Therapeutically Infused Redirected T Cells Targeting WT1 Successfully Inhibited Leukemia Stem Cells in Vivo

Recent findings regarding leukemia stem cell (LSC) have emphasized the importance of suppression of LSC for the achievement of durable remission, the first requisite to establish cure of leukemia. For this subject, successful graft-vs.-leukemia (GvL) effect in allogeneic hematopoietic stem cell transplantation (allo-HSCT) against human leukemias has strongly illustrated the importance of anti-leukemia immunity. Additionally, WT1, one of well-known leukemia-associated antigens, has been obviously demonstrated to be expressed by LSCs in bone marrow niche (Saito Y et al, Sci Transl Med.2010). On the other hand, cell-cycle quiescence of LSCs in bone marrow niche is importantly implicated in their chemoresistance. Taking all above, in this study, we set out to answer questions whether therapeutically adopted T-cell immunity towards WT1 enabled to suppress LSC in vivo, and whether cell-cycle status of leukemia cells affected the sensitivity to cyotocidal activity mediated by WT1-specific cytotoxic T cells (CTLs).

Approval for this study was obtained from the Institutional Review Board of Ehime University Hospital. Written informed consent was given by all patients, healthy volunteers in accordance with the Declaration of Helsinki. Peripheral CD8⁺ T cells obtained from AML or ALL patients in complete remission (CR) or healthy individuals were gene-modified to express HLA-A*2402-restricted and WT1_235–243 nonamer -specific T-cell receptor (TCR) using our unique TCR-a/b gene expression vector carrying silencers for endogenous TCRs (WT1-siTCR vector) were generated as effector cells. Bone marrow CD34⁺ leukemia (L-BMCD34⁺) cells isolated using immunomagnetic beads from HLA-A*2402 positive or negative patients with AML or ALL were serially transplanted into NOD/scid/γc^null (NOG) mice as previously reported (Ochi T et al. Blood, 2011). 12 weeks later, engrafted leukemia cells in murine bone marrow were examined using a flowcytometry. In some experiments, after engrafted, first transplanted mice were treated with intraperitoneal administration of high dose (150mg/kg) of cytosine arabinoside (Ara-C). A week later, those mice received intravenous administration of gene-modified autologous CD8⁺ T cells to express WT1-specific TCR or non-gene-modified (NGM) ones in combination with intraperioneal administration of 500u of IL-2 every 2 days. A week after therapeutic T-cell infusion, bone marrow cells were harvested, and transplantation into second mice. 12 weeks later, engrafted human leukemia cells in murine bone marrow were assessed. Next, using a time-lapse photo assay and fluorescent ubiqutination-based cell-cycle indicator (Fucci)-labeled K562-A24 cells which are known to produce high amounts of WT1 mRNA and are positive for HLA-A*2402, we directly assessed the impact of cell-cycle status of leukemia cells on their sensitivity to redirected CTL towards WT1 in vitro.

Using isolated L-BMCD34⁺ cells, LSCs were detectable as leukemia initiating cell in serially transplanted NOG mice. High dose of Ara-C treatment alone was unable to eradicate LSCs. An experiment using samples from a patient with HLA-A*2402⁺ ALL revealed that intravenously infused gene-modified autologous peripheral CD8⁺ T cells in CR successfully reduced leukemia burden in bone marrow which were refractory to high dose of Ara-C. In serial transplantation experiments using samples from AML patients, therapeutic infusion of redirected CD8⁺ T cells to express WT1-specific TCR, but not NGM ones in nadir state successfully eradicated LSCs out of murine bone marrow. In vitro time-lapse photo assay directly illustrated that retargeted CD8⁺T cells towards WT1 killed fucci-labeled K562-A24 cells irrelevantly to cell-cycle status of target leukemia cells.

In this study, when leukemia mass burden was reduced, therapeutically infused gene-modified CD8⁺ T cells targeting WT1 successfully enabled to inhibit LSCs in vivo. Furthermore cell-cycle status of leukemia cells which is importantly implicated in their chemoresistance in bone marrow niche, did not affect WT1-specific cytocidal activity mediated by genetically redirected CTLs at all. Although it is preliminary, our observation encourages us to actively introduce redirected T cell-based antileukemia adoptive immunotherapy, aiming at a cure of leukemias.

No relevant conflicts of interest to declare.