Role of paraoxonase 3 in regulating ENaC-mediated Na+ transport in the distal nephron.

Paraoxonase 3 (PON3) is expressed in the aldosterone-sensitive distal nephron, where filtered Na+ is reabsorbed mainly via the epithelial Na+ channel (ENaC) and Na+ -coupled co-transporters. We previously showed that PON3 negatively regulates ENaC through a chaperone mechanism. The present study aimed to determine the physiological role of PON3 in renal Na+ and K+ homeostasis. Pon3 knockout (KO) mice had higher amiloride-induced natriuresis and lower plasma [K+ ] at baseline. Single channel recordings in split-open tubules showed that the number of active channels per patch was significantly higher in KO mice, resulting in a higher channel activity in the absence of PON3. Although whole kidney abundance of ENaC subunits was not altered in Pon3 KOs, ENaC gamma subunit was more apically distributed within the connecting tubules and cortical collecting ducts of Pon3 KO kidneys. Additionally, small interfering RNA-mediated knockdown of PON3 in cultured mouse cortical collecting duct cells led to an increased surface abundance of ENaC gamma subunit. As a result of lower plasma [K+ ], sodium chloride co-transporter phosphorylation was enhanced in the KO kidneys, a phenotype that was corrected by a high K+ diet. Finally, PON3 expression was upregulated in mouse kidneys under dietary K+ restriction, potentially providing a mechanism to dampen ENaC activity and associated K+ secretion. Taken together, our results show that PON3 has a role in renal Na+ and K+ homeostasis through regulating ENaC functional expression in the distal nephron. KEY POINTS: Paraoxonase 3 (PON3) is expressed in the distal nephron of mouse kidneys and functions as a molecular chaperone to reduce epithelial Na+ channel (ENaC) expression and activity in heterologous expression systems. We examined the physiological role of PON3 in renal Na+ and K+ handling using a Pon3 knockout (KO) mouse model. At baseline, Pon3 KO mice had lower blood [K+ ], more functional ENaC in connecting tubules/cortical collecting ducts, higher amiloride-induced natriuresis, and enhanced sodium chloride co-transporter (NCC) phosphorylation. Upon challenge with a high K+ diet, Pon3 KO mice had normalized blood [K+ ] and -NCC phosphorylation but lower circulating aldosterone levels compared to their littermate controls. Kidney PON3 abundance was altered in mice under dietary K+ loading or K+ restriction, providing a potential mechanism for regulating ENaC functional expression and renal Na+ and K+ homeostasis in the distal nephron.

Control of ENaC ubiquitination.

Ubiquitination influences the expression of the epithelial Na+ channel (ENaC). We assessed the mechanisms of selective ubiquitination of the mature, cleaved form of γENaC in both native rodent kidneys and Fisher rat thyroid (FRT) cells expressing the channel heterologously. In both models, singly cleaved and fully cleaved γENaCs were strongly ubiquitinated, implying that the second cleavage releasing an inhibitory peptide was not essential for the process. To see whether location of the protein in or near the apical membrane rather than cleavage per se influences ubiquitination, we studied mutants of γENaC in which cleavage sites are abolished. These subunits were ubiquitinated only when coexpressed with α- and ßENaC, facilitating trafficking through the Golgi apparatus. To test whether reaching the apical surface is necessary we performed in situ surface biotinylation and measured ENaC ubiquitination in the apical membrane of rat kidney. Ubiquitination of cleaved γENaC was similar in whole kidney and surface fractions, implying that both apical and subapical channels could be modified. In FRT cells, inhibiting clathrin-mediated endocytosis with Dyngo-4a increased both total and ubiquitinated γENaC at the cell surface. Finally, we tested the idea that increased intracellular Na+ could stimulate ubiquitination. Administration of amiloride to block Na+ entry through the channels did not affect ubiquitination of γENaC in either FRT cells or the rat kidney. However, presumed large increases in cellular Na+ produced by monensin in FRT cells or acute Na+ repletion in rats increased ubiquitination and decreased overall ENaC expression.NEW & NOTEWORTHY We have explored the mechanisms underlying the ubiquitination of the γ subunit of epithelial Na+ channel (ENaC), a process believed to control channel internalization and degradation. We previously reported that the mature, cleaved form of the subunit is selectively ubiquitinated. Here we show that this specificity arises not from the cleavage state of the protein but from its location in the cell. We also show that under some conditions, increased intracellular Na+ can stimulate ENaC ubiquitination.

