Hematopoietic chimerism establishes donor-specific tolerance to solid organ allografts. Targeting recipient immune cells (T, NK cells and macrophages) can effectively reduce the dose of total body irradiation (TBI) required to establish mixed chimerism in a mouse bone marrow transplantation (BMT) model. In this study, we hypothesized that immune signalling pathways may be novel targets for promoting chimerism. B6 (H2^b) mice were transplanted with 15×10⁶ bone marrow cells from MHC-disparate BALB/c (H2^d) donors after nonmyeloablative conditioning with 300cGy TBI (day 0) and cyclophosphamide (CyP, day +2) in combination with different pathway inhibitors (day 0 to +9). With 300cGy/CyP, 100% engraftment occurred in recipients treated with Janus kinase (JAK)1/2 inhibitor [ruxolitinib (Ruxo)], but not with inhibitors of MEK, BTK, PI3K/mTOR, or S1P/S1P1. JAK is a family of intracellular, nonreceptor tyrosine kinases that transduce cytokine-mediated signals via the JAK-STAT pathway. They play a fundamental role in regulation of cell growth and differentiation. Activation of the JAK-STAT pathway occurs at the cell membrane level and the transcriptional response occurs in the nucleus. With Ruxo, 87.5% and 60% of recipients engrafted with 200 and 100 cGy TBI plus CyP, respectively. Donor chimerism was 38.8±14.0%, 18.2±10.4%, and 1.9±1.8% with 300, 200, 100cGy TBI plus Ruxo/CyP at 1 month, respectively. When recipient T cells were depleted with anti-αβTCR in addition to Ruxo/CyP, 100% engraftment was achieved with 100cGy TBI with 26.1±16.9% donor chimerism at 1 month post BMT. This was significantly higher (P<0.0001) than Ruxo/100cGy/CyP conditioning. Engrafted animals exhibited donor-derived multilineage cell production, including donor T, B, NK cells, macrophages, and granulocytes. Chimerism remained stable up to 6 months. Moreover, specific Vβ5.[frac12] and Vβ11 clonal deletion was detected in host T cells in all chimeric mice, indicating central tolerance to donor alloantigens. This JAK1/2 inhibitor totally inhibited B6 and BALB/c alloimmune responses in in vitro MLR and partially inhibited humoral responses in vivo. Targeting JAK1/2 with Ruxo can significantly decrease the TBI dose necessary to achieve mixed chimerism. Ruxo and T cell depletion have a synergistic effect to enhance alloengraftment. These findings may have a significant impact on the use of immune-based nonmyeloablative conditioning strategies for tolerance induction.

CITATION INFORMATION: Xu H., Merchak A., Kahn L., Chhabra A., Wen Y., Huang Y., Ildstad S. A Novel Conditioning Approach to Establish Chimerism Using Manipulation of JAK-STAT Pathways Am J Transplant. 2017;17 (suppl 3).

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