CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity.

Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR-Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.

MR1-dependent immune surveillance of the skin contributes to pathogenesis and is a photobiological target of UV light therapy in a mouse model of atopic dermatitis.

BACKGROUND: Mucosal-associated invariant T (MAIT) cells are unconventional T cells which recognize microbial metabolites presented by the major histocompatibility complex class I-related molecule MR1. Although MAIT cells have been shown to reside in human and murine skin, their contribution to atopic dermatitis (AD), an inflammatory skin disease associated with barrier dysfunction and microbial translocation, has not yet been determined. METHODS: Genetic deletion of MR1 and topical treatment with inhibitory MR1 ligands, which result in the absence and functional inhibition of MAIT cells, respectively, were used to investigate the role of MR1-dependent immune surveillance in a MC903-driven murine model of AD. RESULTS: The absence or inhibition of MR1 arrested AD disease progression through the blockade of both eosinophil activation and recruitment of IL-4- and IL-13-producing cells. In addition, the therapeutic efficacy of phototherapy against MC903-driven AD could be increased with prior application of folate, which photodegrades into the inhibitory MR1 ligand 6-formylpterin. CONCLUSION: We identified MAIT cells as sentinels and mediators of cutaneous type 2 immunity. Their pathogenic activity can be inhibited by topical application or endogenous generation, via phototherapy, of inhibitory MR1 ligands.

Nitric oxide added to the sweep gas of the oxygenator during cardiopulmonary bypass in infants: A pilot randomized controlled trial.

Our objective was to assess the effect of nitric oxide added to the sweep gas of the oxygenator during cardiopulmonary bypass (CPB) in infants on platelet count, platelet function, clinical outcomes, and safety. A randomized, double-blinded, placebo-controlled clinical trial in infants less than a year of age undergoing cardiac surgery requiring CPB was undertaken. Nitric oxide at a dose of 20 ppm was added to the sweep gas in the treatment group. Blood was collected at baseline and prior to separation from CPB to measure platelet count and function as determined by responsiveness to specific agonists. Clinical outcomes were observed through hospital discharge. Methemoglobin levels were measured preoperatively, at the conclusion of CPB, and upon admission to the ICU. Forty patients consented and were randomized in the trial. Eighteen patients were randomized to the treatment group and 22 were included in the placebo group. The groups were similar in terms of age, weight, gender, and surgical complexity. No significant differences were found in measures of platelet count, platelet response to agonist, or clinical outcomes. Patients in the treatment group had higher methemoglobin levels after receiving nitric oxide, but no levels approached toxicity (maximum 2.4%). Nitric oxide added to the sweep gas of the oxygenator during CPB in infants did not have an appreciable effect on the preservation of platelet count, platelet responsiveness to agonist, or clinical outcomes. Methemoglobin levels were increased after receiving nitric oxide but were far below a toxic level of 15%.

Isolation and characterization of NY-ESO-1-specific T cell receptors restricted on various MHC molecules.

Tumor-specific T cell receptor (TCR) gene transfer enables specific and potent immune targeting of tumor antigens. Due to the prevalence of the HLA-A2 MHC class I supertype in most human populations, the majority of TCR gene therapy trials targeting public antigens have employed HLA-A2-restricted TCRs, limiting this approach to those patients expressing this allele. For these patients, TCR gene therapy trials have resulted in both tantalizing successes and lethal adverse events, underscoring the need for careful selection of antigenic targets. Broad and safe application of public antigen-targeted TCR gene therapies will require (i) selecting public antigens that are highly tumor-specific and (ii) targeting multiple epitopes derived from these antigens by obtaining an assortment of TCRs restricted by multiple common MHC alleles. The canonical cancer-testis antigen, NY-ESO-1, is not expressed in normal tissues but is aberrantly expressed across a broad array of cancer types. It has also been targeted with A2-restricted TCR gene therapy without adverse events or notable side effects. To enable the targeting of NY-ESO-1 in a broader array of HLA haplotypes, we isolated TCRs specific for NY-ESO-1 epitopes presented by four MHC molecules: HLA-A2, -B07, -B18, and -C03. Using these TCRs, we pilot an approach to extend TCR gene therapies targeting NY-ESO-1 to patient populations beyond those expressing HLA-A2.

Coronavirus Disease among Persons with Sickle Cell Disease, United States, March 20-May 21, 2020.

