Transient T-cell depletion performed in the Townes model of SCD. (A) Donor leukocyte chimerism. Transient TCD achieved donor cell chimerism that was higher after transplantation with FL LDMCs than with TCD BM. (B) High-performance liquid chromatography. Shown is the percentage of hemoglobin (Hb) that is derived from Balb/c donor erythrocytes as opposed to the homozygous Townes recipient. A high level %Balb/c (donor) Hb is indicative of a high percentage of donor-derived erythrocytes in the PB. This was observed in some animals injected with TCD BM + anti-CD3 mAb and in all animals injected with FL LDMCs + anti-CD3 mAb. (C) Donor engraftment among BM LSK cells. BM LSK is a population enriched for HSCs. (D) Hb concentration. (E) Reticulocyte percentage. Anemia with an associated elevation in percentage reticulocyte is characteristic of untreated homozygous Townes mice. Both were improved after the injection of TCD BM + anti-CD3 mAb and fully corrected after the injection of FL LDMCs + anti-CD3 mAb. (F) Images of reticulocytes after ex vivo hypoxia. (G) Quantification of sickling after ex vivo hypoxia. Sickling was significantly reduced in neonates injected with TCD BM + anti-CD3 mAb and FL LDMCs + anti-CD3 mAb. (H) Quantification of sickling after in vivo hypoxia-reoxygenation. In vivo sickling was similarly reduced in neonates undergoing immune modulation at the time of transplantation. (I) Serum chemistry after in vivo hypoxia-reoxygenation. Uninjected homozygous mice demonstrate profound hyperbilirubinemia after in vivo hypoxia-reoxygenation due to erythrolysis and impaired liver function. This was significantly improved in the successfully treated mice. (J) Quantification of hepatosplenomegaly (HSM). Injection of FL LDMCs + anti-CD3 mAb prevented HSM in all recipients, whereas injection of TCD BM + anti-CD3 mAb prevented HSM in some but not all recipients. (K) Gross histology of the spleen and liver at terminal analysis. Healthy/corrected mice showed normal spleen and liver sizes with a smooth capsule. (L) Microscopic histology at terminal analysis. Shown are H&E preparations imaged at 10× original magnification. Diseased spleens showed significant expansion of the red pulp with pooling of sinusoidal erythrocytes and total loss of lymphoid follicular structure (green arrows). Healthy/corrected spleens showed normal splenic red and white pulps without pooling of sickle erythrocytes or infarcts. Diseased livers show focal areas of necrosis (blue arrows) and congestion of the intrahepatic vasculature with aggregates of sickled red blood cells (RBCs). Erythroid progenitors are evident in the sinusoid, consistent with extramedullary hematopoiesis (red arrows). There was also hemosiderin deposition resulting from Kupffer cell phagocytosis. In contrast, focal areas of necrosis and aggregation of sickled erythrocytes were not observed in healthy/corrected livers, and extramedullary hematopoiesis and hemosiderin deposition were absent. Diseased kidneys show engorgement and occlusion of blood vessels with sickled erythrocytes (orange arrows), resulting in vascular, tubular, and glomerular changes. Healthy/corrected kidneys appeared normal and free of disruptive vascular RBC pooling and hemosiderin deposition. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.