*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 flow crossmatch. Group 1 received a desensitization protocol consisting of bortezomib (0.75mg/kg i.p.) twice and CTLA4-Ig (500µg i.p.) once per week for a total of 4 weeks before orthotopic hindlimb transplantation (day 0) together with a tolerance induction protocol consisting of anti-Thy1.2 (2mg/kg i.p.) and total body irradiation (249.7cGy) on day -1 as well as cyclophosphamide (200mg/kg i.p.) on day +3. Animals in group 2 received the desensitization protocol after hindlimb transplantation and group 3 did not receive desensitization treatment whilst otherwise following the same protocol.

*Results: Four weeks after skin transplantation, all animals developed a significantly increase in DSA levels (15.5 ±7.7-fold increase; p<0.0001). Despite the four week course of combined desensitization, animals in group 1 did not display a significant reduction in DSA levels. After hindlimb transplantation and application of the tolerance protocol, DSA levels significantly dropped to baseline levels (2.2±1.8-fold increase; p<0.0001) in all groups. In the naïve setting this tolerance protocol allows for indefinite graft survival, in animals of group 3 however, median graft survival was limited to 37 days. Combined desensitization did not improve graft survival regardless if applied before (group 1) or after (group2) hind limb transplantation, contrarily it even shortened graft survival (median 15.5 and 19 days, respectively; p=0.26). In addition, allograft rejection seemed to correlate with a rebound in DSA levels in group 1 (p=0.0037) but not in groups 2 and 3.

*Conclusions: Although effective in solid organ transplantation a combined desensitization protocol with CTLA4-Ig and bortezomib failed to show reduced DSA levels and improved allograft survival in a stringent VCA model.

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