Sickle cell disease (SCD) disproportionately affects Black or African American persons in the United States and can cause multisystem organ damage and reduced lifespan. Among 178 persons with SCD in the United States who were reported to an SCD-coronavirus disease case registry, 122 (69%) were hospitalized and 13 (7%) died.

Cystic Fibrosis Plasma Blunts the Immune Response to Bacterial Infection.

Cystic fibrosis (CF) is caused by mutations of the gene encoding the CF transmembrane conductance regulator. It remains unclear whether the abnormal immune response in CF involves extrinsic signals released from the external or internal environment. We sought to characterize the peripheral immune signatures in CF and its association with clinical phenotypes. Healthy peripheral blood mononuclear cells (PBMCs) were cultured with plasma from CF probands (CFPs) or healthy control subjects (HCs) followed by nCounter gene and microRNA (miRNA) profiling. A discovery cohort of 12 CFPs and 12 HCs and a validation cohort of 103 CFPs and 31 HCs (our previous microarray data [GSE71799]) were analyzed to characterize the composition of cultured immune cells and establish a miRNA‒mRNA network. Cell compositions and miRNA profiles were associated with clinical characteristics of the cohorts. Significantly differentially expressed genes and abundance of myeloid cells were downregulated in PMBCs after culture with CF plasma (P < 0.05). Top-ranked miRNAs that increased in response to CF plasma (adjusted P < 0.05) included miR-155 and miR-146a, which target many immune-related genes, such as IL-8. Pseudomonas aeruginosa infection was negatively associated with abundance of monocytes and the presence of those regulatory miRNAs. Extrinsic signals in plasma from patients with CF led to monocyte inactivation and miRNA upregulation in PBMCs. An improved understanding of the immune effects of extrinsic factors in CF holds great promise for integrating immunomodulatory cell therapies into current treatment strategies in CF.

Identification of molecular signatures of cystic fibrosis disease status with plasma-based functional genomics.

Although cystic fibrosis (CF) is attributed to dysfunction of a single gene, the relationships between the abnormal gene product and the development of inflammation and progression of lung disease are not fully understood, which limits our ability to predict an individual patient's clinical course and treatment response. To better understand CF progression, we characterized the molecular signatures of CF disease status with plasma-based functional genomics. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured with plasma samples from CF patients ( n = 103) and unrelated, healthy controls ( n = 31). Gene expression levels were measured with an Affymetrix microarray (GeneChip Human Genome U133 Plus 2.0). Peripheral blood samples from a subset of the CF patients ( n = 40) were immunophenotyped by flow cytometry, and the data were compared with historical data for age-matched healthy controls ( n = 351). Plasma samples from another subset of CF patients ( n = 56) and healthy controls ( n = 16) were analyzed by multiplex enzyme-linked immunosorbent assay (ELISA) for numerous cytokines and chemokines. Principal component analysis and hierarchical clustering of induced transcriptional data revealed disease-specific plasma-induced PBMC profiles. Among 1,094 differentially expressed probe sets, 51 genes were associated with pancreatic sufficient status, and 224 genes were associated with infection with Pseudomonas aeruginosa. The flow cytometry and ELISA data confirmed that various immune modulators are relevant contributors to the CF molecular signature. This study provides strong evidence for distinct molecular signatures among CF patients. An understanding of these molecular signatures may lead to unique molecular markers that will enable more personalized prognoses, individualized treatment plans, and rapid monitoring of treatment response.

The good, the (not so) bad and the ugly of immune homeostasis in melanoma.

Within the immune system multiple mechanisms balance the need for efficient pathogen recognition and destruction with the prevention of tissue damage by excessive, inappropriate or even self-targeting (auto)immune reactions. This immune homeostasis is a tightly regulated system which fails during tumor development, often due to the hijacking of its essential self-regulatory mechanisms by cancer cells. It is facilitated not only by tumor intrinsic properties, but also by the microbiome, host genetics and other factors. In certain ways many cancers can therefore be considered a rare failure of immune control rather than an uncommon or rare disease of the tissue of origin, as the acquisition of potentially oncogenic traits through mutation occurs constantly in most tissues during proliferation. Normally, aberrant cells are well-controlled by cell intrinsic (repair or apoptosis) and extrinsic (immune) mechanisms. However, occasionally oncogenic cells survive and escape control. Melanoma is one of the first cancer types where treatments aimed at restoring and enhancing an immune response to regain control over the tumor have been used with various success rates. With the advent of "modern" immunotherapeutics such as anti-CTLA-4 or anti-PD-1 antibodies that both target negative immune-regulatory pathways on immune cells resulting in durable responses in a proportion of patients, the importance of the interplay between the immune system and cancer has been established beyond doubt.

