T cell-mediated elimination of cancer cells by blocking CEACAM6-CEACAM1 interaction.

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a cell surface receptor, is expressed on normal epithelial tissue and highly expressed in cancers of high unmet medical need, such as non-small cell lung, pancreatic, and colorectal cancer. CEACAM receptors undergo homo- and heterophilic interactions thereby regulating normal tissue homeostasis and angiogenesis, and in cancer, tumor invasion and metastasis. CEACAM6 expression on malignant plasma cells inhibits antitumor activity of T cells, and we hypothesize a similar function on epithelial cancer cells. The interactions between CEACAM6 and its suggested partner CEACAM1 on T cells were studied. A humanized CEACAM6-blocking antibody, BAY 1834942, was developed and characterized for its immunomodulating effects in co-culture experiments with T cells and solid cancer cells and in comparison to antibodies targeting the immune checkpoints programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and T cell immunoglobulin mucin-3 (TIM-3). The immunosuppressive activity of CEACAM6 was mediated by binding to CEACAM1 expressed by activated tumor-specific T cells. BAY 1834942 increased cytokine secretion by T cells and T cell-mediated killing of cancer cells. The in vitro efficacy of BAY 1834942 correlated with the degree of CEACAM6 expression on cancer cells, suggesting potential in guiding patient selection. BAY 1834942 was equally or more efficacious compared to blockade of PD-L1, and at least an additive efficacy was observed in combination with anti-PD-1 or anti-TIM-3 antibodies, suggesting an efficacy independent of the PD-1/PD-L1 axis. In summary, CEACAM6 blockade by BAY 1834942 reactivates the antitumor response of T cells. This warrants clinical evaluation.

pyQms enables universal and accurate quantification of mass spectrometry data.

Quantitative mass spectrometry (MS) is a key technique in many research areas (1), including proteomics, metabolomics, glycomics, and lipidomics. Because all of the corresponding molecules can be described by chemical formulas, universal quantification tools are highly desirable. Here, we present pyQms, an open-source software for accurate quantification of all types of molecules measurable by MS. pyQms uses isotope pattern matching that offers an accurate quality assessment of all quantifications and the ability to directly incorporate mass spectrometer accuracy. pyQms is, due to its universal design, applicable to every research field, labeling strategy, and acquisition technique. This opens ultimate flexibility for researchers to design experiments employing innovative and hitherto unexplored labeling strategies. Importantly, pyQms performs very well to accurately quantify partially labeled proteomes in large scale and high throughput, the most challenging task for a quantification algorithm.

VE-Cadherin Phosphorylation Regulates Endothelial Fluid Shear Stress Responses through the Polarity Protein LGN.

Fluid shear stress due to blood flow on the vascular endothelium regulates blood vessel development, remodeling, physiology, and pathology [1, 2]. A complex consisting of PECAM-1, VE-cadherin, and vascular endothelial growth factor receptors (VEGFRs) that resides at endothelial cell-cell junctions transduces signals important for flow-dependent vasodilation, blood vessel remodeling, and atherosclerosis. PECAM-1 transduces forces to activate src family kinases (SFKs), which phosphorylate and transactivate VEGFRs [3-5]. By contrast, VE-cadherin functions as an adaptor that interacts with VEGFRs through their respective cytoplasmic domains and promotes VEGFR activation in flow [6]. Indeed, shear stress triggers rapid increases in force across PECAM-1 but decreases the force across VE-cadherin, in close association with downstream signaling [5]. Interestingly, VE-cadherin cytoplasmic tyrosine Y658 can be phosphorylated by SFKs [7], which is maximally induced by low shear stress in vitro and in vivo [8]. These considerations prompted us to address the involvement of VE-cadherin cytoplasmic tyrosines in flow sensing. We found that phosphorylation of a small pool of VE-cadherin on Y658 is essential for flow sensing through the junctional complex. Y658 phosphorylation induces dissociation of p120ctn, which allows binding of the polarity protein LGN. LGN is then required for multiple flow responses in vitro and in vivo, including activation of inflammatory signaling at regions of disturbed flow, and flow-dependent vascular remodeling. Thus, endothelial flow mechanotransduction through the junctional complex is mediated by a specific pool of VE-cadherin that is phosphorylated on Y658 and bound to LGN.

