Person:

Dana, Reza

Purpose

To develop and compare effective strategies for depleting graft-derived passenger leukocytes which include antigen-presenting cells from corneal buttons, and to assess the effectiveness of this strategy in promoting graft survival using a high-risk (HR) model of corneal transplantation.

Methods

Corneal buttons harvested from C57BL/6 mice were used in three ex vivo strategies of passenger leukocyte depletion. Two strategies involved storage in Optisol-GS medium at different temperatures for prolonged periods. A third strategy utilized complement-dependent cytotoxicity (CDC) by treating the buttons with anti-CD45 mAb plus complement. Whole-mount corneal buttons or cells from enzyme digested corneas were analyzed using confocal microscopy or flow cytometry, respectively, for the pan-leukocyte surface marker CD45. HR host beds were created and used to evaluate the efficacy of passenger leukocyte depletion on transplant survival.

Results

Passenger leukocyte numbers in the buttons were significantly reduced by all three treatments. CDC was the most efficient strategy for passenger leukocyte depletion with 39% reduction (P < 0.00005) of CD45+ cells, and negligible damage to the endothelial layer, achievable within 24 h. However, 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.

Purpose.Resolvins and lipoxins are lipid mediators generated from essential polyunsaturated fatty acids that are the first dual anti-inflammatory and pro-resolving signals identified in the resolution phase of inflammation. Here the authors investigated the potential of aspirin-triggered lipoxin (LX) A4 analog (ATLa), resolving (Rv) D1, and RvE1, in regulating angiogenesis in a murine model.

Methods.ATLa and RvE1 receptor expression was tested in different corneal cell populations by RT-PCR. Corneal neovascularization (CNV) was induced by suture or micropellet (IL-1β, VEGF-A) placement. Mice were then treated with ATLa, RvD1, RvE1, or vehicle, subconjunctivally at 48-hour intervals. Infiltration of neutrophils and macrophages was quantified after immunofluorescence staining. The mRNA expression levels of inflammatory cytokines, VEGFs, and VEGFRs were analyzed by real-time PCR. CNV was evaluated intravitally and morphometrically.

Results.The receptors for LXA4, ALX/Fpr-rs-2 and for RvE1, ChemR23 were each expressed by epithelium, stromal keratocytes, and infiltrated CD11b+ cells in corneas. Compared to the vehicle-treated eye, ATLa-, RvD1-, and RvE1-treated eyes had reduced numbers of infiltrating neutrophils and macrophages and reduced mRNA expression levels of TNF-α, IL-1α, IL-1β, VEGF-A, VEGF-C, and VEGFR2. Animals treated with these mediators had significantly suppressed suture-induced or IL-1β–induced hemangiogenesis (HA) but not lymphangiogenesis. Interestingly, only the application of ATLa significantly suppressed VEGF-A–induced HA.

Conclusions.ATLa, RvE1, and RvD1 all reduce inflammatory corneal HA by early regulation of resolution mechanisms in innate immune responses. In addition, ATLa directly inhibits VEGF-A–mediated angiogenesis and is the most potent inhibitor of NV among this new genus of dual anti-inflammatory and pro-resolving lipid mediators.

Purpose.

Chemokine receptor 7 (CCR7) is a key homing molecule for immune cell trafficking, including corneal antigen-presenting cell (APC) migration from the inflamed cornea to draining lymph nodes (LNs). Here, the authors investigated the effect of CCR7-facilitated donor APC trafficking on allosensitization, regulatory T-cell (Treg) function, and graft survival in corneal transplantation.

Methods.

CCR7−/− or wild-type (WT) allogeneic corneal grafts were transplanted onto the neovascularized high-risk recipient beds. Two weeks later, the frequency of directly alloprimed host T cells was measured by the IFN-γ ELISPOT assay. Treg function was tested by a coculture suppression assay and an IFN-γ ELISPOT assay. Kaplan-Meier analysis was performed to evaluate graft survival.

Results.

The recipients of CCR7−/− grafts had fewer migrated donor APCs and lower frequency of IFN-γ–producing T cells in the draining LNs. However, there was no statistically significant difference in transplant survival between recipients of CCR7−/− and those of WT grafts. Tregs from the CCR7−/− graft recipient group showed reduced regulatory potential for the suppression of proliferation of naive T cells and direct alloprimed T cells and expressed lower Foxp3 levels. In vitro studies confirmed that mature CCR7+ major histocompatibility complex class II+ CD86+ graft-derived dendritic cells were critical for Treg function.

Conclusions.

Not only is CCR7-mediated donor-derived APC trafficking to the draining LNs important in the initiation of host T-cell priming, it is crucial for Treg-mediated tolerance.