Increased Expression of Plasma-Induced ABCC1 mRNA in Cystic Fibrosis.

The ABCC1 gene is structurally and functionally related to the cystic fibrosis transmembrane conductance regulator gene (CFTR). Upregulation of ABCC1 is thought to improve lung function in patients with cystic fibrosis (CF); the mechanism underlying this effect is unknown. We analyzed the ABCC1 promoter single nucleotide polymorphism (SNP rs504348), plasma-induced ABCC1 mRNA expression levels, and ABCC1 methylation status and their correlation with clinical variables among CF subjects with differing CFTR mutations. We assigned 93 CF subjects into disease severity groups and genotyped SNP rs504348. For 23 CF subjects and 7 healthy controls, donor peripheral blood mononuclear cells (PBMCs) stimulated with plasma underwent gene expression analysis via qRT-PCR. ABCC1 promoter methylation was analyzed in the same 23 CF subjects. No significant correlation was observed between rs504348 genotypes and CF disease severity, but pancreatic insufficient CF subjects showed increased colonization with any form of Pseudomonas aeruginosa (OR = 3.125, 95% CI: 1.192-8.190) and mucoid P. aeruginosa (OR = 5.075, 95% CI: 1.307-28.620) compared to the pancreatic sufficient group. A significantly higher expression of ABCC1 mRNA was induced by CF plasma compared to healthy control plasma (p < 0.001). CF subjects with rs504348 (CC/CG) also had higher mRNA expression compared to those with the ancestral GG genotype (p < 0.005). ABCC1 promoter was completely unmethylated; therefore, we did not detect any association between methylation and CF disease severity. In silico predictions suggested that histone modifications are crucial for regulating ABCC1 expression in PBMCs. Our results suggest that ABCC1 expression has a role in CFTR activity thereby increasing our understanding of the molecular underpinnings of the clinical heterogeneity in CF.

A Pilot Study of Antithrombin Replacement Prior to Cardiopulmonary Bypass in Neonates.

Neonates have low levels of antithrombin. Inadequate anticoagulation during cardiopulmonary bypass (CPB) due to low antithrombin activity may result in a poor preservation of the coagulation system during bypass. We hypothesize that antithrombin replacement to neonates prior to CPB will preserve the hemostatic system and result in less postoperative bleeding. A randomized, double-blinded, placebo-controlled pilot study of antithrombin replacement to neonates prior to CPB was conducted. Preoperative antithrombin levels determined the dose of recombinant antithrombin or placebo to be given. Antithrombin levels were measured following the dosing of the antithrombin/placebo, after initiation of bypass, near the completion of bypass, and upon intensive care unit admission. Eight subjects were enrolled. No subject had safety concerns. Mediastinal exploration occurred in two antithrombin subjects and one placebo subject. Antithrombin activity levels were significantly higher in the treated group following drug administration; levels continued to be higher than preoperatively but not different from the placebo group at all other time points. Total heparin administration was less in the antithrombin group; measurements of blood loss were similar in both groups. A single dose of recombinant antithrombin did not maintain 100% activity levels throughout the entire operation. Although no safety concerns were identified in this pilot study, a larger trial is necessary to determine clinical efficacy.

Flt3 ligand expands CD4+ FoxP3+ regulatory T cells in human subjects.

CD4(+) CD25(+) FoxP3(+) naturally occurring regulatory T (Treg) cells play a crucial role in the maintenance of immune tolerance and in preventing autoimmune pathology. Interventions that expand Treg cells are highly desirable, as they may offer novel treatment options in a variety of autoimmune and transplantation settings. Paralleling previous preclinical studies, we demonstrate here that administration of the hematopoietic growth factor Flt3L to human subjects increases the frequency and absolute number of Treg cells, and reduces the ratio of CD8(+) T cells to Treg cells in the peripheral blood. The increase in Treg cells was due to enhanced Treg-cell proliferation rather than release of Treg cells from the thymus. Further studies revealed that Flt3L-induced proliferation of Treg cells was an indirect effect that occurred via the interaction of Treg cells with the Flt3L-expanded pool of CD1c(+) myeloid dendritic cells. On the basis of these findings, Flt3L may represent a promising agent for promoting immune tolerance in a variety of clinical settings.

