The purinergic receptor antagonist oxidized adenosine triphosphate suppresses immune-mediated corneal allograft rejection

Abstract

Adenosine triphosphate (ATP) is released into the extracellular environment during transplantation, and acts via purinergic receptors to amplify the alloimmune response. Here, using a well-established murine model of allogeneic corneal transplantation, we investigated the immunomodulatory mechanisms of the purinergic receptor antagonist oxidized ATP (oATP). Corneal transplantation was performed using C57BL/6 donors and BALB/c hosts. oATP or sterile saline was administered via intraperitoneal injection for 2 weeks postoperatively. Frequencies of CD45⁺ leukocytes, CD11b⁺MHCII⁺ antigen presenting cells (APCs), CD4⁺IFN-γ⁺ effector Th1 cells and CD4⁺Foxp3⁺ regulatory T cells (Tregs) were evaluated by flow cytometry. Slit-lamp microscopy was performed weekly for 8 weeks to evaluate graft opacity and determine transplant rejection. Treatment with oATP was shown to significantly reduce graft infiltration of CD45⁺ leukocytes, decrease APC maturation and suppress effector Th1 cell generation relative to saline-treated control. No difference in Treg frequencies or Foxp3 expression was observed between the oATP-treated and control groups. Finally, oATP treatment was shown to reduce graft opacity and increase graft survival. This report demonstrates that oATP limits the alloimmune response by regulating APC maturation and suppressing the generation of alloreactive Th1 immunity.

Figure 1

Inhibition of the purinergic pathway with oATP reduces corneal infiltration of leukocytes and suppresses APC maturation. (A) Schematic diagram depicting the time points of corneal transplantation, oATP administration and tissue harvesting. (B) Representative flow cytometric dot plots showing the gating strategy for, and frequencies of, CD45⁺ inflammatory cells in the corneas of oATP-treated mice, relative to saline-treated and naïve mice. (C) Representative flow cytometric dot plots (left) showing the gating strategy for selecting CD11b⁺MHCII⁺ cells in the cornea. Bar chart (right) summarizes the frequencies of CD11b⁺MHCII⁺ cells in the corneas of oATP-treated mice at 14 days after transplantation, relative to saline-treated mice. (D) Representative flow cytometric dot plots (left) and bar chart (right) depicting the frequencies of CD11b⁺MHCII^hi cells in the draining lymph nodes of oATP-treated mice, relative to saline-treated and naïve mice. n = 5–7/group. Representative data from three independent experiments are shown, and data are depicted as mean ± SEM. *p < 0.05; **p < 0.01.

Figure 2

Treatment with oATP suppresses CD4⁺ effector T cell generation and graft infiltration. (A) Representative flow cytometric dot plots (left) and cumulative bar chart (right) showing the frequencies of CD4⁺IFNγ⁺ cells (gated on CD4⁺ cells) in the draining lymph nodes of oATP-treated mice, compared to saline-treated and naïve mice. (B) Representative flow cytometric dot plots (left) showing the gating strategy for selecting CD4⁺ cells in the cornea. Bar chart (right) summarizes the frequencies of CD4⁺ cells in the cornea at 2 weeks post-transplantation, relative to saline-treated and naïve mice. n = 5–7/group. Representative data from three independent experiments are shown, and data are depicted as mean ± SEM. *p < 0.05; **p < 0.01.

Figure 3

oATP treatment does not promote the significant expansion of regulatory T cell frequencies following corneal transplantation. (A) Representative flow cytometric dot plots (left; with Foxp3 MFI) and cumulative bar chart (right) showing the frequencies of CD4⁺Foxp3⁺ cells in the draining lymphoid tissue of oATP-treated recipients, saline-treated recipients and naïve mice. (B) Flow cytometric dot plots demonstrating the gating strategy for enumerating CD4⁺Foxp3⁺ cells (gated on CD4⁺ cells) in the cornea (left), with protein expression (median fluorescence intensity; MFI) of Foxp3 detailed. Bar chart (right) summarizes the frequencies of corneal CD4⁺Foxp3⁺ cells at day 14 post-transplantation in oATP-treated and saline-treated recipients, relative to naïve mice. n = 5–7/group. Representative data from three independent experiments are shown, and data are depicted as mean ± SEM.

Figure 4

Treatment with oATP decreases graft opacity and promotes corneal allograft survival. Allogeneic corneal transplantation was performed and oATP (10 μg/g mouse body weight) or saline (control) were administered via intraperitoneal injection once daily for 14 days starting from the day of transplantation. Graft opacity was evaluated for eight weeks following transplantation, and graft survival was determined according to an established scoring system. (A) Schematic diagram depicting the time points of slit lamp examination of corneal graft opacity. (B) Graft opacity scores were significantly decreased in the oATP-treated group from week 4 until week 8 (p < 0.05). (C) Kaplan-Meier survival curve of oATP-treated graft recipients vs. saline-treated controls. Logrank test demonstrates significantly improved survival in the oATP-treated group relative to control (p = 0.032). Data shown is pooled from two independent experiments (n = 6–10/group). Data is represented as mean ± SEM.