Background: MSCs possess strong immunoregulatory properties and play a central role in maintaining immune homeostasis by limiting inflammatory responses. Their function is highly plastic and influenced by environmental cues, including viral signals. How SARS-CoV-2-derived antigens affect MSC immunoregulation remains incompletely understood. This study aimed to investigate the impact of SARS-CoV-2 peptides on MSC-mediated immune modulation of T-cells. Methods: MSCs were stimulated directly with SARS-CoV-2 spike protein S peptides or cocultured with SARS-CoV-2 peptide-activated T-cells. TLR4 surface expression and receptor downstream signaling were assessed to evaluate pathway activation. MSC immunoregulatory function was analyzed by measuring suppression of TNF-α and IFN-γ expression and induction of CD4+FOXP3+ regulatory T-cells. TLR4 inhibition and lipopolysaccharide (LPS) stimulation were used to examine pathway specificity and interaction. Results: SARS-CoV-2 peptides activated TLR4-associated signaling in MSCs, increasing TLR4 expression and NF-κB phosphorylation. Peptide-treated MSCs showed impaired suppression of pro-inflammatory cytokines and reduced induction of regulatory T-cells. TLR4 inhibition prevented these effects. LPS induced similar effects, while combining LPS and peptide stimulation partially restored physiological T-cell cytokine suppression. Conclusions: SARS-CoV-2 peptides modulate MSC immunoregulatory function on T-cells via TLR4-dependent mechanisms.

Highlights What are the main findings? We validated the feasibility of using a 3D self-assembled blood–brain barrier (BBB) model compromised of different brain cell types, to study BBB disruption induced by both inflammation (TNF-α, IFN-γ) and ischemia–reperfusion injury (oxygen-glucose deprivation-reoxygenation model, OGD-R). Organoids showed increased permeability to fluorescent dextrans, decreased tight-junction gene expression, elevated ROS/hypoxia signals, and upregulated cell adhesion molecules such as ICAM1 and VCAM1.We demonstrated the ability to detect changes in immune cell transmigration across the BBB into the organoids under both conditions of inflammation and OGD-R. Increased immune cell infiltration after cytokine treatment and reperfusion was blocked by anti-cell adhesion molecule antibodies. What are the implications of the main findings? This 3D human BBB organoid provides a biologically relevant platform for modeling neuroimmune interactions, enabling studying how inflammation and ischemia–reperfusion injury alters barrier integrity, adhesion molecule expression, and immune cell entry in a human-specific context.The model offers a powerful predictive preclinical screening tool that bridges the gap between simple, physiologically irrelevant 2D models and expensive in vivo animal models and enables high throughput screening of various anti-cells adhesion molecule therapies. In vitro model representative of human tissue complexity accelerates the translation of drugs into clinical practice. AbstractNumerous central nervous system pathological conditions involve blood–brain barrier (BBB) disruption and the egress of immune cells in the brain. Controlling immune cell transmigration into the brain represents a potential therapeutic target. This study describes the application of a 3D human BBB spheroidal model that consists of six major brain cell types to test the transmigration of immune cells under normal and pathological conditions of inflammation and ischemia–reperfusion injury (IRI). The cell types in the BBB organoid include brain microvascular endothelial cells (HBMVECs) and pericytes at the spheroids’ surface, surrounding a core of astrocytes, microglia, oligodendrocytes, and neural progenitor cells. The model recapitulates the interaction of CD4+ T-cells and immunomodulators with HBMVECs at the BBB including changes in cell adhesion molecules expressed on their surface. This study demonstrated that the human 3D BBB model recapitulates many features of the barrier under normal and pathological conditions of inflammation and hypoxia-reperfusion injury. Proinflammatory cytokines and hypoxia disrupt the barrier and increase its permeability, decreasing the expression of tight junctions. Proinflammatory cytokines and reperfusion increase the expression of cell adhesion molecules and increase immune cell transmigration. Immune cell transmigration could be reduced with anti-cell adhesion molecule antibodies, further validating the model for studying neuroimmune interactions and for conducting high-throughput screening of therapeutic targets that modulate immune cell transmigration into the brain.

BACKGROUND: Neuroblastoma (NBL) is the most common extracranial solid tumor in children, with a 50% survival rate in high-risk cases. Chemotherapy combination with immunotherapy is central to HR-NBL treatment. AIM: This study aimed to explore how Irinotecan influences the immune landscape of NBL cells, particularly focusing on the modulation of immune checkpoints relevant to future chemo-immunotherapy strategies. METHODS: Two neuroblastoma cell lines, SK-N-BE(2) and SH-SY5Y, were exposed to sub-lethal doses of Irinotecan and IFN-γ. Flow cytometry assessed the expression of immune markers, including GD2, CD47, MHC I, and PD-L1. We also examined phagocytosis by macrophages and T-cell-mediated killing. In vivo studies using a neuroblastoma mouse model assessed the effects of Irinotecan and PBMC injections. Soluble CD47 was also measured in cell culture supernatants and exploratory patient plasma samples. RESULTS: Treatment exposure was associated with modulation of several immune-related markers, including GD2, CD47, PD-L1, and MHC class I. While Irinotecan reduced macrophage-mediated phagocytosis, this effect was reversed by the addition of anti-CD47 antibodies. T-cell-mediated killing was enhanced when NBL cells were pre-treated with Irinotecan. In vivo studies showed that the combination of PBMCs and Irinotecan resulted in reduced tumor burden compared to either treatment alone. Furthermore, CD47 expression was detectable in-patient plasma, suggesting that soluble CD47 may warrant exploration in larger future cohorts. CONCLUSION: This pilot study suggests that irinotecan may modulate key immune checkpoints in neuroblastoma. These results supports further investigation of rational chemo-immunotherapy combinations, including GD2 and CD47-targeted combination strategies.