Evolving role of tumor antigens for future melanoma therapies.

Human tumor rejection antigens recognized by T lymphocytes were first defined in the early 1990s and the identification of shared tumor-restricted antigens sparked hopes for the development of a therapeutic vaccination to treat cancer, including melanoma. Despite decades of intense preclinical and clinical research, the success of anticancer vaccines based on these antigens has been limited. While melanoma is a highly immunogenic tumor, the ability to prime immunity with vaccines has not generally translated into objective disease regression. However, with the development of small molecules targeting oncogenic proteins, such as V600-mutated BRAF, and immune checkpoint inhibitors with demonstrable long-lasting clinical benefit, new opportunities for antigen-targeted directed therapies are emerging.

Early Impacts of COVID-19 on Select Hospices: Operations, Care Delivery, and Service Utilization.

OBJECTIVE: The COVID-19 public health emergency (PHE) has important implications for health care service delivery. Little is understood about how the PHE impacted community-based hospice providers and service delivery to hospice-eligible beneficiaries. The aim of this study was to describe hospice response to the PHE and correlated impacts on beneficiary receipt of hospice support services delivered to hospice-eligible beneficiaries participating in the Centers for Medicare & Medicaid Services (CMS) Medicare Care Choices Model (MCCM), a national model testing the provision of certain hospice-like supportive services with concurrent usual care among seriously ill, community-residing Medicare beneficiaries that have not elected to receive hospice care. METHODS: We employed descriptive analysis using concurrent qualitative and quantitative data sources, consisting of provider surveys, beneficiary-level encounter data submitted by hospices, and Medicare administrative claims describing beneficiary service utilization. The sample included both hospice providers (N = 82) and beneficiaries (N = 2294) voluntarily participating in MCCM. RESULTS: Nearly all participating MCCM hospices adopted operational changes to address their staff and beneficiaries' safety during the COVID-19 PHE. We report changes to service delivery, including declines in total encounters as well as service modality, and the types of services provided. CONCLUSIONS: While the analyses reported indicate that seriously ill Medicare beneficiaries participating in MCCM were directly impacted by the PHE, we are still unclear whether changes in the service modality and encounters by provider type and the decline in average service counts per beneficiary are driven more by hospices or by beneficiary decisions to minimize exposure. Future research should attempt to disentangle these factors.

Human kallikrein 4 signal peptide induces cytotoxic T cell responses in healthy donors and prostate cancer patients.

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201+ KLK4 + cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.

CFTR-mediated monocyte/macrophage dysfunction revealed by cystic fibrosis proband-parent comparisons.

Cystic fibrosis (CF) is an inherited disorder caused by biallelic mutations of the CF transmembrane conductance regulator (CFTR) gene. Converging evidence suggests that CF carriers with only 1 defective CFTR copy are at increased risk for CF-related conditions and pulmonary infections, but the molecular mechanisms underpinning this effect remain unknown. We performed transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) of CF child-parent trios (proband, father, and mother) and healthy control (HC) PBMCs or THP-1 cells incubated with the plasma of these participants. Transcriptomic analyses revealed suppression of cytokine-enriched immune-related genes (IL-1ß, CXCL8, CREM), implicating lipopolysaccharide tolerance in innate immune cells (monocytes) of CF probands and their parents. These data suggest that a homozygous as well as a heterozygous CFTR mutation can modulate the immune/inflammatory system. This conclusion is further supported by the finding of lower numbers of circulating monocytes in CF probands and their parents, compared with HCs, and the abundance of mononuclear phagocyte subsets, which correlated with Pseudomonas aeruginosa infection, lung disease severity, and CF progression in the probands. This study provides insight into demonstrated CFTR-related innate immune dysfunction in individuals with CF and carriers of a CFTR mutation that may serve as a target for personalized therapy.

Comorbidities are risk factors for hospitalization and serious COVID-19 illness in children and adults with sickle cell disease.

