Achieving a cure is an urgent need for patients with advanced solid tumors. Here, we discover that oncolytic virus (OV) infection enhances IL-18 receptor expression but fails to increase IL-18 ligand expression. Therefore, we engineer armed oncolytic alphavirus M1 expressing wild-type IL-18 (wtIL-18) or a mutant variant (mutIL-18) that evades IL-18 binding protein (IL-18BP) while maintaining IL-18 receptor (IL-18R) binding. Intravenous administration of M1-mutIL-18 suppresses the growth of multiple advanced solid tumors in C57BL/6 and BALB/c mouse models and promotes long-term systemic immune memory. Mechanistically, armed M1-mutIL-18 enhances directed clonal expansion and differentiation of CD8+ T cells and sustains IFN-γ production. Thus, armed M1-mutIL-18 promotes dendritic cell (DC) activation, priming and activation of CD8+ T cells in lymphatic organs, and infiltration of IL-18R+ CD8+ T cells in the tumor microenvironment, establishing a positive feedback loop. We further show that a PD-L1 inhibitor enhances the anti-tumor efficacy of mutIL-18 OVs. These results highlight the importance of the IL-18 pathway in oncolytic virus therapy and implicate reprogramming ligand-receptor interaction as an effective strategy for immunotherapy.
© 2025. The Author(s).

Multiple genetic associations suggest a causative relationship between Th17-related genes coding for proteins, such as IL-17A, IL-23 and STAT3, and psoriasis. Further support for this link comes from the findings that neutralizing antibodies directed against IL-17A, IL-17RA and IL-23 are efficacious in diseases like psoriasis, psoriatic arthritis and ankylosing spondylitis. RORγt is a centrally positioned transcription factor driving Th17 polarization and cytokine secretion and modulation of RORγt may thus provide additional benefit to patients. However, RORγt also plays a role in the normal development of T cells in the thymus and genetic disruption of RORγt in the mouse leads to the development of lymphoma originating in the thymus. Whilst it is not established that down-regulation of RORγt activity would lead to the same consequence in humans, further understanding of the thymus effects is desirable to support progress of this target as a potential treatment of Th17-driven disease. Herein we present the characterisation of recently disclosed RORγt inverse agonists demonstrating target engagement and efficacy in vitro and in vivo against Th17 endpoints but requiring higher concentrations in vitro to affect thymocyte apoptosis.
Copyright: © 2025 Collins et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Molecular crowding of agonist peptide/MHC class II complexes (pMHCIIs) with structurally similar, yet per se non-stimulatory endogenous pMHCIIs is postulated to sensitize T-cells for the recognition of single antigens on the surface of dendritic cells and B-cells. When testing this premise with the use of advanced live cell microscopy, we observe pMHCIIs as monomeric, randomly distributed entities diffusing rapidly after entering the APC surface. Synaptic TCR engagement of highly abundant endogenous pMHCIIs is low or non-existent and affects neither TCR engagement of rare agonist pMHCII in early and advanced synapses nor agonist-induced TCR-proximal signaling. Our findings highlight the capacity of single freely diffusing agonist pMHCIIs to elicit the full T-cell response in an autonomous and peptide-specific fashion with consequences for adaptive immunity and immunotherapeutic approaches.
© 2023 The Authors. Published under the terms of the CC BY 4.0 license.

The neurovascular unit (NVU), composed of endothelial cells, pericytes, juxtaposed astrocytes and microglia together with neurons, is essential for proper central nervous system functioning. The NVU critically regulates blood-brain barrier (BBB) function, which is impaired in several neurological diseases and is therefore a key therapeutic target. To understand the extent and cellular source of BBB dysfunction, simultaneous isolation and analysis of NVU cells is needed. Here, we describe a protocol for the EPAM-ia method, which is based on flow cytometry for simultaneous isolation and analysis of endothelial cells, pericytes, astrocytes and microglia. This method is based on differential processing of NVU cell types using enzymes, mechanical homogenization and filtration specific for each cell type followed by combining them for immunostaining and fluorescence-activated cell sorting. The gating strategy encompasses cell-type-specific and exclusion markers for contaminating cells to isolate the major NVU cell types. This protocol takes ~6 h for two sets of one or two animals. The isolation part requires experience in animal handling, fresh tissue processing and immunolabeling for flow cytometry. Sorted NVU cells can be used for downstream applications including transcriptomics, proteomics and cell culture. Multiple cell-type analyses using UpSet can then be applied to obtain robust targets from single or multiple NVU cell types in neurological diseases associated with BBB dysfunction. The EPAM-ia method is also amenable to isolation of several other cell types, including cancer cells and immune cells. This protocol is applicable to healthy and pathological tissue from mouse and human sources and to several cell types compared with similar protocols.
© 2023. Springer Nature Limited.

MLKL and RIPK3 are the core signaling proteins of the inflammatory cell death pathway, necroptosis, which is a known mediator and modifier of human disease. Necroptosis has been implicated in the progression of disease in almost every physiological system and recent reports suggest a role for necroptosis in aging. Here, we present the first comprehensive analysis of age-related histopathological and immunological phenotypes in a cohort of Mlkl-/- and Ripk3-/- mice on a congenic C57BL/6 J genetic background. We show that genetic deletion of Mlkl in female mice interrupts immune system aging, specifically delaying the age-related reduction of circulating lymphocytes. -Seventeen-month-old Mlkl-/- female mice were also protected against age-related chronic sterile inflammation in connective tissue and skeletal muscle relative to wild-type littermate controls, exhibiting a reduced number of immune cell infiltrates in these sites and fewer regenerating myocytes. These observations implicate MLKL in age-related sterile inflammation, suggesting a possible application for long-term anti-necroptotic therapy in humans.
© 2023. The Author(s).