941 Stromal cell sialylation suppresses T cells in inflammatory tumour microenvironments: a new tumour stromal cell immune checkpoint?

BackgroundImmunosuppressive tumour microenvironments (TME) reduce the effectiveness of immune responses in cancer. Non-haematopoietic mesenchymal stromal cells, precursors to cancer-associated fibroblasts (CAFs), dictate tumour progression by enhancing immune cell suppression. Sialic acids, which exist as terminal sugars of glycans (known as sialoglycans), are highly expressed on cancer cells and hyper-sialylation of glycans is known to promote immune evasion in cancer. Sialoglycans are recognized by sialic acid-binding immunoglobulin-like lectins (Siglecs), a family of immunomodulatory receptors, which are analogous to the immune checkpoint inhibitor PD-1.1 The role of sialyation in stromal cell-mediated immunosuppression, however, is unknown. Using models of solid (colorectal cancer - CRC) and haematological (multiple myeloma - MM) stromal-rich tumours in both mouse and human, the aim of this study was to investigate if stromal cell sialylation contributes to enhanced immunosuppression in the TME.MethodsFlow cytometric analysis of sialic acid expression was performed initially on bone marrow-derived stromal cells isolated from healthy human donor bone marrow aspirates, from wild-type Balb/c mice or from 5T33 multiple myeloma mice. Stromal cells were also isolated and expanded from colorectal cancer patient tumour biopsies (CAFs) with matched controls isolated from tumour-adjacent non-cancerous tissue (normal-associated fibroblasts - NAFs) or from whole blood from primary multiple myeloma bone aspirates. Informed consent was obtained from all patients prior to sampling. Immunosuppression assays were performed using these stromal cells with or without exposure to the tumour cell secretome from the mouse and human CRC cell lines CT26 or HCT116 and HT29, respectively, co-cultured with either murine lymphocytes or healthy human donor-derived peripheral blood mononuclear cells (PBMCs).ResultsOur results showed that tumour conditioned stromal cells have increased levels of sialyltransferase gene expression, α2,3/α2,6-linked sialic acid and Siglec ligands. Co-culture assays revealed that CAFs induced significantly higher frequencies of Siglec 7 and Siglec 9-expressing CD8 T cells, as well as Tim-3 and PD-1-expressing CD8 T cells, compared to NAFs. Inhibition of sialyltransferase activity using the inhibitor 3FAXNeu5Ac reversed these CAF-induced effects. Interestingly, sialyltransferase inhibition had no observed effects on T cells co-cultured with NAFs.ConclusionsThese results demonstrate that targeting stromal cell sialylation can reverse immune cell suppression and reactivate exhausted T cells. These novel data support a rationale for the assessment of stromal cell sialylation and Siglec ligand expression in order to better stratify patients for immunotherapeutic combination treatments that aim to reactivate exhausted T cells in stromal-enriched tumour microenvironments.AcknowledgementsThe authors would like to thank the Blood Cancer Network of Ireland Biobank for providing bone marrow aspirates.ReferenceGray MA, Stanczak MA, Mantuano NR, Xiao H, Pijnenborg JFA, Malaker SA, Miller CL, Weidenbacher PA, Tanzo JT, Ahn G, Woods EC, Läubli H, Bertozzi CR. Targeted glycan degradation potentiates the anticancer immune response in vivo. Nat Chem Biol 2020;16:1376–1384.Ethics ApprovalColorectal tumor and adjacent normal mucosal tissue were obtained from patients undergoing colon tumor resection at University Hospital Galway under an ethically approved protocol (Clinical Research Ethics Committee, Ref: C.A. 2074). Samples were collected and isolated by the Blood Cancer Network of Ireland under an ethically approved protocol. Written informed explicit consent was obtained from all patients prior to sampling. Mice were housed and maintained following the conditions approved by the Animals Care Research Ethics Committee of the National University of Ireland, Galway (NUIG) and procedures were conducted under individual and project authorisation licenses from the Health Products Regulatory Authority (HPRA) of Ireland or from the Ethical Committee for Animal Experiments, Vrije Universiteit Brussel (license no. LA1230281, 16-281-6).

Post-translational polymodification of β1-tubulin regulates motor protein localisation in platelet production and function

In specialised cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviours. The mechanisms by which β1-tubulin, the platelet and megakaryocyte (MK) lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational tubulin polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived MKs, and healthy human donor platelets. We find distinct patterns of polymodification in MKs and platelets, mediated by the antagonistic activities of the cell specific expression of Tubulin Tyrosine Ligase Like (TTLLs) and Cytosolic Carboxypeptidase (CCP) enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganisation required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved.

Fibrinogen function achieved through multiple covalent states

Disulfide bonds link pairs of cysteine amino acids and their formation is assumed to be complete in the mature, functional protein. Here, we test this assumption by quantifying the redox state of disulfide bonds in the blood clotting protein fibrinogen. The disulfide status of fibrinogen from healthy human donor plasma and cultured human hepatocytes are measured using differential cysteine alkylation and mass spectrometry. This analysis identifies 13 disulfide bonds that are 10–50% reduced, indicating that fibrinogen is produced in multiple disulfide-bonded or covalent states. We further show that disulfides form upon fibrin polymerization and are required for a robust fibrin matrix that withstands the mechanical forces of flowing blood and resists premature fibrinolysis. The covalent states of fibrinogen are changed by fluid shear forces ex vivo and in vivo, indicating that the different states are dynamic. These findings demonstrate that fibrinogen exists and functions as multiple covalent forms.

The first versatile human iPSC-based model of ectopic virus induction allows new insights in RNA-virus disease

A detailed description of pathophysiological effects that viruses exert on their host is still challenging. For the first time, we report a highly controllable viral expression model based on an iPS-cell line from a healthy human donor. The established viral model system enables a dose-dependent and highly localized RNA-virus expression in a fully controllable environment, giving rise for new applications for the scientific community.

Direct demonstration of 25- and 50-μm arteriovenous pathways in healthy human and baboon lungs

Postmortem microsphere studies in adult human lungs have demonstrated the existence of intrapulmonary arteriovenous pathways using nonphysiological conditions. The aim of the current study was to determine whether large diameter (>25 and 50 μm) intrapulmonary arteriovenous pathways are functional in human and baboon lungs under physiological perfusion and ventilation pressures. We used fresh healthy human donor lungs obtained for transplantion and fresh lungs from baboons ( Papio c. anubis). Lungs were ventilated with room air by using a peak inflation pressure of 15 cmH2O and a positive end-expiratory pressure of 5 cmH2O. Lungs were perfused between 10 and 20 cmH2O by using a phosphate-buffered saline solution with 5% albumin. We infused a mixture of 25- and 50-μm microspheres (0.5 and 1 million total for baboons and human studies, respectively) into the pulmonary artery and collected the entire pulmonary venous outflow. Under these conditions, evidence of intrapulmonary arteriovenous anastomoses was found in baboon ( n = 3/4) and human ( n = 4/6) lungs. In those lungs showing evidence of arteriovenous pathways, 50-μm microspheres were always able to traverse the pulmonary circulation, and the fraction of transpulmonary passage ranged from 0.0003 to 0.42%. These data show that intrapulmonary arteriovenous pathways >50 μm in diameter are functional under physiological ventilation and perfusion pressures in the isolated lung. These pathways provide an alternative conduit for pulmonary blood flow that likely bypasses the areas of gas exchange at the capillary-alveolar interface that could compromise both gas exchange and the ability of the lung to filter out microemboli.