously reported in DCs, which were upregulated in DCs presenting the same MUC1 peptide as a foreign Ag. In this article, we examined the self-antigen-tolerized DC phenotype, function, and mechanisms responsible for inducing or maintaining their tolerized state. Tolerized DCs share some characteristics with immature DCs, such as a less inflammatory cytokine/chemokine profile, deficient activation of NF-κB, and sustained expression of zDC and CCR2. However, tolerized DCs demonstrated a novel inducible expression of aldehyde dehydrogenase 1/2 and phospho-STAT3. Suppressed expression of one of the pancreatic enzymes, trypsin, in these DC impeded their ability to degrade extracellular matrix, thus affecting their motility. Suppressed metallopeptidases, reflected in low expression of carboxypeptidase B1, prevented optimal Ag-specific CD4(+) T cell proliferation suggesting their role in Ag processing. Tolerized DCs were not refractory to maturation after stimulation with a TLR3 agonist, demonstrating that this tolerized state is not terminally differentiated and that tolerized DCs can recover their ability to induce immunity to foreign Ags.

Pharmacomechanical catheter-directed thrombolysis for pregnancy-related iliofemoral deep vein thrombosis.

PURPOSE: Pharmacomechanical catheter-directed thrombolysis (PCDT) is relatively contraindicated during pregnancy and postpartum. The purpose of this study was to evaluate outcomes of PCDT in this population. MATERIALS AND METHODS: Data for 11 consecutive patients (aged 21-35 y) undergoing PCDT at a tertiary center for symptomatic pregnancy-related iliofemoral deep vein thrombosis (DVT) were retrospectively reviewed. Details regarding patient presentation, location and extent of thrombus, the PCDT procedure, outcomes, frequency of postthrombotic syndrome (PTS), and subsequent pregnancies were recorded. Two patients who presented in the first trimester terminated their pregnancies after PCDT, 2 patients who presented in the third trimester delayed PCDT until after delivery, and 7 patients who presented with postpartum DVT underwent immediate PCDT. RESULTS: Thrombus extended into the inferior vena cava in 5 patients (45%) and into popliteal/tibial veins in 7 (64%). Four patients (36%) had synchronous pulmonary embolism and three had May-Thurner compression. Median interval from diagnosis to PCDT was 5 days (range, 2-68 d); median duration of urokinase infusion was 27 hours (range, 16-72 h). Greater than 90% clot lysis was achieved in 9 of 11 patients (82%). Metal stents were placed in 8 of 11 patients (73%). A self-limiting popliteal hematoma developed in 1 patient, and 2 had early recurrent thrombosis requiring repeat PCDT. At median 20-month follow-up, nonocclusive thrombus was seen in 5 patients. No patient developed PTS. Three patients, all with stents, had uneventful pregnancies after PCDT. CONCLUSIONS: Pharmacomechanical catheter-directed thrombolysis achieved encouraging initial outcomes in this series. Validation in prospective trials with larger enrollment and longer follow-up is needed.

Antigen choice determines vaccine-induced generation of immunogenic versus tolerogenic dendritic cells that are marked by differential expression of pancreatic enzymes.

Dendritic cells (DC) elicit immunity to pathogens and tumors while simultaneously preserving tolerance to self. Efficacious cancer vaccines have been a challenge because they are based on tumor Ags, some of which are self-Ags and thus subject to self-tolerance. One such Ag is the tumor-associated mucin MUC1. Preclinical testing of MUC1 vaccines revealed existence of peripheral tolerance to MUC1 that compromises their efficacy. To identify mechanisms that act early postvaccination and might predict vaccine outcome, we immunized human MUC1 transgenic mice (MUC1.Tg) i.v. with a MUC1 peptide vaccine against which they generate weak immunity and wild-type (WT) mice that respond strongly to the same peptide. We analyzed differences in splenic DC phenotype and function between the two mouse strains at 24 and 72 h postvaccination and also performed unbiased total gene expression analysis of the spleen. Compared to WT, MUC1.Tg spleens had significantly fewer DC, and they exhibited significantly lower expression of costimulatory molecules, decreased motility, and preferential priming of Ag-specific Foxp3(+) regulatory T cells. This tolerogenic DC phenotype and function was marked by a new putative biomarker revealed by the microarray: a cohort of pancreatic enzymes (trypsin, carboxypeptidase, elastase, and others) not previously reported in DC. These enzymes were strongly upregulated in the splenic DC from vaccinated WT mice and suppressed in the splenic DC of vaccinated MUC1.Tg mice. Suppression of the enzymes was dependent on regulatory T cells and on signaling through the IL-10R and correlated with global downregulation of DC immunostimulatory phenotype and function.

Vaccines based on abnormal self-antigens as tumor-associated antigens: immune regulation.