Kcnma1 alternative splicing in mouse kidney: regulation during development and by dietary K+ intake.

The pore-forming α-subunit of the large-conductance K+ (BK) channel is encoded by a single gene, KCNMA1. BK channel-mediated K+ secretion in the kidney is crucial for overall renal K+ homeostasis in both physiological and pathological conditions. BK channels achieve phenotypic diversity by various mechanisms, including substantial exon rearrangements at seven major alternative splicing sites. However, KCNMA1 alternative splicing in the kidney has not been characterized. The present study aims to identify the major splice variants of mouse Kcnma1 in whole kidney and distal nephron segments. We designed primers that specifically cross exons within each alternative splice site of mouse Kcnma1 and performed real-time quantitative RT-PCR (RT-qPCR) to quantify relative abundance of each splice variant. Our data suggest that Kcnma1 splice variants within mouse kidney are less diverse than in the brain. During postnatal kidney development, most Kcnma1 splice variants at site 5 and the COOH terminus increase in abundance over time. Within the kidney, the regulation of Kcnma1 alternative exon splicing within these two sites by dietary K+ loading is both site and sex specific. In microdissected distal tubules, the Kcnma1 alternative splicing profile, as well as its regulation by dietary K+, are distinctly different than in the whole kidney, suggesting segment and/or cell type specificity in Kcnma1 splicing events. Overall, our data provide evidence that Kcnma1 alternative splicing is regulated during postnatal development and may serve as an important adaptive mechanism to dietary K+ loading in mouse kidney.NEW & NOTEWORTHY We identified the major Kcnma1 splice variants that are specifically expressed in the whole mouse kidney or aldosterone-sensitive distal nephron segments. Our data suggest that Kcnma1 alternative splicing is developmentally regulated and subject to changes in dietary K+.

Secukinumab in enthesitis-related arthritis and juvenile psoriatic arthritis: a randomised, double-blind, placebo-controlled, treatment withdrawal, phase 3 trial.

BACKGROUND: Treatment options in patients with enthesitis-related arthritis (ERA) and juvenile psoriatic arthritis (JPsA) are currently limited. This trial aimed to demonstrate the efficacy and safety of secukinumab in patients with active ERA and JPsA with inadequate response to conventional therapy. METHODS: In this randomised, double-blind, placebo-controlled, treatment-withdrawal, phase 3 trial, biologic-naïve patients (aged 2 to <18 years) with active disease were treated with open-label subcutaneous secukinumab (75/150 mg in patients <50/≥50 kg) in treatment period (TP) 1 up to week 12, and juvenile idiopathic arthritis (JIA) American College of Rheumatology 30 responders at week 12 were randomised 1:1 to secukinumab or placebo up to 100 weeks. Patients who flared in TP2 immediately entered open-label secukinumab TP3 that lasted up to week 104. Primary endpoint was time to disease flare in TP2. RESULTS: A total of 86 patients (median age, 14 years) entered open-label secukinumab in TP1. In TP2, responders (ERA, 44/52; JPsA, 31/34) received secukinumab or placebo. The study met its primary end point and demonstrated a statistically significant longer time to disease flare in TP2 for ERA and JPsA with secukinumab versus placebo (27% vs 55%, HR, 0.28; 95% CI 0.13 to 0.63; p<0.001). Exposure-adjusted incidence rates (per 100 patient-years (PY), 95% CI) for total patients were 290.7/100 PY (230.2 to 362.3) for adverse events and 8.2/100 PY (4.1 to 14.6) for serious adverse events in the overall JIA population. CONCLUSIONS: Secukinumab demonstrated significantly longer time to disease flare than placebo in children with ERA and JPsA with a consistent safety profile with the adult indications of psoriatic arthritis and axial spondyloarthritis. TRIAL REGISTRATION NUMBER: NCT03031782.

Estimating Phosphorescent Emission Energies in IrIII Complexes Using Large-Scale Quantum Computing Simulations.

