Age-associated B cells predict impaired humoral immunity after COVID-19 vaccination in patients receiving immune checkpoint blockade.

Age-associated B cells (ABC) accumulate with age and in individuals with different immunological disorders, including cancer patients treated with immune checkpoint blockade and those with inborn errors of immunity. Here, we investigate whether ABCs from different conditions are similar and how they impact the longitudinal level of the COVID-19 vaccine response. Single-cell RNA sequencing indicates that ABCs with distinct aetiologies have common transcriptional profiles and can be categorised according to their expression of immune genes, such as the autoimmune regulator (AIRE). Furthermore, higher baseline ABC frequency correlates with decreased levels of antigen-specific memory B cells and reduced neutralising capacity against SARS-CoV-2. ABCs express high levels of the inhibitory FcγRIIB receptor and are distinctive in their ability to bind immune complexes, which could contribute to diminish vaccine responses either directly, or indirectly via enhanced clearance of immune complexed-antigen. Expansion of ABCs may, therefore, serve as a biomarker identifying individuals at risk of suboptimal responses to vaccination.

ADGRL4/ELTD1 Expression in Breast Cancer Cells Induces Vascular Normalization and Immune Suppression.

ELTD1/ADGRL4 expression is increased in the vasculature of a number of tumor types and this correlates with a good prognosis. Expression has also been reported in some tumor cells with high expression correlating with a good prognosis in hepatocellular carcinoma (HCC) and a poor prognosis in glioblastoma. Here we show that 35% of primary human breast tumors stain positively for ELTD1, with 9% having high expression that correlates with improved relapse-free survival. Using immunocompetent, syngeneic mouse breast cancer models we found that tumors expressing recombinant murine Eltd1 grew faster than controls, with an enhanced ability to metastasize and promote systemic immune effects. The Eltd1-expressing tumors had larger and better perfused vessels and tumor-endothelial cell interaction led to the release of proangiogenic and immune-modulating factors. M2-like macrophages increased in the stroma along with expression of programmed death-ligand 1 (PD-L1) on tumor and immune cells, to create an immunosuppressive microenvironment that allowed Eltd1-regulated tumor growth in the presence of an NY-ESO-1-specific immune response. Eltd1-positive tumors also responded better to chemotherapy which could explain the relationship to a good prognosis observed in primary human cases. Thus, ELTD1 expression may enhance delivery of therapeutic antibodies to reverse the immunosuppression and increase response to chemotherapy and radiotherapy in this subset of tumors. ELTD1 may be useful as a selection marker for such therapies. IMPLICATIONS: ELTD1 expression in mouse breast tumors creates an immunosuppressive microenvironment and increases vessel size and perfusion. Its expression may enhance the delivery of therapies targeting the immune system.

A functional assay for serum detection of antibodies against SARS-CoV-2 nucleoprotein.

The humoral immune response to SARS-CoV-2 results in antibodies against spike (S) and nucleoprotein (N). However, whilst there are widely available neutralization assays for S antibodies, there is no assay for N-antibody activity. Here, we present a simple in vitro method called EDNA (electroporated-antibody-dependent neutralization assay) that provides a quantitative measure of N-antibody activity in unpurified serum from SARS-CoV-2 convalescents. We show that N antibodies neutralize SARS-CoV-2 intracellularly and cell-autonomously but require the cytosolic Fc receptor TRIM21. Using EDNA, we show that low N-antibody titres can be neutralizing, whilst some convalescents possess serum with high titres but weak activity. N-antibody and N-specific T-cell activity correlates within individuals, suggesting N antibodies may protect against SARS-CoV-2 by promoting antigen presentation. This work highlights the potential benefits of N-based vaccines and provides an in vitro assay to allow the antibodies they induce to be tested.

Elevated expression of the adhesion GPCR ADGRL4/ELTD1 promotes endothelial sprouting angiogenesis without activating canonical GPCR signalling.

