Highly inflamed and neovascularized corneal graft beds are known as high-risk (HR) environments for transplant survival. One of the primary factors leading to this rejection is reduction in the suppressive function of regulatory T cells (Treg). Our results show that myeloid-derived suppressor cells (MDSC) counteract interleukin-6-mediated Treg dysfunction by expressing interleukin-10. Additionally, MDSC maintain forkhead box P3 stability and their ability to suppress IFN-γ⁺ Th1 cells. Administering MDSC to HR corneal transplant recipients demonstrates prolonged graft survival via promotion of Treg while concurrently suppressing IFN-γ⁺ Th1 cells. Moreover, MDSC-mediated donor-specific immune tolerance leads to long-term corneal graft survival as evidenced by the higher survival rate or delayed survival of a second-party C57BL/7 (B6) graft compared to those of third-party C3H grafts observed in contralateral low-risk or HR corneal transplantation of BALB/c recipient mice, respectively. Our study provides compelling preliminary evidence demonstrating the effectiveness of MDSC in preventing Treg dysfunction, significantly improving graft survival in HR corneal transplantation, and showing promising potential for immune tolerance induction.

Corneal transplantation is the most common form of solid tissue grafting, with an approximately 80% to 90% success rate. However, success rates may decline when donor tissues are derived from patients with a history of diabetes mellitus (DM). To evaluate the underlying immunopathologic processes that cause graft rejection, we used streptozotocin-induced type 1 DM (DM1) and transgenic Lep^ob/ob type 2 DM (DM2) diabetic murine models as donors and nondiabetic BALB/c as recipients. DM resulted in an increased frequency of corneal antigen-presenting cells (APCs) with an acquired immunostimulatory phenotype. Following transplantation, recipients that received either type of diabetic graft showed increased APC migration and T helper type 1 alloreactive cells, impaired functional regulatory T cells, and graft survival. Insulin treatment in streptozotocin-induced diabetic mice led to an increased tolerogenic profile of graft APC, lower T helper type 1 sensitization, and a higher frequency of functional regulatory T cells with high suppressive capacity, reflected in increased graft survival. We conclude that both DM1 and DM2 in donors can impact corneal APC functional phenotype, rendering the tissue more immunogenic and thereby increasing the risk of graft failure.

Efficient ocular drug delivery remains a significant challenge in treating eye inflammation due to physiological barriers such as the tear film and frequent blinking, which lead to rapid drug clearance. Commercial eyedrops, like Oceanside® (0.5% loteprednol etabonate (LE) ophthalmic suspension), suffer from low ocular bioavailability and require frequent dosing to maintain therapeutic levels. To address these limitations, we developed a mucoadhesive micellar drug delivery system to enhance the bioavailability and retention of LE on the cornea. Our system employed polymeric micelles (MCs) functionalized with phenylboronic acid (PBA), which exhibited high conjugation efficiency (>67%) to enable strong binding to the mucin-rich corneal layer. These MCs were synthesized using PBA-functionalized poly (ethylene glycol)-b-poly(N-(2-hydroxypropyl) methacrylamide-oligolactate) (PBA-PEG-b-p(HPMA-Lac_m)) and subsequently dispersed into a shear-thinning matrix solution to form a micellar eyedrop formulation. The resulting PBA-MC-LE eyedrop demonstrated a sustained LE release over 12 days, enabling prolonged therapeutic exposure. In vitro, ex vivo, and in vivo studies confirmed enhanced mucoadhesion and extended corneal retention. The formulation was biocompatible with human corneal epithelial cells and demonstrated ocular safety in mice. In a murine model of electrocautery-induced corneal inflammation, a once-daily administration of LE-loaded PBA-MC eyedrops significantly reduced corneal opacity, preserved corneal structure, and lowered immune cell infiltration and cytokine levels. Notably, the therapeutic efficacy of the LE-loaded PBA-MC eyedrops matched that of commercial Oceanside®, which required four daily doses. These findings suggest that the engineered PBA-MC eyedrops could serve as a promising platform for ocular drug delivery, addressing the challenges associated with treating eye inflammation effectively. STATEMENT OF SIGNIFICANCE: Mucoadhesive nanoparticles used for ocular drug delivery often suffer from low attachment efficiency, limiting their effectiveness. Additionally, the lack of in vivo comparisons with commercial eye drops hinders evaluating their clinical benefits. To address these issues, we developed phenylboronic acid (PBA)-functionalized polymeric micelles to enhance the bioavailability of loteprednol etabonate (LE) by increasing its retention on the corneal mucin layer. These micelles showed high PBA conjugation efficiency (>67%), a 12-day sustained release of LE, strong mucin adhesion, in vitro and in vivo biocompatibility. In a mouse model of corneal inflammation, a once-daily LE-loaded micellar eyedrop matched the efficacy of the commercial LE eyedrop (Oceanside®, 0.5%), which is dosed four times daily, reducing corneal opacity, preserving corneal structure, and decreasing inflammation.