Here we calculate T1 →S0 transition energies in nine phosphorescent iridium complexes using the iterative qubit coupled cluster (iQCC) method to determine if quantum simulations have any advantages over classical methods. These simulations would require a gate-based quantum computer with at least 72 fully-connected logical qubits. Since such devices do not yet exist, we demonstrate the iQCC method using a purpose-built quantum simulator on classical hardware. The results are compared to a selection of common DFT functionals, ab initio methods, and empirical data. iQCC is found to match the accuracy of the best DFT functionals, but with a better correlation coefficient, demonstrating that it is better at predicting the structure-property relationship. Results indicate that the iQCC method has the required accuracy to design organometallic complexes when deployed on emerging quantum hardware and sets an industrially relevant target for demonstrating quantum advantage.

COM902, a novel therapeutic antibody targeting TIGIT augments anti-tumor T cell function in combination with PVRIG or PD-1 pathway blockade.

Immune checkpoint inhibitors (ICIs) have emerged as promising therapies for the treatment of cancer. However, existing ICIs, namely PD-(L)1 and CTLA-4 inhibitors, generate durable responses only in a subset of patients. TIGIT is a co-inhibitory receptor and member of the DNAM-1 family of immune modulating proteins. We evaluated the prevalence of TIGIT and its cognate ligand, PVR (CD155), in human cancers by assessing their expression in a large set of solid tumors. TIGIT is expressed on CD4+ and CD8+ TILs and is upregulated in tumors compared to normal tissues. PVR is expressed on tumor cells and tumor-associated macrophages from multiple solid tumors. We explored the therapeutic potential of targeting TIGIT by generating COM902, a fully human anti-TIGIT hinge-stabilized IgG4 monoclonal antibody that binds specifically to human, cynomolgus monkey, and mouse TIGIT, and disrupts the binding of TIGIT with PVR. COM902, either alone or in combination with a PVRIG (COM701) or PD-1 inhibitor, enhances antigen-specific human T cell responses in-vitro. In-vivo, a mouse chimeric version of COM902 in combination with an anti-PVRIG or anti-PD-L1 antibody inhibited tumor growth and increased survival in two syngeneic mouse tumor models. In summary, COM902 enhances anti-tumor immune responses and is a promising candidate for the treatment of advanced malignancies.

PVRIG is a novel natural killer cell immune checkpoint receptor in acute myeloid leukemia

This study explored the novel immune checkpoint poliovirus receptor-related immunoglobulin domain-containing (PVRIG) in acute myeloid leukemia (AML). We showed that AML patient blasts consistently expressed the PVRIG ligand (poliovirus receptor-related 2, PVRL2). Furthermore, PVRIG blockade significantly enhanced NK cell killing of PVRL2+, poliovirus receptor (PVR)lo AML cell lines, and significantly increased NK cell activation and degranulation in the context of patient primary AML blasts. However, in AML patient bone marrow, NK cell PVRIG expression levels were not increased. To understand how PVRIG blockade might potentially be exploited therapeutically, we investigated the biology of PVRIG and revealed that NK cell activation resulted in reduced PVRIG expression on the cell surface. This occurred whether NK cells were activated by tumour cell recognition, cytokines (IL-2 and IL-12) or activating receptor stimulation (CD16 and NKp46). PVRIG was present at higher levels in the cytoplasm than on the cell surface, particularly on CD56bright NK cells, which further increased cytoplasmic PVRIG levels following IL-2 and IL-12 activation. PVRIG was continually transported to the cell surface via the endoplasmic reticulum (ER) and Golgi in both unstimulated and activated NK cells. Taken together, our findings suggest that anti- PVRIG blocking antibody functions by binding to surface-bound PVRIG, which undergoes rapid turnover in both unstimulated and activated NK cells. We conclude that the PVRIGPVRL2 immune checkpoint axis can feasibly be targeted with PVRIG blocking antibody for NK-mediated immunotherapy of PVRL2+ AML.

Effect of Canakinumab vs Placebo on Survival Without Invasive Mechanical Ventilation in Patients Hospitalized With Severe COVID-19: A Randomized Clinical Trial.

