Anna Dimberg

Cerebral cavernous malformations (CCMs) constitute the second most common type of intracranial vascular malformations. Cavernomas arise because of somatic or autosomal dominant inherited loss-of-function mutations in CCM genes, forming fragile mulberry-like malformed blood vessel structures that are prone to leak and break. Treatment is limited to surgical removal of solitary cavernomas, and because patients often present with multiple lesions, pharmacological alternatives to surgery are needed. We performed a high-throughput screen of 5200 bioactive molecules in programmed cell death 10 (PDCD10 or CCM3)–deficient mouse endothelial cells, coupled to automated image analysis. This screen identified dabrafenib, a clinically approved BRAF inhibitor, as a compound that restored cell-cell junctions and vascular barrier properties of CCM3-deficient endothelial cells. The potential translatability of dabrafenib was tested in acute and chronic CCM mouse models, where it reduced cerebral lesion burden and suppressed lesion permeability. Dabrafenib also rescued abnormal structural defects present in the developing retina vasculature in an acute CCM mouse model. Mechanistically, dabrafenib targeted extracellular regulated kinase (ERK)5–Krüppel-like factor (KLF)2/4 signaling and suppressed F-actin stress fiber formation in human brain endothelial cells, two potentially independent and central molecular processes involved in CCM pathology. Silencing BRAF did not prevent dabrafenib-induced KLF2/4 inhibition or restoration of cell-cell junctions, suggesting that its dual therapeutic outcome is independent of BRAF pathway inhibition. In conclusion, dabrafenib is a low–molecular weight compound that inhibits cavernoma pathogenesis, suggesting that CCMs could be treated with dabrafenib to block cavernoma growth and vascular leakage.

Idiopathic nephrotic syndrome (INS) is a podocyte disease triggered by immune-derived factors. Endothelial activation occurs in this context, but whether the activated endothelium contributes to podocyte injury is unknown. We tested the hypothesis that CD93, a protein primarily expressed in the endothelium, is a contributory factor of podocyte injury. We studied 460 patients with INS and 150 with other podocytopathies. CD93 was analyzed in kidney tissue, urine, and serum samples. We tested the efficacy of CD93 blockade in vitro and in vivo and investigated the relationship between soluble CD93 and clinical outcomes in human INS. CD93 was highly expressed by glomerular endothelial cells (GEnCs) in human INS, and INS sera stimulated cultured human GEnCs to release CD93. Mechanistically, soluble CD93 mediated podocyte activation via β1 integrin/FAK signaling in cultured human podocytes. CD93 blockade mitigated the activation of cultured human podocytes and albumin permeability in human GEnC-podocyte cocultures as well as albuminuria, glomerulosclerosis, and podocyte loss in two models of nephrotic syndrome: podocyte-specific transforming growth factor–β1 signaling ( PodTgfbr1 ) mice and adriamycin-treated mice. Cd93 knockout mice showed less proteinuria and glomerulosclerosis, compared with controls, after adriamycin injection. In patients with INS, soluble CD93 was high in urine in ~90% and 50% of patients in relapse and remission, respectively. High urinary CD93 was associated with faster decline in kidney function and slower response to immunosuppression. Soluble and glomerular CD93 was also elevated in other podocytopathies. We conclude that soluble CD93 contributes to podocyte injury.

Trogocytosis is an exchange of membrane-associated molecules between cells that can either halt or boost immune responses. However, the mechanism that regulates trogocytosis in T cells and its consequences are not yet clear. Here, we demonstrate that T cells can exchange chimeric antigen receptors (CARs) by trogocytosis, thereby arming recipient T cells with the capacity to respond to tumor antigens by up-regulating proteins associated with a cytotoxic response and killing of target cells. We demonstrate that although trogocytosis is dependent on cell-cell contact, the exchange of a specific cell membrane protein does not require a cognate binding partner on the surface of recipient cells. Instead, the probability that a protein is exchanged by trogocytosis is determined by its transmembrane domain. This finding opens new avenues for modulating this process in CAR-T cells.

Semliki Forest virus (SFV) is a neuropathogenic alphavirus which is of interest both as a model neurotropic alphavirus and as an oncolytic virus with proven potency in preclinical cancer models. In laboratory mice, peripherally administered SFV infiltrates the central nervous system (CNS) and causes encephalitis of varying severity. The route of SFV CNS entrance is poorly understood but has been considered to occur through the blood-brain barrier. Here we show that neuroinvasion of intravenously administered SFV is strictly dependent on very-low-density-lipoprotein receptor (VLDLR) which acts as an entry receptor for SFV. Moreover, SFV primarily enters the CNS through the blood-cerebrospinal fluid (B-CSF) barrier via infecting choroid plexus epithelial cells which show distinctly high expression of VLDLR. This is the first indication of neurotropic alphavirus utilizing choroid plexus for CNS entry, and VLDLR playing a specific and crucial role for mediating SFV entry through this pathway.

The central nervous system (CNS) constitutes a tightly regulated milieu, where immune responses are strictly controlled to prevent neurological damage. This poses considerable challenges to the therapeutic management of CNS pathologies, such as autoimmune disorders and cancer. Tertiary lymphoid structures (TLS) are ectopic, lymph node-like structures containing B- and T-cells, often associated with chronic inflammation or cancer, which have been shown to be detrimental in autoimmunity but beneficial in cancer. In-depth studies of TLS induction in CNS disorders, as well as their precise role in regulating adaptive immune responses in this context, will be paramount to the development of novel TLS-targeting therapies. In the present chapter, we review the anatomical and physiological peculiarities shaping TLS formation in the CNS, their relevance in autoimmunity and cancer, as well as their implications for the development of novel therapeutic modalities for these patients.

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates associated with improved prognosis in numerous tumors. To date, their prognostic value in central nervous system cancers and the events underlying their formation remain unclear. Here, we find that TLS correlate with improved survival in glioblastoma patients. Furthermore, combining spatial transcriptomics of human tissues with longitudinal studies in murine glioma, we establish that the assembly of T cell-rich clusters is a prerequisite for the development of canonical TLS, and show that CD4 T cells play a central role in this process. Indeed, we provide evidence that IL7R ⁺ CCR7 ⁺ Th1 lymphocytes with lymphoid tissue-inducing potential are recruited to TLS nucleation sites, where they can initiate TLS assembly by expressing lymphotoxin β. Our work defines TLS as a potential prognostic factor in glioblastoma and identifies cellular and molecular mechanisms of TLS formation. These findings have broader implications for the development of TLS-inducing therapies for cancer. Graphical

Compromised vascular integrity facilitates extravasation of cancer cells and promotes metastatic dissemination. CD93 has emerged as a target for antiangiogenic therapy, but its importance for vascular integrity in metastatic cancers has not been evaluated. Here, we demonstrate that CD93 participates in maintaining the endothelial barrier and reducing metastatic dissemination. Primary melanoma growth was hampered in CD93 ^–/– mice, but metastatic dissemination was increased and associated with disruption of adherens and tight junctions in tumor endothelial cells and elevated expression of matrix metalloprotease 9 at the metastatic site. CD93 directly interacted with vascular endothelial growth factor receptor 2 (VEGFR2) and its absence led to VEGF-induced hyperphosphorylation of VEGFR2 in endothelial cells. Antagonistic anti-VEGFR2 antibody therapy rescued endothelial barrier function and reduced the metastatic burden in CD93 ^–/– mice to wild-type levels. These findings reveal a key role of CD93 in maintaining vascular integrity, which has implications for pathological angiogenesis and endothelial barrier function in metastatic cancer.