To explore the potential of expanded healthy donor-derived allogeneic CD4 and CD8 double-negative cells (DNT) as a novel cellular immunotherapy for leukemia patients. Clinical-grade DNTs from peripheral blood of healthy donors were expanded and their antileukemic activity and safety were examined using flow cytometry-based 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. Expanded DNTs from healthy donors target a majority (36/46) of primary AML cells, including 9 chemotherapy-resistant patient samples , 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. 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. .

Despite the demonstration of potent immunosuppressive function of T cell receptor (TCR)-αβ double-negative regulatory T cells (DN T ), scarce numbers and lack of effective expansion method limit their clinical applications. Here we describe an approach that allows for ∼3500-fold ex-vivo expansion of human DN T within 3 weeks with > 97% purity. Ex-vivo-expanded DN T suppress proliferation of polyclonally stimulated autologous T and B cells in vitro through direct cell-to-cell contact. In vivo, we demonstrate for the first time that infusion of human DN T delayed an onset of xenogeneic graft-versus-host disease (GVHD) significantly in a humanized mouse model. Furthermore, preincubation of ex-vivo-expanded DN T with a mechanistic target of rapamycin (mTOR) inhibitor rapamycin enhanced their immune regulatory function further. Taken together, this study demonstrates that human DN T can be expanded ex vivo to therapeutic numbers. The expanded DN T can suppress proliferation of T and B cells and attenuate GVHD, highlighting the potential clinical use of DN T to mitigate GVHD.

<p>Table S1. Clinical information of the 46 AML patients who provided AML cells for in vitro or in vivo assays.</p>

<p>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.</p>