Importance: Effective treatments for patients with severe COVID-19 are needed. Objective: To evaluate the efficacy of canakinumab, an anti-interleukin-1ß antibody, in patients hospitalized with severe COVID-19. Design, Setting, and Participants: This randomized, double-blind, placebo-controlled phase 3 trial was conducted at 39 hospitals in Europe and the United States. A total of 454 hospitalized patients with COVID-19 pneumonia, hypoxia (not requiring invasive mechanical ventilation [IMV]), and systemic hyperinflammation defined by increased blood concentrations of C-reactive protein or ferritin were enrolled between April 30 and August 17, 2020, with the last assessment of the primary end point on September 22, 2020. Intervention: Patients were randomly assigned 1:1 to receive a single intravenous infusion of canakinumab (450 mg for body weight of 40-<60 kg, 600 mg for 60-80 kg, and 750 mg for >80 kg; n = 227) or placebo (n = 227). Main Outcomes and Measures: The primary outcome was survival without IMV from day 3 to day 29. Secondary outcomes were COVID-19-related mortality, measurements of biomarkers of systemic hyperinflammation, and safety evaluations. Results: Among 454 patients who were randomized (median age, 59 years; 187 women [41.2%]), 417 (91.9%) completed day 29 of the trial. Between days 3 and 29, 198 of 223 patients (88.8%) survived without requiring IMV in the canakinumab group and 191 of 223 (85.7%) in the placebo group, with a rate difference of 3.1% (95% CI, -3.1% to 9.3%) and an odds ratio of 1.39 (95% CI, 0.76 to 2.54; P = .29). COVID-19-related mortality occurred in 11 of 223 patients (4.9%) in the canakinumab group vs 16 of 222 (7.2%) in the placebo group, with a rate difference of -2.3% (95% CI, -6.7% to 2.2%) and an odds ratio of 0.67 (95% CI, 0.30 to 1.50). Serious adverse events were observed in 36 of 225 patients (16%) treated with canakinumab vs 46 of 223 (20.6%) who received placebo. Conclusions and Relevance: Among patients hospitalized with severe COVID-19, treatment with canakinumab, compared with placebo, did not significantly increase the likelihood of survival without IMV at day 29. Trial Registration: ClinicalTrials.gov Identifier: NCT04362813.

Tissue-resident Eomes(hi) T-bet(lo) CD56(bright) NK cells with reduced proinflammatory potential are enriched in the adult human liver.

The adult human liver is enriched with natural killer (NK) cells, accounting for 30-50% of hepatic lymphocytes, which include tissue-resident hepatic NK-cell subpopulations, distinct from peripheral blood NK cells. In murine liver, a subset of liver-resident hepatic NK cells have altered expression of the two highly related T-box transcription factors, T-bet and eomesodermin (Eomes). Here, we investigate the heterogeneity of T-bet and Eomes expression in NK cells from healthy adult human liver with a view to identifying human liver-resident populations. Hepatic NK cells were isolated from donor liver perfusates and biopsies obtained during orthotopic liver transplantation (N = 28). Hepatic CD56(bright) NK cells were Eomes(hi) T-bet(lo) , a phenotype virtually absent from peripheral blood. These NK cells express the chemokine receptor CXCR6 (chemokine (C-X-C motif) receptor 6), a marker of tissue residency, which is absent from hepatic CD56(dim) and blood NK cells. Compared to blood populations, these hepatic CD56(bright) NK cells have increased expression of activatory receptors (NKp44, NKp46, and NKG2D). They show reduced ability to produce IFN-γ but enhanced degranulation in response to challenge with target cells. This functionally distinct population of hepatic NK cells constitutes 20-30% of the total hepatic lymphocyte repertoire and represents a tissue-resident immune cell population adapted to the tolerogenic liver microenvironment.

Efficacy and Safety of Intravenous Secukinumab for the Treatment of Active Psoriatic Arthritis: Results From a Randomized, Placebo-Controlled Phase 3 Study.