ADGRL4/ELTD1 is an orphan adhesion GPCR (aGPCR) expressed in endothelial cells that regulates tumour angiogenesis. The majority of aGPCRs are orphan receptors. The Stachel Hypothesis proposes a mechanism for aGPCR activation, in which aGPCRs contain a tethered agonist (termed Stachel) C-terminal to the GPCR-proteolytic site (GPS) cleavage point which, when exposed, initiates canonical GPCR signalling. This has been shown in a growing number of aGPCRs. We tested this hypothesis on ADGRL4/ELTD1 by designing full length (FL) and C-terminal fragment (CTF) ADGRL4/ELTD1 constructs, and a range of potential Stachel peptides. Constructs were transfected into HEK293T cells and HTRF FRET, luciferase-reporter and Alphascreen GPCR signalling assays were performed. A stable ADGRL4/ELTD1 overexpressing HUVEC line was additionally generated and angiogenesis assays, signalling assays and transcriptional profiling were performed. ADGRL4/ELTD1 has the lowest GC content in the aGPCR family and codon optimisation significantly increased its expression. FL and CTF ADGRL4/ELTD1 constructs, as well as Stachel peptides, did not activate canonical GPCR signalling. Furthermore, stable overexpression of ADGRL4/ELTD1 in HUVECs induced sprouting angiogenesis, lowered in vitro anastomoses, and decreased proliferation, without activating canonical GPCR signalling or MAPK/ERK, PI3K/AKT, JNK, JAK/HIF-1α, beta catenin or STAT3 pathways. Overexpression upregulated ANTXR1, SLC39A6, HBB, CHRNA, ELMOD1, JAG1 and downregulated DLL4, KIT, CCL15, CYP26B1. ADGRL4/ELTD1 specifically regulates the endothelial tip-cell phenotype through yet undefined signalling pathways.

Five-year review of corticosteroid duration and complications in the management of immune checkpoint inhibitor-related diarrhoea and colitis in advanced melanoma.

BACKGROUND: Immune-related diarrhoea/colitis (ir-D/C) is a common adverse event of immune checkpoint inhibitor (ICI) therapy. Guidelines recommend corticosteroid (CS) treatment; however, the average treatment duration for ir-D/C remains poorly defined. METHODS: All advanced melanoma patients treated with ICI therapy at the Royal Marsden Hospital between 2011 and 2016 were reviewed to identify ir-D/C cases alongside clinical variables. RESULTS: 117 any-grade ir-D/C episodes occurred in 109 (21%) patients out of a total of 519 patients treated (ipilimumab=77 episodes, anti-PD1=17 (nivolumab or pembrolizumab), ipilimumab and nivolumab=23 (ipi+nivo)) (seven patients had ir-D/C more than once on different lines of treatment) and >/=grade 3 ir-D/C occurred most frequently (63/519 patients (12%) vs 29/519 (5%) grade 1, and 25/519 (5%) grade 2). Median onset (days) of all-grade ir-D/C after starting ICI therapy was 41 for ipilimumab (IQR 24 to 59, n=77), 91 for anti-PD1 (IQR 46 to 355, n=17) and 45 for ipi+nivo (IQR 24 to 67, n=23). In 71/117 (61%) patients, ir-D/C episodes were treated with CS (17% grade 2; 79% grade 3/4): 54 being steroid-responsive; 17 being steroid-refractory and received additional anti-tumor necrosis factor (TNF) treatment. Median grade 3 ir-D/C CS duration was similar across treatments, averaging 58 days. Median overall CS duration (days) was longer in the grade 3/4 D/C steroid-refractory group (94 vs 45 days). Infection developed in 11/71 (15%) CS recipients and in 6/17 (35%) anti-TNF recipients. In 65/117 (55%) patients, ir-D/C episodes were investigated with flexible sigmoidoscopy. Of these patients, 38/65 (58%) had macroscopic colitis and 12/65 (18%) had microscopic colitis. The steroid-refractory group had more macroscopic changes, 13/17 (76%), than the steroid-responsive group, 22/41 (54%). CONCLUSION: Rates of grade 3 ir-D/C were higher than reported in clinical trials. The 58-day median duration of CS therapy for grade 3 ir-D/C places a significant number of patients at risk of complications. We demonstrate that microscopic colitis is an important subgroup, advocating biopsies in ir-D/C even with macroscopically normal bowel.

ADGRL4/ELTD1 Silencing in Endothelial Cells Induces ACLY and SLC25A1 and Alters the Cellular Metabolic Profile.

Adhesion G Protein-Coupled Receptor L4 (ADGRL4/ELTD1) is an endothelial cell adhesion G protein-coupled receptor (aGPCR) which regulates physiological and tumour angiogenesis, providing an attractive target for anti-cancer therapeutics. To date, ADGRL4/ELTD1's full role and mechanism of function within endothelial biology remains unknown, as do its ligand(s). In this study, ADGRL4/ELTD1 silencing, using two independent small interfering RNAs (siRNAs), was performed in human umbilical vein endothelial cells (HUVECS) followed by transcriptional profiling, target gene validation, and metabolomics using liquid chromatography-mass spectrometry in order to better characterise ADGRL4/ELTD1's role in endothelial cell biology. We show that ADGRL4/ELTD1 silencing induced expression of the cytoplasmic metabolic regulator ATP Citrate Lyase (ACLY) and the mitochondria-to-cytoplasm citrate transporter Solute Carrier Family 25 Member 1 (SLC25A1) but had no apparent effect on pathways downstream of ACLY (fatty acid and cholesterol synthesis or acetylation). Silencing induced KIT expression and affected the Notch signalling pathway, upregulating Delta Like Canonical Notch Ligand 4 (DLL4) and suppressing Jagged Canonical Notch Ligand 1 (JAG1) and Hes Family BHLH Transcription Factor 2 (HES2). The effect of ADGRL4/ELTD1 silencing on the cellular metabolic profile was modest but several metabolites were significantly affected. Cis-aconitic acid, uridine diphosphate (UDP)-glucoronate, fructose 2,6-diphosphate, uridine 5-diphosphate, and aspartic acid were all elevated as a result of silencing and phosphocreatine, N-acetylglutamic acid, taurine, deoxyadenosine triphosphate, and cytidine monophosphate were depleted. Metabolic pathway analysis implicated ADGRL4/ELTD1 in pyrimidine, amino acid, and sugar metabolism. In summary, this study shows that ADGRL4/ELTD1 impacts core components of endothelial metabolism and regulates genes involved in endothelial differentiation/homeostasis and Notch signalling.

