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Delayed Tolerance Induction Protocol for Vascularized Composite Allografts in Non-Human Primates: The Immunomodulatory Effect of Donor Bone Marrow Transplantation Does Not Prevent the Development of Chronic Rejection in the Absence of Durable Mixed Chimerism
Alexandre G. Lellouch, MD, Zhi Yang Ng, MD, Ivy A. Rosales, MD, Robert B. Colvin, MD, Amon-Ra Gama, BS, Ilse M. Schol, BS, Luke Geoghegan, BS, Josef M. Kurtz, PhD, Curtis L. Cetrulo, Jr., MD.
Massachusetts General Hospital, Boston, MA, USA.

PURPOSE: Our laboratory is currently developing a delayed tolerance induction protocol for vascularized composite allografts (VCAs) in a clinically relevant non-human primate (NHP) model using donor bone marrow transplantation (DBMT) after VCA to induce durable mixed chimerism and tolerance. DBMT has a demonstrated immunomodulatory effect in our previous experimental work and in clinical VCA, enabling some patients to be maintained on low-dose tacrolimus monotherapy. With longer-term follow-up of clinical VCA, recent reports of chronic rejection have emerged. We sought to investigate in further detail the immunomodulatory effect of DBMT on acute and chronic rejection of VCAs in NHPs.
METHODS: Following VCA transplantation and maintenance on standard triple immunosuppression (IS) for 2 months, donor bone marrow cells that were previously harvested from the vertebrae, minimally processed and cryopreserved were thawed and infused into MHC-mismatched recipient NHPs (n=6) conditioned with irradiation (total body and thymic), T cell depletion, co-stimulatory blockade and anti-inflammation (with anti-IL-6 receptor monoclonal antibody). A bridging course of calcineurin inhibition (CNI) was given for 4 weeks before IS withdrawal and assessment of the VCA for tolerance. Observed rejection episodes of the VCA were biopsied and treated with CNI and a tapering course of steroids before IS withdrawal after 2 weeks. Systemic immune function was assessed by CFSE-based mixed lymphocyte reaction (MLR) proliferation assays and allo-sensitization was evaluated by the detection of serum allo-antibody formation. Evidence for mixed chimerism was assayed in peripheral blood.
RESULTS: Durable mixed chimerism (only detected transiently at 6 weeks post-DBMT) did not develop but recipients managed to come off all IS for 4-5 weeks before acute rejection (Banff II) developed between POD115-POD126. Rejection could be treated and reversed both clinically and histologically. However, following subsequent re-withdrawal of IS, Banff II rejection developed again within 2 weeks (on POD172), could not be reversed despite treatment (Banff II on POD195) and ultimately culminated in necrosis of the VCA (on POD224). Final histology showed severe acute cell-mediated and antibody-mediated rejection with C4d deposition in subcutaneous arteries and arterioles as well as chronic allograft vasculopathy (Figure 1). Despite these findings, corresponding serial MLR assays demonstrate unresponsiveness after DBMT and no allo-antibody formation was detected.
CONCLUSION: While DBMT demonstrated systemic immunomodulation based on MLR, in the absence of durable mixed chimerism, this effect was not enough to prevent acute projection episodes and the development of chronic rejection in this cohort of animals. These rejection episodes may be related to the subsequent development of chronic rejection. The presence of vascular lesions (based on C4d deposition) throughout all time points following DBMT may predate clinical progression to eventual chronic allograft vasculopathy and rejection, and may represent a potential prognostic factor in clinical VCA.


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