Abnormal expression of "self" antigens on tumors compared with normal cells provides opportunities and challenges for development of cancer vaccines. We review recent work in pre-clinical transgenic mouse models and in clinical trials that has elucidated multiple regulatory mechanisms that interfere with the induction of effective immunity. We discuss these as being either part of the normal function of the immune system or being driven by the tumor microenvironment. Collectively this work shows that it is possible to design vaccines based on tumor-associated antigens and elicit effective immunity against abnormal expression of these antigens on tumors without causing autoimmunity.

Urine scRNAseq reveals new insights into the bladder tumor immune microenvironment.

Due to bladder tumors' contact with urine, urine-derived cells (UDCs) may serve as a surrogate for monitoring the tumor microenvironment (TME) in bladder cancer (BC). However, the composition of UDCs and the extent to which they mirror the tumor remain poorly characterized. We generated the first single-cell RNA-sequencing of BC patient UDCs with matched tumor and peripheral blood mononuclear cells (PBMC). BC urine was more cellular than healthy donor (HD) urine, containing multiple immune populations including myeloid cells, CD4+ and CD8+ T cells, natural killer (NK) cells, B cells, and dendritic cells (DCs) in addition to tumor and stromal cells. Immune UDCs were transcriptionally more similar to tumor than blood. UDCs encompassed cytotoxic and activated CD4+ T cells, exhausted and tissue-resident memory CD8+ T cells, macrophages, germinal-center-like B cells, tissue-resident and adaptive NK cells, and regulatory DCs found in tumor but lacking or absent in blood. Our findings suggest BC UDCs may be surrogates for the TME and serve as therapeutic biomarkers.

Natural Killer Cell Dysfunction In Human Bladder Cancer Is Caused By Tissue-Specific Suppression of SLAMF6 Signaling.

NK cells are innate lymphocytes critical for surveillance of viruses and tumors, however the mechanisms underlying NK cell dysfunction in cancer are incompletely understood. We assessed the effector function of NK cells from bladder cancer patients and found severe dysfunction in NK cells derived from tumors versus peripheral blood. While both peripheral and tumor-infiltrating NK cells exhibited conserved patterns of inhibitory receptor over-expression, this did not explain the observed defects in NK surveillance in bladder tumors. Rather, TME-specific TGF-ß and metabolic perturbations such as hypoxia directly suppressed NK cell function. Specifically, an oxygen-dependent reduction in signaling through SLAMF6 was mechanistically responsible for poor NK cell function, as tumor-infiltrating NK cells cultured ex vivo under normoxic conditions exhibited complete restoration of function, while deletion of SLAMF6 abrogated NK cell cytolytic function even under normoxic conditions. Collectively, this work highlights the role of tissue-specific factors in dictating NK cell function, and implicates SLAMF6 signaling as a rational target for immuno-modulation to improve NK cell function in bladder cancer.

TIM-3 and TIGIT are possible immune checkpoint targets in patients with bladder cancer.

The resurgence of immunotherapy as an effective anticancer strategy has been coupled with more mature understandings of the underlying immune pathways and the development of novel immune checkpoint targets. The clinical development of antibodies first directed against cytotoxic T-lymphocyte-associated antigen 4, and later against program death 1, achieved durable disease control in a subset of patients across a large number of tumor types. Previous work demonstrates that targeting the programmed death 1 pathway alone does not result in complete restoration of T cell function and in some cancers, targeting this axis does not restore T cell function at all, suggesting a need to identify other molecules and inhibitory pathways that are involved in T cell exhaustion. In a comprehensive immune profiling study of patients with bladder cancer, we demonstrate T-cell immunoglobulin domain and mucin domain-containing molecule and T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain as possible targets as perhaps monotherapy or in combination with other immune checkpoint inhibitors.

Oncological Screening of Kidney Tumors After Renal Transplantation.

BACKGROUND: Among renal transplant recipients, renal cell carcinoma in the native kidneys represents the most common solid tumor. At the Department of Surgery, Transplantation and Gastroenterology of Semmelweis University annual control abdominal ultrasound examination is recommended for transplant patients. Our goal was to evaluate the effectiveness of the ultrasound screening program at our institute and to learn about the characteristics of shrunken kidney tumors. METHODS: Retrospectively, we processed the results of abdominal and pelvic ultrasound examinations of 1687 kidney transplant patients, which were performed at our institute between January 1, 2012 and December 31, 2016. RESULTS: A total of 26 tumors were detected during the abovementioned period of time, of which 18 were renal cancers. Renal cancer was significantly (P = 0.029) more common in men. Seventeen renal cancers were classified as stage I and one as stage IV disease. The mean time of dialysis was 37.73 ± 24.37 months. The mean time between kidney transplantation and tumor recognition was 7.9 ± 6.29 years. The 5-year survival was 66%; however, it should be noted that only 1 patient lost his life due to his tumor disease. The mean time between the last 2 ultrasound examinations was 27.8 ± 23.89 months. Only 57% of tumors were detected by screening. No significant differences in tumor size, stage, and survival could be detected between screened and nonscreened renal cancer patients. CONCLUSIONS: Ultrasound examination at least every 2 years is an effective tool for the early detection of renal cell carcinoma of the shrunken kidneys.

