Figure S1. Characterization of ex vivo expanded healthy donor (HD) DNTs; Figure S2. Allogeneic DNTs induce dose- and time- dependent cytotoxicity against leukemic cells; Figure S3. Correlation between the susceptibility of AML patient blasts to DNT-mediated cytotoxicity and clinical features; Figure S4. Potency of anti-leukemic function mediated by allogeneic DNTs; Figure S5. Cytotoxic function of allogeneic DNTs on normal peripheral blood myeloid cells; Figure S6. NKp30, NKp44, and NKp46 are not involved in DNT-mediated killing of AML; Figure S7. Level of IFNγ production by DNTs corresponds with the level of cytotoxicity mediated by DNTs; Figure S8. DNAM-1 and NKG2D blocking abrogates IFNγ production by DNTs; Figure S9. IFNγ treatment does not affect the NKG2D and DNAM-1 ligand expression on normal PBMC; Figure S10. Cytotoxic activity of DNTs against leukemia cell lines derived from different types of leukemia and lymphoma; Figure S11. Effect of cryopreservation on the viability and anti-leukemic activity of ex vivo expanded DNTs.
ARTICLE ABSTRACT
Purpose: To explore the potential of ex vivo expanded healthy donor–derived allogeneic CD4 and CD8 double-negative cells (DNT) as a novel cellular immunotherapy for leukemia patients.Experimental Design: Clinical-grade DNTs from peripheral blood of healthy donors were expanded and their antileukemic activity and safety were examined using flow cytometry–based in vitro killing assays and xenograft models against AML patient blasts and healthy donor–derived hematopoietic cells. Mechanism of action was investigated using antibody-mediated blocking assays and recombinant protein treatment assays.Results: Expanded DNTs from healthy donors target a majority (36/46) of primary AML cells, including 9 chemotherapy-resistant patient samples in vitro, and significantly reduce the leukemia load in patient-derived xenograft models in a DNT donor–unrestricted manner. Importantly, allogeneic DNTs do not attack normal hematopoietic cells or affect hematopoietic stem/progenitor cell engraftment and differentiation, or cause xenogeneic GVHD in recipients. Mechanistically, DNTs express high levels of NKG2D and DNAM-1 that bind to cognate ligands preferentially expressed on AML cells. Upon recognition of AML cells, DNTs rapidly release IFNγ, which further increases NKG2D and DNAM-1 ligands’ expression on AML cells. IFNγ pretreatment enhances the susceptibility of AML cells to DNT-mediated cytotoxicity, including primary AML samples that are otherwise resistant to DNTs, and the effect of IFNγ treatment is abrogated by NKG2D and DNAM-1–blocking antibodies.Conclusions: This study supports healthy donor–derived allogeneic DNTs as a therapy to treat patients with chemotherapy-resistant AML and also reveals interrelated roles of NKG2D, DNAM-1, and IFNγ in selective targeting of AML by DNTs. Clin Cancer Res; 24(2); 370–82. ©2017 AACR.