Patients with sickle cell disease (SCD) are at high risk of developing serious infections, therefore, understanding the impact that severe acute respiratory syndrome coronavirus 2 infection has on this population is important. We sought to identify factors associated with hospitalization and serious COVID-19 illness in children and adults with SCD.We established the international SECURE-SCD Registry to collect data on patients with SCD and COVID-19 illness. We used multivariable logistic models to estimate the independent effects of age, sex, genotype, hydroxyurea, and SCD-related and -nonrelated comorbidities on hospitalization, serious COVID-19 illness, and pain as a presenting symptom during COVID-19 illness. As of 23 March 2021, 750 COVID-19 illness cases in patients with SCD were reported to the registry. We identified history of pain (relative risk [RR], 2.15; P < .0001) and SCD heart/lung comorbidities (RR, 1.61; P = .0001) as risk factors for hospitalization in children. History of pain (RR, 1.78; P = .002) was also a risk factor for hospitalization in adults. Children with history of pain (RR, 3.09; P = .009), SCD heart/lung comorbidities (RR, 1.76; P = .03), and SCD renal comorbidities (RR, 3.67; P < .0001) and adults with history of pain (RR 1.94, P = .02) were at higher risk of developing serious COVID-19 illness. History of pain and SCD renal comorbidities also increased risk of pain during COVID-19 in children; history of pain, SCD heart/lung comorbidities, and female sex increased risk of pain during COVID-19 in adults. Hydroxyurea showed no effect on hospitalization and COVID-19 severity, but it lowered the risk of presenting with pain in adults during COVID-19.

Ropporin-1 and 1B Are Widely Expressed in Human Melanoma and Evoke Strong Humoral Immune Responses.

Antibodies that block immune regulatory checkpoints (programmed cell death 1, PD-1 and cytotoxic T-lymphocyte-associated antigen 4, CTLA-4) to mobilise immunity have shown unprecedented clinical efficacy against cancer, demonstrating the importance of antigen-specific tumour recognition. Despite this, many patients still fail to benefit from these treatments and additional approaches are being sought. These include mechanisms that boost antigen-specific immunity either by vaccination or adoptive transfer of effector cells. Other than neoantigens, epigenetically regulated and shared antigens such as NY-ESO-1 are attractive targets; however, tissue expression is often heterogeneous and weak. Therefore, peptide-specific therapies combining multiple antigens rationally selected to give additive anti-cancer benefits are necessary to achieve optimal outcomes. Here, we show that Ropporin-1 (ROPN1) and 1B (ROPN1B), cancer restricted antigens, are highly expressed and immunogenic, inducing humoral immunity in patients with advanced metastatic melanoma. By multispectral immunohistochemistry, 88.5% of melanoma patients tested (n = 54/61) showed ROPN1B expression in at least 1 of 2/3 tumour cores in tissue microarrays. Antibody responses against ROPN1A and ROPN1B were detected in 71.2% of melanoma patients tested (n = 74/104), with increased reactivity seen with more advanced disease stages. Thus, ROPN1A and ROPN1B may indeed be viable targets for cancer immunotherapy, alone or in combination with other cancer antigens, and could be combined with additional therapies such as immune checkpoint blockade.

Distinct Dysfunctional States of Circulating Innate-Like T Cells in Metabolic Disease.

The immune system plays a significant role in controlling systemic metabolism. Innate-like T (ILT) cells in particular, such as mucosal-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells and γδ T cell receptor expressing cells, have been reported to promote metabolic homeostasis. However, these different ILT cell subsets have, to date, been generally studied in isolation. Here we conducted a pilot study assessing the phenotype and function of circulating MAIT, iNKT, and Vδ2+ T cells in a small cohort of 10 people with obesity and type 2 diabetes (T2D), 10 people with obesity but no diabetes, and 12 healthy individuals. We conducted phenotypic analysis by flow cytometry ex vivo, and then functional analysis after in vitro stimulation using either PMA/ionomycin or synthetic agonists, or precursors thereof, for each of the cell-types; use of the latter may provide important knowledge for the development of novel therapeutics aimed at activating human ILT cells. The results of our pilot study, conducted on circulating cells, show clear dysfunction of all three ILT cell subsets in obese and obese T2D patients, as compared to healthy controls. Importantly, while both iNKT and Vδ2+ T cell dysfunctions were characterized by diminished IL-2 and interferon-γ production, the distinct dysfunctional state of MAIT cells was instead defined by skewed subset composition, heightened sensitivity to T cell receptor engagement and unchanged production of all measured cytokines.