Gating strategies for HSPC analysis were as described previously (15). Phosphoflow analysis. For phospho-Stat1 analysis, whole blood was stimulated for 15 minutes with 100 ng/mL murine IFN- at Lappaconite HBr 37C, then immediately fixed with 1 mL Lyse/Fix Buffer (BD) for 10 minutes at 37C. the promise of immunotherapy to improve the safety of HSCT for treating hematologic diseases. test (B, CD45-SAP), 1-way ANOVA (B, cKit-SAP), and repeated measures ANOVA (C) were used for statistical comparisons. * 0.05; *** 0.001; **** 0.0001. In a syngeneic HSCT model with GFP-tagged B6 HSCs transplanted to WT B6 recipients (B6-GFPB6), 75 g CD45-SAP was well tolerated and permitted stable, high-level donor engraftment comparable to that reported previously (ref. 14 and Figure 1C). Although 10 g cKit-SAP depleted HSCs as effectively as 75 g CD45-SAP, it was somewhat less effective at promoting engraftment. However, when 50 Lappaconite HBr g cKit-SAP was used, overall donor engraftment was equivalent to that seen with CD45-SAP. CBCs largely remained within reference limits during these experiments (Figure 1D), and donor chimerism in lymphoid organs mirrored that observed in peripheral blood (Supplemental Figure 2A). Finally, donor marrow from CD45-SAPC and cKit-SAPCconditioned primary transplant recipients was successfully transplanted into secondary recipients, confirming engraftment of functional HSCs (Figure 1E and Supplemental Figure 2B). Taken together, these studies confirm the efficacy Lappaconite HBr of CD45-SAP Lappaconite HBr and cKit-SAP as conditioning for HSCT in the absence of immunologic barriers. CD45-SAP plus in vivo T cell depletion enables engraftment in miHA- and MHC-mismatched allo-HSCT. To investigate the efficacy of ADCs for allo-HSCT conditioning, we used 2 transplant models (Figure 2A): an miHA-mismatched model (BALB/c-Ly5.1DBA/2) and a haploidentical F1-to-parent (CB6F1B6) model mismatched for H-2d in the host-versus-graft direction. We focused on CD45-SAP for conditioning in these studies to leverage its lymphodepleting activity to overcome graft rejection. However, CD45-SAP alone failed to allow alloengraftment, likely due to its incomplete ablation of host T cells. This suggested further immunosuppression was needed to achieve alloengraftment. Open in a separate window Figure 2 TCD in CD45-SAPCconditioned mice permits engraftment in miHA- and MHC-mismatched allo-HSCT.(A) Schematic for miHA- and MHC-mismatched allo-HSCT models utilizing CD45-SAP plus TCD. (B and C) Peripheral blood donor chimerism for individual mice in the miHA-mismatched (B) and MHC-mismatched allo-HSCT models (C) pooled from 2 to 3 3 experiments. (D and E) Serial CBCs for miHA- (D) and MHC-mismatched (E) models. X indicates mouse euthanized for severe head tilt unrelated to the experimental treatment. Data points and error bars represent mean SEM. Repeated measures ANOVA or mixed effects model (for group with missing data due to mouse loss) was used for statistical comparisons of overall donor chimerism. ** 0.01; *** 0.001; **** 0.0001. We therefore treated CD45-SAPCconditioned animals with antibodies depleting CD4+ and/or CD8+ T cells throughout the peritransplant period (Figure 2A) and followed donor chimerism longitudinally (Supplemental Figure 3, ACC, and ref. 22). In the miHA model, in vivo CD8+ T cell depletion (TCD), but not CD4+ TCD, was sufficient to produce engraftment in most (7/9) recipients. CD4+ and CD8+ pan-TCD of CD45-SAPCconditioned mice resulted in multilineage engraftment in all treated mice, albeit with significant variability in donor chimerism (Figure 2B). Gradual loss of donor chimerism was noted in 1 of 10 pan-TCD mice, with the shorter-lived myeloid cells showing Rabbit polyclonal to PDK4 the most rapid decline, a pattern suggesting failure of long-term HSC persistence. Low-level donor T cell engraftment was observed in pan-TCD mice conditioned with an inactive ADC. Finally, serial transplantation studies using marrow from CD45-SAPCconditioned, pan-TCD recipients confirmed engraftment of functional donor-derived HSCs (Supplemental Figure 3D). In the F1-to-parent model, pan-TCD was required for engraftment (Figure 2C). High-level donor B cell and myeloid lineage chimerism and lower T cell chimerism were routinely observed in this system. Although all pan-TCD animals showed donor engraftment initially, 5 of 9 mice showed evidence of graft loss, with one showing a sudden, multilineage loss of donor-derived cells indicative of rejection. Serial CBCs in both models remained largely stable within reference limits (Figure 2, D and E). Although the miHA model has potential for bidirectional alloreactivity, we observed neither overt graft rejection nor GvHD, suggesting achievement of stable mixed chimerism. To directly.
