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Mare endometrosis is a chronic degenerative condition of the endometrium, primarily characterized by fibrosis, involving interactions among fibroblasts, immune cells, and epithelial cells regulated by cytokines and growth factors. T helper (Th)1 and Th2 cells seem to play a pivotal role in fibrosis. However, their roles in equine endometrial fibrosis remain unknown. This study explores Th1 and Th2 cell distribution across different stages of endometrium histopathological Kenney and Doig categories; and evaluated their secretome effects on non-fibrotic endometrium derived fibroblast functional characteristics, extracellular matrix (ECM)-associated mRNA transcription, and transcriptomic profiles. Th1 and Th2 cells, along with cytokines (IFN-¦Ã, IL-4, IL-13) and their receptors, were present in mare endometria at all endometrium stages. Th1 secretome influenced genes enriched in metabolism, cell cycle, and ECM-related pathways, while Th2 secretome regulated genes enriched in tissue remodeling and signaling pathways, suggesting their role in the development of fibrosis in the endometrosis progression.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-20152-0.

Chimeric antigen receptor (CAR) T cells have become an established immunotherapy with promising results for the treatment of hematological malignancies. However, modulation of the targeted antigen¡¯s surface level in cancer cells affects the quality and safety of CAR-T cell therapy. Here we present an engineered yeast-based antigen system for simulation of cancer cells with precise regulation of surface-antigen densities, providing a tool for controlled activation of CAR T cells and systematic assessment of antigen density effects. This Synthetic Cellular Advanced Signal Adapter (SCASA) system uses G protein-coupled receptor signaling to control cancer antigen densities on the yeast surface and provides a customizable platform allowing selectable signal inputs and modular pathway engineering for precise output fine-tuning. In relation to CD19+ cancers, we demonstrate synthetic cellular communication between CD19-displaying yeast and human CAR T cells as well as applications in high-throughput characterization of different CAR designs. We show that yeast is an alternative to conventional technologies (e.g. microbeads) and can provide higher activation control of clinically derived CAR T cells in vitro, relative to cancer cells. In summary, we present a customizable yeast-based platform for high-throughput characterization of CAR-T cell functionality and show potential applications within therapeutic T cells in clinical settings. Chimeric antigen receptor T (CAR-T) cell therapy uses engineered donor T cells to recognize and eliminate cancer cells through cognate antigen-dependent activation. Here authors develop an alternative to bead-based or cancer-cell-induced CAR-T cell activation by presenting the antigen on the surface of engineered yeast cells, which allows precise regulation of antigen density.

Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination (HR), with efficiency improved by CRISPR/Cas9 technology. Circular single-stranded DNA (cssDNA) has been used as a genome engineering catalyst (GATALYST) for efficient and safe gene knock-in. Here, we introduce enGager, an enhanced GATALYST associated genome editor system that increases transgene integration efficiency by tethering cssDNA donors to nuclear-localized Cas9 fused with single-stranded DNA binding peptide motifs. This approach further improves targeted integration and expression of reporter genes at multiple genomic loci in various cell types, showing up to 6-fold higher efficiency compared to unfused Cas9, especially for large transgenes in primary cells. Notably, enGager enables efficient integration of a chimeric antigen receptor (CAR) transgene in 33% of primary human T cells, enhancing anti-tumor functionality. This ¡®tripartite editor with ssDNA optimized genome engineering (TESOGENASE) offers a safer, more efficient alternative to viral vectors for therapeutic gene modification. Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination. Here the authors present the TESOGENASE system which enhances CRISPR-based gene integration by tethering circular single-stranded DNA to Cas9.