Inhibition of human lymphocyte proliferation by small inhibitory molecules after stimulation with porcine cells in vitro

B.B.F. Alavi¹, D. Mihalicz¹, P. Buchwald², R. Rajotte¹, G. Rayat^1
1 University of Alberta, Surgery, Edmonton, Canada; ² University of Miami, Miami, USA

Objective: Small inhibitory molecules have long been an attractive area of research in transplant immunology as they are able to block protein to protein interactions by binding to small but high affinity areas called hot spots. Recently, a number of small inhibitory molecules were identified to block the CD154/CD40 interaction in vitro. These molecules include Suramin and Direct Red 80 (DR80). Suramin is a symmetric polysulfonated naphthylamine-benzamide urea derivative that is approved for the treatment of trypanosomiasis and oncocerciasis. DR80 is an azo dye that is used as colorant for textile, paper and leather. We determined the effect of Suramin and DR80 on the proliferation of human lymphocytes in vitro.

Methods: Human PBMCs (5x10⁵) were stimulated with either Concanavalin A (10 µg/ml, n=6) or gamma irradiated porcine spleen cells (5x10⁵, n=6) with or without the addition of various concentrations of Suramin or DR80. After 2 days of Concanavalin A stimulation or 4 days of stimulation with porcine cells, the human PBMCs were incubated with [³H]-thymidine for 18 hours and incorporation of [³H]-thymidine was measured using a beta counter.

Results: Human PBMCs proliferated robustly after stimulation with Concanavalin A (93,881±10,490 counts/minute) or gamma irradiated porcine spleen cells (51,282±3,297 counts/minute). Suramin and DR80 inhibited the proliferation of human PBMCs in a dose-dependent manner. At 125 µg/ml of Suramin and DR80, the proliferation of human PBMCs after stimulation with Concanavalin A was reduced to 50% and 56%, respectively. While at 31 µg/ml of Suramin and DR80, the proliferation of human PBMCs after stimulation with porcine spleen cells was reduced to 24% and 53%, respectively.

Conclusion: These results indicate that Suramin and DR80 small inhibitory molecules could inhibit the activation of human lymphocytes in vitro and may have the potential to prevent rejection of neonatal porcine islet xenografts mediated by human lymphocytes in vivo.