To determine whether CD45+ cells emigrated from the corneas into the medium, both adherent cells and non-adherent cells were collected from the medium of culture dishes. passenger leukocyte depletion failed to improve HR graft longevity. Conclusions Anti-CD45 antibody plus complement-mediated targeting of donor tissue is the most efficient way to deplete corneal passenger leukocytes and can considerably reduce the time required for cell depletion. However, depletion of graft passenger leukocytes does not have a significant effect on promoting graft survival even in the HR setting. INTRODUCTION Despite the successful outcome of corneal transplantation in non-vascularized, or so-called low-risk (LR), recipient beds corneal transplantation performed in vascularized and inflamed high-risk (HR) host beds has shown little improvement in survival over the past several decades. Indeed, while systemic corticosteroids and immunosuppressive agents may be partially effective in preventing graft rejection, their use is limited because of a wide range of side effects, including infection, cataract, and glaucoma. Moreover, even with potent immune suppression, rejection rates in HR corneal transplantation can be as high as 50C90%.1, 2 There is, therefore, a compelling need to improve the survival of HR grafts in a manner that minimizes the impact on host immune competence. The most common cause of corneal graft failure remains allograft rejection, during which host alloreactive T cells are activated and once peripheralized to AKT-IN-1 the graft, can result in graft destruction.3 Allograft rejection is triggered by two distinct, but ARPC1B not mutually exclusive pathways of allorecognition mediated by either donor-derived antigen-presenting cells (APCs) or recipient APCs the direct and indirect pathways of allosensitization respectively. In the direct pathway, donor APCs present intact major histocompatibility complex (MHC) class II molecules residing on their surface to T cells. In contrast, in the indirect pathway recipient APCs present processed MHC or minor antigens to T cells.4C8 The direct and indirect pathways play distinct roles in corneal alloimmunity. Using a murine orthotopic corneal transplantation model, Huq et al. demonstrated that T cells activated via the direct pathway are detected and donor-derived APCs are functionally capable of priming host T cells in HR graft recipients. They also found that transplantation using MHC class II knockout donor tissue leads to significantly improved survival of HR allografts.6 Relatedly, Simon et al. discovered that prolonged storage of donor corneas prevented allograft rejection particularly in HR corneal transplantation but offered no mechanistic information to explain this finding, although they postulated that depletion of donor APCs through prolonged storage could reduce graft immunogenicity.9 CD45 is a transmembrane molecule found on the surface of all bone marrow-derived nucleated hematopoietic cells and their precursors, hence, a leukocyte common antigen.10 It is well known that the cornea contains a heterogeneous population of bone marrow (BM)-derived cells which express the leukocyte common antigen, CD45.11, 12, AKT-IN-1 13, 14 Given the capacity of CD45+ cells to potentially prime T cells, and thereby initiate alloimmune responses, it is of interest to investigate the effect of graft passenger leukocyte depletion on corneal transplant survival. To this end, complement-dependent cytotoxicity (CDC) is a mechanism of killing cells in which antibody binds to the receptor of a target cell, then fixes and activates the complement system. The end result is the formation of a membrane attack complex that makes a hole within the cell membrane, causing cell lysis and death. CDC can potentially provide a powerful strategy for depleting graft passenger leukocytes prior to corneal transplantation, and has been shown to be effective to this end in renal transplantation15. The current study was conducted to compare the efficiency of donor passenger leukocyte depletion using CDC and other strategies, as well as to AKT-IN-1 investigate the acceptance of the APC-depleted corneal grafts in HR corneal transplants. METHODS Mice C57BL/6 and BALB/c mice (male, 6C14 weeks) were purchased from Taconic Farms (Germantown, NY). Corneal buttons from C57BL/6 mice were used as sources of donor tissue because they are MHC and multiple minor H disparate to BALB/c mice which were used as recipients in the corneal transplantation experiments. For the experiments, mice were euthanized by cervical dislocation and the corneal buttons were harvested. All protocols were approved by the Schepens Eye Research Institute Animal.