ADGRL4/ELTD1 is a highly conserved angiogenesis-associated orphan adhesion GPCR that emerged with the first vertebrates and comprises 3 evolutionary variants.

BACKGROUND: Our laboratory identified ADGRL4/ELTD1, an orphan GPCR belonging to the adhesion GPCR (aGPCR) family, as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. Little is known about how ADGRL4/ELTD1 (and aGPCRs in general) function, a problem compounded by a lack of known ligands or means of activation. With this in mind, we turned to computational evolutionary biology with the aim of better understanding ADGRL4/ELTD1. RESULTS: We identified ADGRL4/ELTD1 as a highly conserved early angiogenic gene which emerged in the first true vertebrates (bony fish) approximately 435 million years ago (mya), evolving alongside key angiogenic genes VEGFR2 and DLL4. We identified 3 evolutionary ADGRL4/ELTD1 variants based on EGF domain deletions with variant 2 first emerging 101 mya (95% CI 96-105) in Afrotheria and 82 mya (95% CI 76-89) in Primates. Additionally, conservation mapping across all orthologues reveals highest level conservation in EGF Ca binding domain 1, suggesting that this motif plays an essential role, as well as specific regions of the GAIN domain, GPS motif and 7TM domain, suggesting possible activation mechanisms and ligand binding positions. Additionally, we found that ADGRL4/ELTD1 (a member aGPCR family 1) is possibly ancestral to members of aGPCR family 2. CONCLUSION: This work establishes ADGRL4/ELTD1's evolution, sheds light on its possible activation and ligand binding zones, and establishes the first temporal references for the emergence of ADGRL4/ELTD1 variants during vertebrate evolution. Our approach is applicable to the greater aGPCR family and opens up new avenues for future experimental work.

Theodor Billroth: A Surgeon for the 21st Century.

This perspective article describes unique aspects of Theodor Billroth's personality and life, which enabled him to succeed and become a leading figure of 19th century medicine. These qualities are discussed with today's developing surgeon in mind.

A review of ELTD1, a pro-angiogenic adhesion GPCR.

Epidermal growth factor, latrophilin and seven-transmembrane domain-containing 1 (ELTD1), an orphan G-protein-coupled receptor (GPCR) belonging to the adhesion GPCR family, has recently been identified as a potential cancer biomarker and a novel regulator of angiogenesis. In this mini-review, we present an overview of the current literature on ELTD1 and present bioinformatics data showing ELTD1's sequence conservation, its expression in cancer cell lines and its mutational frequency in human cancers. Additionally, we present sequence homology alignment results confirming ELTD1 to be a hybrid comprising motifs shared with individual members in both adhesion GPCR subfamilies 1 and 2. Finally, we discuss why tumour endothelial ELTD1 expression may confer a good prognosis yet still represent a therapeutic target.

VEGF sticky-trap: the first report of a non-systemically acting angiogenesis inhibitor.

Current therapeutic anti-angiogenic biologics used for the treatment of pathological ocular angiogenesis such as in diabetic retinopathy and wet macular degeneration often lead to detrimental side effects due to their interference with normal blood vessel physiology. In this issue of EMBO Molecular Medicine, Michael et al report on a novel angiogenesis inhibitor with unique properties that allow for local inhibition of angiogenesis without detectable systemic side effects.

The Students' Health and Welfare Centres Organisation (SHAWCO) of the University of Cape Town: a review of the past 69 years.

The Students' Health and Welfare Centres Organisation (SHAWCO) is a student-run non-profit community development organisation based at the University of Cape Town (UCT). In 2012, SHAWCO celebrates its 69th anniversary, making it the oldest active student-run free clinic in South Africa. Over the past 7 decades, SHAWCO has become an integral part of the UCT Faculty of Health Sciences. This article reviews its history, current activities, and plans for the future.