OBJECTIVE: The aim of this study was to evaluate the efficacy and safety of intravenous (IV) secukinumab in patients with active psoriatic arthritis (PsA). METHODS: INVIGORATE-2 (NCT04209205) was a randomized, placebo-controlled, phase 3 trial. Patients with active PsA were randomized 1:1 to receive IV secukinumab (6 mg/kg at baseline followed by 3 mg/kg every four weeks [q4w]) or placebo. At week 16, patients randomized to placebo were switched to IV secukinumab (3 mg/kg q4w), and patients who received IV secukinumab continued treatment through week 52. The primary efficacy endpoint was achievement of 50% improvement in American College of Rheumatology response criteria (ACR50) at week 16. Efficacy and safety were evaluated through weeks 52 and 60, respectively. RESULTS: Among 191 patients randomized to IV secukinumab and 190 to placebo/IV secukinumab, 177 (92.7%) and 170 (89.5%) completed the entire study period, respectively. A significantly higher proportion of patients who received IV secukinumab versus placebo achieved ACR50 at week 16 (31.4% vs 6.3%; adjusted P < 0.0001). All secondary efficacy endpoints were met at week 16 (all adjusted P < 0.05 using the predefined hypothesis-testing hierarchy). Patients who switched from placebo to secukinumab at week 16 showed rapid improvements in ACR50 rates; by week 52, both treatment arms experienced similar improvements in efficacy outcomes. No new or unexpected safety signals were observed with receiving IV secukinumab. One death was reported in the placebo group before week 16. CONCLUSION: IV secukinumab led to rapid and sustained improvements in clinical measures of PsA, and the safety profile was consistent with that of secukinumab administered subcutaneously.

Glutathione synthesis in the mouse liver supports lipid abundance through NRF2 repression.

Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we have developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are highest in liver tissue, which is also a hub for lipid production. While the loss of GSH does not cause liver failure, it decreases lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we find that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver's balance of redox buffering and triglyceride production.

Validation of commercially available antibodies directed against subunits of the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is traditionally composed of three subunits, although non-canonical expression has been found in various tissues including the vasculature, brain, lung, and dendritic cells of the immune system. Studies of ENaC structure and function have largely relied on heterologous expression systems, often with epitope-tagged channel subunits. Relevant in vivo physiological studies have used ENaC inhibitors, mice with global or tissue specific knockout of subunits, and anti-ENaC subunit antibodies generated by investigators or by commercial sources. Availability of well-characterized, specific antibodies is imperative as we move forward in understanding the role of ENaC in non-epithelial tissues where expression, subunit organization, and electrophysiological characteristics may differ from epithelial tissues. We report that a commonly used commercial anti-α subunit antibody recognizes an intense non-specific band on mouse whole kidney and lung immunoblots, which migrates adjacent to a less intense, aldosterone-induced full length α-subunit. This antibody localizes to the basolateral membrane of aquaporin 2 negative cells in kidney medulla. We validated antibodies against the ß- and γ-subunits from the same commercial source. Our work illustrates the importance of validation studies when using popular, commercially available anti-ENaC antibodies.

Hyperstable Synthetic Mini-Proteins as Effective Ligand Scaffolds.

Small, single-domain protein scaffolds are compelling sources of molecular binding ligands with the potential for efficient physiological transport, modularity, and manufacturing. Yet, mini-proteins require a balance between biophysical robustness and diversity to enable new functions. We tested the developability and evolvability of millions of variants of 43 designed libraries of synthetic 40-amino acid ßαßß proteins with diversified sheet, loop, or helix paratopes. We discovered a scaffold library that yielded hundreds of binders to seven targets while exhibiting high stability and soluble expression. Binder discovery yielded 6-122 nM affinities without affinity maturation and Tms averaging ≥78 °C. Broader ßαßß libraries exhibited varied developability and evolvability. Sheet paratopes were the most consistently developable, and framework 1 was the most evolvable. Paratope evolvability was dependent on target, though several libraries were evolvable across many targets while exhibiting high stability and soluble expression. Select ßαßß proteins are strong starting points for engineering performant binders.

BRAFV600E-mutated serrated colorectal neoplasia drives transcriptional activation of cholesterol metabolism.

BRAF mutations occur early in serrated colorectal cancers, but their long-term influence on tissue homeostasis is poorly characterized. We investigated the impact of short-term (3 days) and long-term (6 months) expression of BrafV600E in the intestinal tissue of an inducible mouse model. We show that BrafV600E perturbs the homeostasis of intestinal epithelial cells, with impaired differentiation of enterocytes emerging after prolonged expression of the oncogene. Moreover, BrafV600E leads to a persistent transcriptional reprogramming with enrichment of numerous gene signatures indicative of proliferation and tumorigenesis, and signatures suggestive of metabolic rewiring. We focused on the top-ranking cholesterol biosynthesis signature and confirmed its increased expression in human serrated lesions. Functionally, the cholesterol lowering drug atorvastatin prevents the establishment of intestinal crypt hyperplasia in BrafV600E-mutant mice. Overall, our work unveils the long-term impact of BrafV600E expression in intestinal tissue and suggests that colorectal cancers with mutations in BRAF might be prevented by statins.

