*Purpose: A recent murine study has shown that successful mixed chimerism is achievable without myelosuppressive treatments by Bcl-2 inhibition. However, the mechanism by which selective Bcl-2 inhibition promotes chimerism was unclear. Since Mcl-1, not Bcl-2, has been reported as a major survival factor for hematopoietic stem cells (HSC), Bcl-2 inhibition is not supposed to deplete HSCs to open a space in the BM niches for allogeneic HSC engraftment. We therefore, tested Venetoclax, an FDA approved a highly selective Bcl-2 inhibitor, to evaluate whether selective Bcl-2 inhibition alone can deplete HSCs in BM niches. Venetoclax was then included in our conditioning regimen for combined BM and kidney transplantation (KTx) with only half dose (1.5Gy) of total body irradiation (TBI) previously required (3 Gy) to induce mixed chimerism.

*Methods: Cynomolgus macaques were treated with 10 mg/kg of Venetoclax for 5 days. BM cells were serially aspirated to evaluate HSC (CD34+CD90+CD45RA-) depletion. Expression of apoptotic cascade proteins (BAX, Cytochrome C, Caspase-3, -7, and -9) on BM cells were also evaluated by flow cytometry. Finally, peri-transplant administration of Venetoclax (10mg/kg for 11 days) was added to our standard conditioning regimen (TBI, thymic irradiation, anti-CD154 mAb, ATG and a one month course of CyA) for combined BM transplantation (BMT) and KTx but with reduced (1.5Gy) TBI.

*Results: After Venetoclax treatment, expression of Caspase-7 and -9 in HSC was significantly increased (167 ± 25% and 135 ± 6%; p<0.01 and p<0.001; Fig. 1a). As a result, HSCs were significantly depleted to 36 ± 15% of baseline levels (Fig.1b). In peripheral lymphocytes, expression of those apoptotic proteins in T cells, B cells, and NK cells also remarkably increased. With 1.5Gy TBI without Venetoclax in our conditioning regimen for BMT, no chimerism was induced in all four recipients and three rejected kidney (58, 100 and 167days). In contrast, all five BMT recipients with Venetoclax consistently developed excellent mixed chimerism, which was significantly superior to those induced by 3 Gy TBI but without Venetoclax (Fig. 2). Three combined KTx recipients also achieved renal allograft tolerance (>313, >946 and >1274 days).

*Conclusions: Bcl-2 appeared to be a critical survival factor for HSCs and its inhibition resulted in promotion of chimerism induction with significantly lower dose of myelosuppressive treatment.

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