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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-¦Á and IFN-¦Ã 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-¦ÊB 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.

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-¦Ã. 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.

BackgroundImmune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, providing durable and even curative responses. However, most patients do not respond and current biomarkers (eg, programmed death 1 ligand 1 [PD-L1]), mismatch repair deficiency [dMMR]/high microsatellite instability [MSI] and tumor mutational burden) lack predictive accuracy. Ex vivo profiling of patient-derived tumor fragments shows promise as a predictive biomarker but relies on substantial surgical tissue to mitigate intra-specimen heterogeneity. Innovations are needed that address these challenges, particularly where limited tissue is available such as in core needle biopsies (CNBs).MethodsLive tumor fragments (LTFs) were generated from 59 human tumor resections and 31 CNBs from patients enrolled in observational clinical trials (ClinicalTrials.gov identifiers: NCT05478538, NCT05520099, NCT06349642) to assess cytokine induction following ICI treatment. LTFs were encapsulated in hydrogel and cultured ex vivo for up to 72 hours. A sequential treatment strategy that applies control and treatment within the same well was used with response to ICI or ¦ÁCD3/¦ÁCD28 assessed using a multiplex secretome assay. Viability was assessed using established metabolic assays and dynamic optical coherence microscopy.ResultsLTFs maintained viability and retained T cells responsive to stimulation throughout ex vivo culture. Multiplex immunofluorescence and immunohistochemistry showed key components of the tumor microenvironment, including relative proportions of CD4+ and CD8+ immune cell populations, were preserved. Specimens positive for PD-L1 or dMMR/MSI-high were enriched for cytokine upregulation, including T-cell response cytokines IFN¦Ã and CXCL10, after ¦ÁPD-1 treatment. To demonstrate clinical applicability of the sequential treatment strategy, CNBs from patients with lung, gastrointestinal or kidney cancer were profiled and differential cytokine induction in response to ICI treatment was observed.ConclusionsThe novel ex vivo platform presented is capable of detecting T-cell response to ICI treatment by using a sequential treatment strategy. This approach addresses challenges associated with cross-well heterogeneity in tissue composition and requires half as much tissue as a cross-well comparison, mitigating tissue limitations typically associated with non-surgical biopsies. Importantly, the platform is compatible with established functional assays as well as non-destructive spatial imaging, enabling researchers to characterize response to ICI longitudinally. Ongoing trials will enable clinicians to assess platform performance in predicting response to immunotherapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-07378-2.