Objectives: In this study, we establish a protocol for evaluating the outcomes of endothelial keratoplasty, including graft survival, rejection, or failure. Additionally, we also evaluate the alloimmune response in graft recipients. Methods: We performed EK using C57BL/6 (allogeneic) and BALB/c (syngeneic) as donors and BALB/c mice as recipients. Slit-lamp examination and optical coherence tomography were performed for clinical evaluations for 16 weeks post-procedure. Criteria for the assessment of corneal opacity were established and the animals were graded weekly. Additionally, we assessed corneal endothelial cell density by harvesting the corneas and staining with zonula occludens-1 (ZO-1). Lastly, lymph nodes were collected, and CD4⁺ T cells were MACS-sorted and co-cultured with syngeneic or allogeneic antigen-presenting cells (APCs) to assess the IFN-γ expression levels by alloreactive Th1 cells (ELISPOT) in response to the direct (donor) or indirect (host) pathways of sensitization. Results: We observed graft failure in four animals, including irreversible corneal opacity, graft detachment, and anterior synechiae in the first four weeks. The remaining animals were graded between 0 and 5 as per the established criteria. The total and graft corneal thickness and endothelial cell density progressively worsened with a higher grade of corneal opacity. The direct allosensitization of Th1 cells was significantly higher in mice with a higher grade of corneal opacity. At 16 weeks follow-up, the grafts remained stable with low opacity scores in syngeneic EK recipients; however, the opacity scores were higher and variable in allogeneic EK recipients. Conclusions: These findings establish a standardized protocol to assess the graft outcomes in a murine model of EK. Furthermore, we delineate the underlying immunological pathway that contributes to the immune-mediated rejection of grafts in this model.

Purpose: To compare graft survival and alloimmune responses in murine endothelial keratoplasty (EK) versus penetrating keratoplasty (PK) and to elucidate the immunological mechanisms that underlie the differential graft outcomes. Methods: Allogeneic EK and PK were performed in BALB/c recipient mice using fully disparate C57BL/6 donors; syngeneic EK recipients served as controls. Graft clarity was monitored over 16 weeks by slitlamp biomicroscopy and scored using standardized opacity grading. Anterior segment optical coherence tomography (AS-OCT) was used to measure central corneal thickness. Graft survival was assessed using Kaplan–Meier analysis. Immunohistochemistry and confocal microscopy were performed to evaluate corneal endothelial cell (CEnC) integrity through ZO-1 staining. T-cell–mediated alloimmunity was assessed using intracellular IFN-γ staining (flow cytometry) and ELISPOT assays targeting both direct and indirect antigen presentation pathways. Results: PK allografts exhibited significantly higher corneal opacity and lower survival (50%) than allogeneic EK grafts (71.4%, P < 0.0001). AS-OCT showed that corneal edema was highest in rejected PK grafts at 4 weeks and in rejected EK grafts at 16 weeks, with EK displaying a more gradual increase in thickness. Flow cytometry revealed significantly greater frequencies of IFN-γ ⁺ CD4 ⁺ T cells in PK recipients compared with EK recipients ( P < 0.001). ELISPOT assays demonstrated a more robust Th1 response in PK through both the direct and indirect sensitization pathways. Corneal endothelial cell (CEnC) density was significantly reduced in rejected EK and PK grafts compared with their respective accepted counterparts ( P < 0.01), whereas CEnC density was comparable between accepted EK and PK grafts. Conclusions: EK grafts exhibit higher graft survival rates and significantly reduced activation of host T-cell responses compared with PK grafts, which may be attributed to lower frequencies of graft-borne antigen presenting cells, thus resulting in a milder Th1-mediated immune response.