Glutathione supports lipid abundance in vivo.

Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver's balance of redox buffering and triglyceride production.

Similar Clinical Outcomes in Patients with Systemic Juvenile Idiopathic Arthritis and Adult-Onset Still's Disease Treated with Canakinumab: Bayesian and Population Model-Based Analyses.

INTRODUCTION: Systemic juvenile idiopathic arthritis (sJIA) and adult-onset Still's disease (AOSD) represent pediatric and adult variants of the Still's disease continuum. To determine whether clinical outcomes between patients with sJIA and AOSD were similar, Bayesian and population model-based analyses were conducted on endpoints from studies of canakinumab in both patient populations. The objective was to further support the efficacy of canakinumab in patients with AOSD. METHODS: A Bayesian analysis of endpoints from a study of canakinumab in AOSD was conducted borrowing information from five pooled sJIA studies using a robust meta-analytic predictive (MAP) approach. Similarity of clinical outcomes across populations was fulfilled if the AOSD study posterior median fell within the 95% predicted credible interval for the outcome of interest, based on the pooled sJIA data. Population model-based analyses (pharmacokinetic [PK] and PK/pharmacodynamic [PKPD]) were conducted to compare the pharmacokinetics and exposure-response relationships between populations. RESULTS: The AOSD study posterior medians for adapted American College of Rheumatology (ACR)30 response, continuous adapted ACR response, number of active joints, C-reactive protein, and absence of fever were within the 95% credible interval for the prediction of the MAP analysis from the pooled sJIA data, supporting the similarity in outcomes between patient populations. PK analysis demonstrated comparable exposure across sJIA age groups and patients with AOSD. PKPD relationships were consistent across patient populations. Analyses indicated that no therapeutic benefit can be expected from a dose increase in patients with AOSD. CONCLUSION: The analyses presented support the similarity of clinical outcomes following treatment with canakinumab in patients with sJIA and AOSD.

Therapeutic Targeting of Checkpoint Receptors within the DNAM1 Axis.

Therapeutic antibodies targeting the CTLA4/PD-1 pathways have revolutionized cancer immunotherapy by eliciting durable remission in patients with cancer. However, relapse following early response, attributable to primary and adaptive resistance, is frequently observed. Additional immunomodulatory pathways are being studied in patients with primary or acquired resistance to CTLA4 or PD-1 blockade. The DNAM1 axis is a potent coregulator of innate and adaptive immunity whose other components include the immunoglobulin receptors TIGIT, PVRIG, and CD96, and their nectin and nectin-like ligands. We review the basic biology and therapeutic relevance of this family, which has begun to show promise in cancer clinical trials. SIGNIFICANCE: Recent studies have outlined the immuno-oncologic ascendancy of coinhibitory receptors in the DNAM1 axis such as TIGIT and PVRIG and, to a lesser extent, CD96. Biological elucidation backed by ongoing clinical trials of single-agent therapy directed against TIGIT or PVRIG is beginning to provide the rationale for testing combination regimens of DNAM1 axis blockers in conjunction with anti-PD-1/PD-L1 agents.

Improvement in health status with once-daily indacaterol/glycopyrronium 110/50 µg in COPD patients: real-world evidence from an observational study in Ireland.

AIMS: Indacaterol/glycopyrronium (IND/GLY) 110/50 µg is a once-daily (o.d.) fixed-dose combination of long-acting ß2-agonist/long-acting muscarinic antagonist approved in over 90 countries, including Ireland, for the management of COPD. The present study was conducted to evaluate health status of COPD patients, initiated on IND/GLY 110/50 µg o.d., using the Clinical COPD Questionnaire (CCQ) tool in a real-world primary care setting in Ireland. METHODS: This was a real-world, prospective, open-label study. COPD patients aged > 40 years and with a smoking history of > 10 pack-years were included and switched to once-daily IND/GLY 110/50 µg. Enrolment of patients into the study occurred only after the decision had been made by the physician to prescribe IND/GLY 110/50 µg. Data were collected at baseline and Week 26. Health status was assessed using the validated CCQ. RESULTS: A total of 200 patients were included in study. The mean CCQ total score decreased from 2.36 at baseline to 1.44 at Week 26 (Δ, 0.92; P < 0.0005). Of the 156 patients who completed study, 113 (72.4%) achieved minimum clinically important difference in CCQ total score with IND/GLY 110/50 µg. CCQ domain scores also decreased during the study. Improvement in health status was observed across all GOLD groups and irrespective of prior COPD treatment. Adverse events were reported by 20% of patients with COPD exacerbation/infected COPD being the most common AE, reported by 11 patients. CONCLUSIONS: In real-life clinical practice in Ireland, IND/GLY 110/50 µg o.d. demonstrated statistically significant and clinically important improvement in health status in patients with COPD.

PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8+ T-cell Function.

Although checkpoint inhibitors that block CTLA-4 and PD-1 have improved cancer immunotherapies, targeting additional checkpoint receptors may be required to broaden patient response to immunotherapy. PVRIG is a coinhibitory receptor of the DNAM/TIGIT/CD96 nectin family that binds to PVRL2. We report that antagonism of PVRIG and TIGIT, but not CD96, increased CD8+ T-cell cytokine production and cytotoxic activity. The inhibitory effect of PVRL2 was mediated by PVRIG and not TIGIT, demonstrating that the PVRIG-PVRL2 pathway is a nonredundant signaling node. A combination of PVRIG blockade with TIGIT or PD-1 blockade further increased T-cell activation. In human tumors, PVRIG expression on T cells was increased relative to normal tissue and trended with TIGIT and PD-1 expression. Tumor cells coexpressing PVR and PVRL2 were observed in multiple tumor types, with highest coexpression in endometrial cancers. Tumor cells expressing either PVR or PVRL2 were also present in numbers that varied with the cancer type, with ovarian cancers having the highest percentage of PVR-PVRL2+ tumor cells and colorectal cancers having the highest percentage of PVR+PVRL2- cells. To demonstrate a role of PVRIG and TIGIT on tumor-derived T cells, we examined the effect of PVRIG and TIGIT blockade on human tumor-infiltrating lymphocytes. For some donors, blockade of PVRIG increased T-cell function, an effect enhanced by combination with TIGIT or PD-1 blockade. In summary, we demonstrate that PVRIG and PVRL2 are expressed in human cancers and the PVRIG-PVRL2 and TIGIT-PVR pathways are nonredundant inhibitory signaling pathways.See related article on p. 244.

Myeloid Engraftment in Humanized Mice: Impact of Granulocyte-Colony Stimulating Factor Treatment and Transgenic Mouse Strain.

Poor myeloid engraftment remains a barrier to experimental use of humanized mice. Focusing primarily on peripheral blood cells, we compared the engraftment profile of NOD-scid-IL2Rγc(-/-) (NSG) mice with that of NSG mice transgenic for human membrane stem cell factor (hu-mSCF mice), NSG mice transgenic for human interleukin (IL)-3, granulocyte-macrophage-colony stimulating factor (GM-CSF), and stem cell factor (SGM3 mice). hu-mSCF and SGM3 mice showed enhanced engraftment of human leukocytes compared to NSG mice, and this was reflected in the number of human neutrophils and monocytes present in these strains. Importantly, discrete classical, intermediate, and nonclassical monocyte populations were identifiable in the blood of NSG and hu-mSCF mice, while the nonclassical population was absent in the blood of SGM3 mice. Granulocyte-colony stimulating factor (GCSF) treatment increased the number of blood monocytes in NSG and hu-mSCF mice, and neutrophils in NSG and SGM3 mice; however, this effect appeared to be at least partially dependent on the stem cell donor used to engraft the mice. Furthermore, GCSF treatment resulted in a preferential expansion of nonclassical monocytes in both NSG and hu-mSCF mice. Human tubulointerstitial CD11c(+) cells were present in the kidneys of hu-mSCF mice, while monocytes and neutrophils were identified in the liver of all strains. Bone marrow-derived macrophages prepared from NSG mice were most effective at phagocytosing polystyrene beads. In conclusion, hu-mSCF mice provide the best environment for the generation of human myeloid cells, with GCSF treatment further enhancing peripheral blood human monocyte cell numbers in this strain.