Blocking neutrophil diapedesis prevents hemorrhage during thrombocytopenia.

Spontaneous organ hemorrhage is the major complication in thrombocytopenia with a potential fatal outcome. However, the exact mechanisms regulating vascular integrity are still unknown. Here, we demonstrate that neutrophils recruited to inflammatory sites are the cellular culprits inducing thrombocytopenic tissue hemorrhage. Exposure of thrombocytopenic mice to UVB light provokes cutaneous petechial bleeding. This phenomenon is also observed in immune-thrombocytopenic patients when tested for UVB tolerance. Mechanistically, we show, analyzing several inflammatory models, that it is neutrophil diapedesis through the endothelial barrier that is responsible for the bleeding defect. First, bleeding is triggered by neutrophil-mediated mechanisms, which act downstream of capturing, adhesion, and crawling on the blood vessel wall and require Gαi signaling in neutrophils. Second, mutating Y731 in the cytoplasmic tail of VE-cadherin, known to selectively affect leukocyte diapedesis, but not the induction of vascular permeability, attenuates bleeding. Third, and in line with this, simply destabilizing endothelial junctions by histamine did not trigger bleeding. We conclude that specifically targeting neutrophil diapedesis through the endothelial barrier may represent a new therapeutic avenue to prevent fatal bleeding in immune-thrombocytopenic patients.

Similarities and differences in the regulation of leukocyte extravasation and vascular permeability.

Leukocyte extravasation is regulated and mediated by a multitude of adhesion and signaling molecules. Many of them enable the capturing and docking of leukocytes to the vessel wall. Others allow leukocytes to crawl on the apical surface of endothelial cells to appropriate sites of exit. While these steps are well understood and the adhesion molecules mediating these interactions are largely identified, a still growing number of adhesion receptors mediate the diapedesis process, the actual migration of leukocytes through the endothelial cell layer, and the underlying basement membrane. In most cases, it is not known which molecular processes they actually mediate, whether they enable the migration of leukocytes through the endothelial cell layer or whether they are involved in the destabilization of endothelial junctions. In addition, leukocytes are able to circumvent junctions and transcytose directly through the body of endothelial cells. While this latter route indeed exists, recent work has highlighted in vivo the junctional pathway as the prevalent way of leukocyte exit in various inflamed tissues. Recent work elucidating molecular mechanisms that regulate endothelial junctions and thereby leukocyte extravasation and vascular permeability will be discussed.

Leukocyte extravasation and vascular permeability are each controlled in vivo by different tyrosine residues of VE-cadherin.

Tyrosine phosphorylation of the adhesion molecule VE-cadherin is assumed to affect endothelial junction integrity. However, it remains unclear whether tyrosine residues of VE-cadherin are required for the induction of vascular permeability and the regulation of leukocyte extravasation in vivo. We found here that knock-in mice expressing a Y685F mutant of VE-cadherin had impaired induction of vascular permeability, but those expressing a Y731F mutant did not. In contrast, mice expressing the Y731F VE-cadherin mutant showed decreased neutrophil-extravasation in cremaster tissue, but those expressing the Y685F mutant did not. Whereas inflammatory mediators induced the phosphorylation of Tyr685 in vivo, Tyr731 showed high baseline phosphorylation. Leukocytes triggered dephosphorylation of Tyr731 via the tyrosine phosphatase SHP-2, which allowed the adaptin AP-2 to bind and initiate endocytosis of VE-cadherin. Thus, Tyr685 and Tyr731 of VE-cadherin distinctly and selectively regulate the induction of vascular permeability or leukocyte extravasation.