*Purpose: Immunosuppression avoidance by induction of immune tolerance represents a primary goal in the field of transplantation. Yet, success of this concept relies on extensive pre-transplant recipient preconditioning which is not feasible in VCA. In this study we therefore explored a novel approach to induce cyclophosphamide-based delayed immune tolerance.

*Methods: Orthotopic hindlimb transplantation from Balb/c to C57BL6 was performed. Allografts were maintained with Rapamycin until POD29. The induction regimen comprised non-myeloablative total body irradiation, anti-thymocyte immune globulin (ATG) on POD30 and high-dose cyclophosphamide on POD34. Additional modification included donor bone marrow transplantation (dBMT) on POD31 with or without combination of post-induction ATG (POD40) and pre-induction fludarabine (POD 27 and 29). Graft survival, chimerism level, and frequency of CD8 memory T cells (Tmem, CD44^hiCD62L^low) were monitored. Multiple 14-color flow panel and subsequent t-SNE analysis were performed in long-term survivor (>200d).

*Results: The induction regimen applied on POD0 led to indefinite graft survival (>200d) in all VCA recipients (n=13) with stable chimerism levels. In contrast, with delayed induction regimen on POD30(n=9), graft survival was 70.1±22.9d with a chimerism level of 2.5±2.3%. With the addition of dBMT alone (n=5), graft survival and chimerism level increased to 83.6±15.9d and 3.4±1.4%, respectively. A significant elevation of post-induction CD8 Tmem was detected in those groups (39.8±25.9% and 51%±14.4%, compared with naïve control 4.5%±0.6%). With further optimization by adding pre-induction fludarabine and post-induction ATG, graft survival increased to 136±58d with a stable chimerism level of 27.2±28.8% (n=6). Long-term survivors (n=3) accepted donor matched secondary skin grafts (>50d) while acutely rejecting skin grafts from FVBN/J (<7d) confirming donor specific tolerance. Post-induction CD8 Tmem levels in those long-term survivors decreased to 7.9%±2.6%. In addition, t-SNE analysis revealed an elevation of exhaustion markers (PD-1 and Tim-3 on CD4 and CD8 T cells).

*Conclusions: Successful delayed tolerance induction can be achieved in a murine model of hindlimb transplantation with intensified conditioning regimens.

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