Non-Myeloablative TLI/ATG + Alkylator Conditioning Augments Bidirectional Immune Tolerance Via Regulatory MDSC in a Robust Murine Model of MHC-Mismatched BMT for Beta-Thalassemia

Allogeneic hematopoietic cell transplantation (allo-HCT) for hemoglobinopathies is restricted by limited matched donors and by graft rejection and graft-versus-host disease (GVHD) after alternative donor HCT.  Based upon our clinical data in aplastic anemia (Pillai et al, ASBMT 2011), we pre-clinically assessed non-myeloablative Total Lymphoid Irradiation (TLI) + Anti-Thymocyte Serum (ATS) +/- cyclophosphamide (CTX) vs. total body irradiation (TBI)/ATS +/- CTX in an MHC-mismatched BALB/c (H-2^d) into HW-80 (H-2^b) β-thalassemia (HW-80 β-thal^+/-) model. Methods: Wild-type (WT) and β-thal^+/- HW-80 mice received TLI (240 cGy x 10) + ATS (TLI/ATS, n = 10),  +/- CTX (200 mg/kg) (TLI/ATS/CTX, n = 15), ATS/CTX alone (n = 10), or ATS/CTX with 1200 cGy, 600 cGy, or 300 cGy TBI (1200 T/A/C, 600 T/A/C, 300 T/A/C; n = 10 each), and 50 x 10⁶ bone marrow + 60 x 10⁶ splenocytes (BMT) from WT BALB/c donors. Survival, weekly weight, day 28 and day 100 tri-lineage engraftment and CBC, and histopathologic GVHD were assessed.  Results: WT HW-80 recipients of either TLI/ATS or TLI/ATS/CTX engrafted through day 100; HW-80 β-thal^+/- mice had > 40% graft rejection after TLI/ATS + BMT, but 100% graft retention after TLI/ATS/CTX + BMT, indicating a disease-associated engraftment barrier overcome by addition of CTX to TLI/ATS. All engrafting β-thal^+/- mice showed stable mixed hematopoietic chimerism and day 100 hematocrit (Hct) correction (P = 0.01 vs untreated β-thal^+/-). However, TLI/ATS/CTX recipients had no GVHD, whereas engrafting TLI/ATS mice developed lethal GVHD by day 100 [cumulative scores: TLI/ATS/CTX: 0.3 +/- 0.2; TLI/ATS: 6.8 +/- 0.9; P < 0.01]. 100% of 1200 T/A/C, 60% of 600 T/A/C, and 40% of 300 T/A/C mice engrafted. In all T/A/C groups, > 80% engrafters had lethal GVHD, whereas > 70% of 100-day survivors showed non-engraftment. We delineated that recipient regulatory MDSC induce tolerance via MHC-independent in vivo expansion of donor Foxp3+ nTreg after TLI/ATS + BMT (van der Merwe et al, J Immunol 2013). We found significant (P < 0.001) increase in recipient CD11b⁺Gr-1(G)^hi MDSC in TLI/ATS/CTX- versus TLI/ATS-conditioned β-thal^+/- mice, with increased number (P < 0.01) and proliferation (P < 0.05) of donor Foxp3+ nTreg and decreased CD8+ T cell number and proliferation (P < 0.01) in spleens at day 6. In adoptive transfers, TLI/ATS/CTX-derived recipient CD11b⁺Gr-1G^hi MDSC regulated otherwise lethal GVHD through day 100 post-BMT in 1200 cGy TBI-conditioned WT B6 recipients of WT BALB/c BMT. Our data indicate that TLI/ATS/CTX facilitates robust recipient <--> donor tolerance, provide pre-clinical support for application of TLI/ATS/CTX prior to (potentially MHC-mismatched/haploidentical) allo-HCT for hemoglobinopathies, and give the first direct evidence for an innate immune mechanism of alkylator-associated transplantation tolerance, which we propose to validate in the context of a clinical trial.