After transplantation, Th1-mediated immune rejection is the predominant cause of graft failure. Th1 cell sensitization occurs through complex and context-dependent interaction among antigen-presenting cell subsets, particularly CD11b ⁺ DCs (DC2) and CD103 ⁺ DCs (DC1). This interaction necessitates further investigation in the context of transplant immunity. We used well-established preclinical models of corneal transplantation and identified distinct roles of migratory CD103 ⁺ DC1 in influencing the outcomes of the grafted tissue. In recipients with uninflamed corneal beds, migratory CD103 ⁺ DC1 demonstrate a tolerogenic phenotype that modulates the immunogenic capacity of CD11b ⁺ DC2 primarily mediated by IL-10, suppressing alloreactive CD4 ⁺ Th1 cells via the PD-L1/PD-1 pathway and enhancing Treg-mediated tolerance via αvβ8 integrin–activated TGF-β1, thus facilitating graft survival. Conversely, in recipients with inflamed and vascularized corneal beds, IFN-γ produced by CD4 ⁺ Th1 cells induced migratory CD103 ⁺ DC1 to adopt an immunostimulatory phenotype, characterized by the downregulation of regulatory markers, including αvβ8 integrin and IL-10, and the upregulation of IL-12 and costimulatory molecules CD80/86, resulting in graft failure. The adoptive transfer of ex vivo induced tolerogenic CD103 ⁺ DC1 (iDC1) effectively inhibited Th1 polarization and preserved the tolerogenic phenotype of their physiological counterparts. Collectively, our findings underscore the essential role played by CD103 ⁺ DC1 in modulating host alloimmune responses.

Purpose: While the first murine endothelial keratoplasty (EK) model established surgical feasibility, manual lamellar dissection and endothelial scraping introduce significant procedural variability. This study aims to develop a standardized murine EK model implementing a controlled stromal peeling technique and quadrant-based host bed preparation to minimize inconsistency. Methods: We introduced 2 critical methodological refinements. First, donor corneas were prepared using a controlled stromal peeling approach to ensure uniform graft thickness. Second, traditional circular scraping of the recipient endothelium was replaced with quadrant-based mechanical removal to standardize the denuded area. Postoperative outcomes were quantified using AS-OCT and a novel time-resolved opacity scoring system designed to distinguish early technical failure from immunologic rejection. Results: Controlled stromal peeling produced posterior lamellar grafts with significantly higher thickness consistency compared with traditional needle dissection. The quadrant-based removal resulted in highly reproducible postoperative corneal edema, reflecting standardized disruption of host endothelial function. Furthermore, the time-resolved grading system allowed more granular, objective differentiation between early graft failure and delayed rejection compared with traditional binary survival assessments. Conclusions: These refinements overcome the inherent variability of the initial murine EK model. By standardizing both the surgical procedure and the interpretive framework, this optimized model provides a robust, reproducible platform for investigating cellular mechanisms of graft outcomes in experimental keratoplasty.

This study evaluates how titanium and polymethyl methacrylate (PMMA) Boston Keratoprosthesis backplate substrates influence human corneal fibroblast proliferation, cytotoxicity, morphology, activation phenotype, and mechanotransductive signaling. Human corneal fibroblasts were cultured on titanium and PMMA, with tissue culture plastic or glass as controls. Proliferation was assessed over 7 days using metabolic assays, and cytotoxicity was measured by lactate dehydrogenase release. Cell morphology and surface coverage were examined by scanning electron microscopy, while immunofluorescence quantified fibroblast-specific protein 1 (FSP-1) and α-smooth muscle actin (α-SMA). Gene expression of α-SMA, collagen I, FSP-1, and focal adhesion kinase (FAK) was analyzed by quantitative PCR. Cells cultured on both substrates maintained stable viability with modest increases in estimated cell numbers and comparable proliferation curves, indicating preserved metabolic activity without growth suppression. Cytotoxicity remained low and similar between groups. SEM demonstrated broader and more continuous cell spreading on titanium, whereas cells on PMMA were more sparsely distributed. Immunofluorescence showed higher FSP-1 expression on titanium and increased α-SMA on PMMA. Gene expression analysis revealed higher FAK transcripts on PMMA, with no significant differences in α-SMA, FSP-1, or collagen I. These results confirm the cytocompatibility of both titanium and PMMA backplates with human corneal fibroblasts and support their use with the Boston Keratoprosthesis.