The potential of novel Direct Red 80 organic dye to prevent rejection of newborn pig islets in mice

D. Mihalicz¹, K. Bayrack¹, F. Alavi¹, C. Le¹, P. Buchwald², D. Williams¹, RV. Rajotte¹, GR. Rayat^1
1 University of Alberta, Surgery, Edmonton, Canada; ² University of Miami, Miami, FL, USA

Objective: Organic dyes such as Direct Red 80 (DR80) have been shown to inhibit the CD40/CD154interaction in vitro. In this study, we aimed to confirm these findings and assess their ability to prevent rejection of xenotransplanted newborn pig islets (NPI) in mice.

Methods: Mitogen stimulation assays were created using C57BL/6 mouse splenocytes, Concavalin A,³HThymidine, and various concentrations of DR80. Both viability and radiation counts were measured. Islets from 3-day-old newborn pigs were isolated using our standard protocol and transplanted under the kidney capsule of chemically induced diabetic C57BL/6 mice. They were treated with either DR80 or anti-LFA-1 monoclonal antibody (mAb) alone, or in combination. Blood glucose levels were measured twice weekly for 150 days. In normoglycemic mice, a survival nephrectomy was performed to demonstrate return to the hyperglycemic state, and the graft was assessed with immunohistochemistry. The mouse splenocytes were then analyzed with flow cytometry for the presence of numerous cell surface markers to characterize the immune cell phenotype.

Results: Mitogen and antigen-specific stimulation assays confirmed the inhibition of T-cell activation, with significant reduction at doses of 31.25μg/mL and greater (p<0.05), with no significant cytotoxicity (p<0.05). None of the untreated mice (n=3), or the mice treated with either DR80 alone (n=10) or anti-LFA-1 mAb alone (n=4) achieved normoglycemia, while 6 of the 10 mice treated with DR80 and anti-LFA-1 mAb achieved normoglycemia. One of the 6 mice rejected the NPI xenograft at 185 days post-transplantation. Flow cytometric analysis reveals a trend towards an increase in the proportion of T-regulatory cells in the normoglycemic mice, suggesting a possible mechanism for protection.

Conclusions: DR80 inhibits T cell activation in vitro with no significant cytotoxicity. It is a promising therapeutic agent to prevent the rejection of porcine islets in mice in combination with anti-LFA-1mAb.