In Vivo Eradication of Human Clonigenic Acute Lymphoblastic Leukemic Cells Expressing the Wilms Tumor Protein, WT-1, by HLA Restricted WT-1 Specific T-Cells in NOD/SCID Mice Monitored by Bioluminescent Imaging.
Authors: Doubrovina Ekaterina , Doubrovin Mikhail , Kanaeva Elena , O’Reilly Richard J. 
Published: October 2019

Abstract
WT-1 is expressed in 60–80% of acute leukemias, CML and high risk forms of MDS. Its expression has been hypothesized to be critical to the growth or survival of leukemic stem cells. Previously, alloreactive HLAA0201− T-cells recognizing a complex of WT-1 peptide and HLA A0201 were reported to prevent growth of leukemic HLA A0201+ CD34+ Ph+CML progenitor cells in NOD/ SCID mice (Transplantation, vol 75, No9, 2003). In this study, we have assessed the capacity of HLA-restricted, WT-1 peptide specific CTL (WT1-CTL) lacking alloreactivity to prevent the outgrowth of a human acute preB-lymphocytic leukemia (B-ALL)in NOD/SCID mice. This leukemia contained 65% of the blasts expressed WT-1 as determined by FACS analysis. For these studies the leukemic cells were transduced to express a luciferase reporter gene, permitting sequential monitoring of growth in vivo by bioluminescent imaging. WT-1 specific T-cells were generated from normal HLA A0201+ donor PBMC by in vitro sensitization with autologous dendritic cells loaded with the immunogenic HLA A0201 binding WT-1 peptide, RMFPNAPYL, and shown to be selectively cytotoxic against HLA A0201+WT-1+ leukemias and peptide loaded PHA blasts. T-cells from the same donor sensitized with autologous EBV BLCL and exhibiting HLA A0201 restricted EBV-specific cytotoxic activity served as controls. WT-1-CTL or EBV CTL were co-incubated in vitro with the WT-1+ HLA A0201+ BALL-LUC at a 4:1 effector target ratio for 7 hours at 37°C. Thereafter, separate groups of 5 NOD/SCID mice received intravenous infusions of cells from each of the co-cultures, at doses providing 12 × 106 WT1 CTL or EBVCTL and 3 × 106 BALL-LUC cells/mouse. A third group received 3×106 BALL-LUC alone. Leukemia growth was monitored at 2–3 day intervals from day 1–45 post infusion. In all 3 groups, BALL-LUC could be detected in the thorax by imaging at day 1. In mice treated with BALL-LUC alone or together with EBV-CTL, signal accumulation in the thorax increased steadily through 45 days of observation. By day 17, BALL-LUC were also detected throughout the head, abdomen and pelvis, and thereafter also increased until sacrifice at day 45. Autopsy confirmed presence of leukemic nodules in the lung and leukemic cells in blood, spleen and marrow as well as other organs. In contrast, in mice treated with WT1-CTL+ BALL LUC, signal intensity in lung decreased by day 4. In 4/5 of these mice, BALL-LUC could not be detected thereafter. In one mouse from this group, BALL-LUC were first detected in the head 31 days post infusion. At autopsy on day 45, this mouse had detectable BALL in the skull but in no other sites. WT-1 expression of residual leukemic cells is being analyzed. The other mice treated with WT-1 CTL had no detectable residual disease. These results suggest that clonogenic BALL cells express WT-1 and are susceptible to eradication in vivo by WT-1 peptide specific cytotoxic T-cells. The elimination of such clonogenic leukemic cells is sufficient to prevent subsequent development of leukemia.