Purpose: Sensitization in form of donor-specific antibodies (DSA) and subsequent antibody-mediated rejection constitute some of the greatest risk factors for allograft rejection and long-term graft failure. Here we investigated the effect of dual targeted desensitization using costimulatory blockade and bortezomib on DSA levels and graft survival after hindlimb transplantation in the mouse.
Methods: C57BL/6 mice were sensitized with Balb/c skin transplants. All animals rejected the graft within 2 weeks and sensitization was confirmed by T cell flow crossmatch. Group 1 received a desensitization protocol consisting of bortezomib (1 mg/kg) twice and CTLA4-Ig (500 µg) once per week for a total of 4 weeks before orthotopic hindlimb transplantation together with a tolerance induction protocol consisting of anti-Thy1.2 (2 mg/kg), total body irradiation (249.7 cGy), and cyclophosphamide (200 mg/kg). Animals in group 2 received an eight weeks course of desensitization and animals in group 3 served as non-desensitized controls. Hindlimb transplants were performed after additional eight weeks of observation whilst otherwise following the same protocol.
Results: Four weeks after skin transplantation, all animals developed a significantly increase in DSA levels. Despite the four-week course of combined desensitization, animals in group 1 did not display a significant reduction in DSA levels. Despite lower overall DSA levels, treated animals had a worse graft survival with 15.5 compared to 37 days in controls. All animals lost their grafts due to antibody mediated rejection. In group 2 desensitization was performed for eight weeks and thereby a significant reduction of DSA levels was achieved (desensitization: median 18.1 [week 1] and 4.2 [week 8] fold increase, p = 0.006; no-desensitization: 7 [week 1] and 6.7 [week 8] fold increase, p > 0.9). Desensitized animals (group 2) displayed superior survival with 28.5 days compared to 18.5 days in untreated controls (group 3). Two animals displayed prolonged survival with 41 and 53 days, and one animals achieved indefinite survival with > 200 days. In all three mid- and long-term surviving animals, mixed chimerism levels could be detected. In the two animals which eventually lost their graft, initial overall chimerism levels were below 10%. The animal with long-term survival displayed almost 20% initial mixed chimerism levels that dropped but were still detectable up to 200 days after transplant.
Conclusion: Eight-week dual targeted desensitization with Bortezomib and CTLA-4 Ig was able to significantly decrease circulating DSA levels without detectable rebound. Despite this decrease of DSA, antibody mediated rejection has been observed in transplanted animals. Prolonged graft survival and tolerance induction was only seen after additional 8 weeks of observation in a small proportion of animals.