Citrullinated fibrinogen forms densely packed clots with decreased permeability.

BACKGROUND: Fibrin, the main scaffold of thrombi, is susceptible to citrullination by PAD (peptidyl arginine deiminase) 4, secreted from neutrophils during the formation of neutrophil extracellular traps. Citrullinated fibrinogen (citFg) has been detected in human plasma as well as in murine venous thrombi, and it decreases the lysability and mechanical resistance of fibrin clots. OBJECTIVE: To investigate the effect of fibrinogen citrullination on the structure of fibrin clots. METHODS: Fibrinogen was citrullinated with PAD4 and clotted with thrombin. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to measure fiber thickness, fiber height/width ratio, and fiber persistence length in clots containing citFg. Fiber density was measured with laser scanning microscopy (LSM) and permeability measurements were carried out to estimate the porosity of the clots. The intra-fiber structure of fibrin was analyzed with small-angle X-ray scattering (SAXS). RESULTS: SEM images revealed a decrease in the median fiber diameter that correlated with the fraction of citFg in the clot, while the fiber width/length ratio remained unchanged according to AFM. With SAXS we observed that citrullination resulted in the formation of denser clots in line with increased fiber density shown by LSM. The permeability constant of citrullinated fibrin decreased more than 3-fold indicating significantly decreased porosity. SAXS also showed largely preserved periodicity in the longitudinal assembly of fibrin monomers. CONCLUSION: The current observations of thin fibers combined with dense packing and low porosity in the presence of citFg can provide a structural framework for the mechanical fragility and lytic resistance of citrullinated fibrin.

Tumor-Infiltrating Myeloid Cells Confer De Novo Resistance to PD-L1 Blockade through EMT-Stromal and Tgfß-Dependent Mechanisms.

SIGNIFICANCE: Most patients with bladder cancer do not respond to ICB targeting of the PD-L1 signaling axis. Our modeling applied a de novo resistance signature to show that tumor-infiltrating myeloid cells promote poor treatment response in a TGFß-dependent mechanism.

NKG2A and HLA-E define an alternative immune checkpoint axis in bladder cancer.

Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1)-blockade immunotherapies have limited efficacy in the treatment of bladder cancer. Here, we show that NKG2A associates with improved survival and responsiveness to PD-L1 blockade immunotherapy in bladder tumors that have high abundance of CD8+ T cells. In bladder tumors, NKG2A is acquired on CD8+ T cells later than PD-1 as well as other well-established immune checkpoints. NKG2A+ PD-1+ CD8+ T cells diverge from classically defined exhausted T cells through their ability to react to human leukocyte antigen (HLA) class I-deficient tumors using T cell receptor (TCR)-independent innate-like mechanisms. HLA-ABC expression by bladder tumors is progressively diminished as disease progresses, framing the importance of targeting TCR-independent anti-tumor functions. Notably, NKG2A+ CD8+ T cells are inhibited when HLA-E is expressed by tumors and partly restored upon NKG2A blockade in an HLA-E-dependent manner. Overall, our study provides a framework for subsequent clinical trials combining NKG2A blockade with other T cell-targeted immunotherapies, where tumors express higher levels of HLA-E.

Bacillus Calmette-Guerin (BCG): Its fight against pathogens and cancer.

Bacillus Calmette-Guerin (BCG) is the only FDA approved first line therapy for patients with nonmuscle invasive bladder cancer. Since the turn of the 20th century BCG has been used as a vaccine for protection against Mycobacterium tuberculosis (Mtb) and has also been found to have protection against nontuberculosis related pathogens. Recently the role of "trained immunity" has been identified as a possible mechanism for BCG vaccine-mediated immunity to Mtb. Similarly, BCG has been used as an immunotherapy for bladder cancer for more than 40 years, and the underlying mechanisms for BCG-mediated anti-tumor activity is poorly characterized. Several studies have shown that multiple immune pathways contribute to the immune response, and efficacy of intravesicle BCG as a cancer therapy. It is vital that we integrate our understanding of BCG as a vaccine and as a cancer therapeutic to facilitate design of future studies in order to maximize the immunotherapeutic potential of BCG. In this review we will outline the role of BCG as a vaccine, the known immune pathways that are activated by intravesical BCG and outline a potential clinical study integrating BCG vaccination prior to intravesicle instillation of BCG.