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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.

Monoclonal antibody-based immune checkpoint inhibitors, which have brought breakthrough effects in cancer treatments, are expected to assist in the treatment of viral diseases. However, antibody therapies may cause immune-related side effects, such as inflammation and pneumonia, due to cytokine storms. Small-molecule PD-1/PD-L1 inhibitors are an alternative to monoclonal antibody-based therapeutics. We have identified a novel small-molecule PD-1/PD-L1 inhibitor having a functional group (disulfide group), namely compound 2 (molecular weight: 456.6), from our library of sulfur-containing protein¨Cprotein interaction inhibitor compounds. Compound 2 selectively bound to PD-L1 over PD-1, with the dissociation rate constant (K D ) of 77.60?¡À?4.44?nM (obtained by affinity analysis) and showed promising T cell activation recovery. A molecular docking simulation study between 2 and PD-L1 suggested that 2 binds to PD-L1 in a binding mode different from those of other small-molecule PD-L1/PD-1 inhibitors. Notably, oral administration of 2 to mice pre-infected with influenza A virus (A/NWS/33, H1N1 subtype) caused a significant increase in the neutralizing antibody titers, as well as recovery from influenza-induced pneumonia. Overall, 2 provides insight for the development of therapeutic drugs against early viral infections, with both virus titer-reducing and antibody titer-boosting effects. Moreover, 2 is widely used as a rubber peptizing agent in the production process of tires and other rubber products. Our findings may provide useful information for investigating its influence on living organisms. The online version contains supplementary material available at 10.1038/s41598-025-17982-3. Subject terms: Drug discovery and development, Pharmacology, Screening, Structure-based drug design

The intestinal epithelium of human infants is developmentally immature compared to that of adults. Exactly how this immaturity affects key epithelial functions and their interactions with nearby immune cells remains an understudied area of research, partly due to limited access to non-diseased infant gut tissues. Human intestinal organoids, or ¡°mini guts¡± generated from tissue stem cells, are promising models for investigating intestinal biology and disease mechanisms. These three-dimensional structures closely mimic their tissue of origin, including cellular physiology and genetics. We have also previously shown that neonatal Th17 cells represent a distinct cell population with a cytokine profile skewed toward IL-22 production rather than IL-17A, as seen in adult Th17 cells. In this study, we sought to model the impact of neonatal-derived Th17 cytokine, namely IL-22 and the intestinal epithelium using infant-derived ileal enteroids. We generated enteroids from ileal biopsies from infants (< 6 months old) and cultured them for seven days with standard organoid growth media, organoid media supplemented with conditioned media from cord-blood-derived Th17 cells, or media supplemented with recombinant IL-22. We assessed morphological changes and conducted transcriptomics profiling via RNAseq. Exposing enteroids to neonatal Th17-cells-derived conditioned media led to enhanced growth, maturation, and differentiation as compared to control media. These effects were ablated when an IL-22 neutralizing antibody was used, while conversely, supplementing with recombinant IL-22 mimicked the Th17 effects, increasing intestinal epithelial cell proliferation and inducing marked differentiation of secretory cells. Our transcriptomic profiling similarly demonstrated significant changes in response to IL-22 with downregulation of Wnt and Notch signaling and upregulation of immune pathways, particularly interferon signaling. The transcriptomic data also suggested that IL-22 treatment led to changes in cell type composition with an increase in stem- and progenitor cells at the expense of enterocytes. Taken together, our data suggests that early-life intestinal development is likely influenced by IL-22-dependent crosstalk between the infant epithelium and exposure to neighboring Th17 cells. This promotes epithelial cell maturation and immune readiness, reflected at both the morphological and molecular levels. Our work also provides a relevant framework for studying healthy infant gut development, which can be further leveraged to examine early-life gastrointestinal disorders, model complex human disease, and therapeutic testing while reducing reliance on animal models.