Background
Vascularized composite allotransplantation (VCA) has become a valid therapeutic option after devastating tissue loss, such as an extremity or face. The costimulatory blocking fusion protein CTLA4-Ig has shown considerable efficacy in preventing rejection of solid organ transplants in preclinical models and clinical trials, however, its effects in VCA are still poorly understood. The current study investigated the immunosuppressive potential of CTLA4-Ig in a novel murine model of hind limb transplantation.
Methods
Fully MHC-mismatched allogeneic, orthotopic hind limb transplants were performed from Balb/c to C57BL/6 mice. Recipient animals in the experimental groups received (1) no treatment; (2) CTLA4-Ig on postoperative days (POD) 0, 2, 4, and 6; (3) CTLA4-Ig plus anti-CD154 mAb on POD 0, 2, 4, and 6; or (4) CTLA4-Ig on POD 0, 2, 4, 6 plus donor splenocytes transfusion (DST)on POD 0. Mixed chimerism and clonal deletion of alloreactive T cells were checked by analyzing PBMC profiles by flow cytometry.
Results
The CTLA4-Ig treated group showed increased graft survival compared to non-treated controls (mean survival time [MST] 15 d vs. 8 days; p<0 .01). Adding DST to CTLA4-Ig increased graft survival compared with the non-treatment group (MST 16.5 d vs. 8 days; p<0 .01) but there was no significant difference compared to the CTLA4-Ig treatment group. Interestingly, adding anti-CD154 mAb with CTLA4-Ig significantly increased graft survival (MST 82 days; p<0 .01). Moreover, increased mixed chimerism was detected in the CTLA4-Ig plus anti-CD154mAb treated group. On POD 13, the percentage of donor derived CD11b+ cells was 10.44% in the CTLA4-Ig plus anti-CD154 mAb group vs. 5.2% in the CTLA4-Ig alone group and this higher level was maintained over POD60. Furthermore, decreased proportions of circulating νβ11+ and νβ5+CD4+ T cells (sign of thymic negative selection) were detected in recipients treated with CTLA4-Ig plus anti-CD154 mAb while control groups failed to delete donor-reactive νβ11+ and νβ5+ CD4+ T cells.
Conclusion
This study shows that costimulatory blockade prevents VCA rejection and significantly prolongs graft survival. Also, these data underscore the potential to design interventions to exploit the tolerogenic potential of mixed chimerism in VCA.