Though immune checkpoint blockade (ICB) against PD-1 has shown success in the treatment of lung cancer, not all patients respond. We have previously shown that adoptive transfer of double negative T (DNT) cells expanded from healthy donors can target leukemia but their role in treating established lung cancer is not clear. Here we explore the role of human DNT cells in targeting late-stage established lung cancer either alone or in combination with Nivolumab (anti-PD-1 antibody) and describe underlying mechanisms. DNT cells from resected lung cancer tissue of patients were analyzed by flow cytometry to determine their infiltration and PD-1 expression. Expansion capacity and anti-tumor function of lung cancer patient and healthy donor DNT cells were compared. Late-stage lung cancer xenograft models were developed to determine the anti-tumor effect of DNT cells alone or in combination with anti-PD-1 antibody, and the level of tumor-infiltrating DNT cells was quantified by histology and characterized by flow cytometry. Patient-derived tumor infiltrating lymphocytes contained a lower frequency of DNT cells with a higher expression of PD-1 relative to normal lung tissue. Ex vivo expanded patient- and healthy donor-derived DNT cells showed similar levels of cytotoxicity against lung cancer cells in vitro. Healthy donor-derived DNT cells significantly inhibited the growth of late-stage lung cancer xenografts, which was further augmented by anti-PD-1 through increased DNT cell tumor infiltration. This study supports the use of DNT cells for adoptive cellular therapy against lung cancer either alone or in combination with anti-PD-1.

While conventional chemotherapy is effective at eliminating the bulk of leukemic cells, chemotherapy resistance in acute myeloid leukemia (AML) is a prevalent problem that hinders conventional therapies and contributes to disease relapse, and ultimately patient death. We have recently shown that allogeneic double negative T cells (DNTs) are able to target the majority of primary AML blasts in vitro and in patient-derived xenograft models. However, some primary AML blast samples are resistant to DNT cell therapy. Given the differences in the modes of action of DNTs and chemotherapy, we hypothesize that DNT therapy can be used in combination with conventional chemotherapy to further improve their anti-leukemic effects and to target chemotherapy-resistant disease. Drug titration assays and flow-based cytotoxicity assays using ex vivo expanded allogeneic DNTs were performed on multiple AML cell lines to identify therapy-resistance. Primary AML samples were also tested to validate our in vitro findings. Further, a xenograft model was employed to demonstrate the feasibility of combining conventional chemotherapy and adoptive DNT therapy to target therapy-resistant AML. Lastly, blocking assays with neutralizing antibodies were employed to determine the mechanism by which chemotherapy increases the susceptibility of AML to DNT-mediated cytotoxicity. Here, we demonstrate that KG1a, a stem-like AML cell line that is resistant to DNTs and chemotherapy, and chemotherapy-resistant primary AML samples both became more susceptible to DNT-mediated cytotoxicity in vitro following pre-treatment with daunorubicin. Moreover, chemotherapy treatment followed by adoptive DNT cell therapy significantly decreased bone marrow engraftment of KG1a in a xenograft model. Mechanistically, daunorubicin increased the expression of NKG2D and DNAM-1 ligands on KG1a; blocking of these pathways attenuated DNT-mediated cytotoxicity. Our results demonstrate the feasibility and benefit of using DNTs as an immunotherapy after the administration of conventional chemotherapy.

With recent clinical breakthroughs, immunotherapy has become the fourth pillar of cancer treatment. Particularly, immune cell-based therapies have been envisioned as a promising treatment option with curative potential for leukemia patients. Hence, an increasing number of preclinical and clinical studies focus on various approaches of immune cell-based therapy for treatment of acute leukemia (AL). However, the use of different immune cell lineages and subsets against different types of leukemia and patient disease statuses challenge the interpretation of the clinical applicability and outcome of immune cell-based therapies. This review aims to provide an overview on recent approaches using various immune cell-based therapies against acute B-, T-, and myeloid leukemias. Further, the apparent limitations observed and potential approaches to overcome these limitations are discussed.

The double negative T cell (DNT) is a unique subset of T cells with potent anti-leukemic potential. Previously, DNT therapy has been shown to effectively target AML cells in patient-derived xenograft (PDX) models. Further, a recently completed phase I/IIa clinical study demonstrated the safety, feasibility, and potential efficacy in AML patients that relapsed after allogeneic hematopoietic stem cell transplantation. However, the persistence and durability of DNT-mediated anti-leukemic response is less well understood. In this study, we characterized the in vivo persistence of DNTs in PDX models. Further, we improved the efficacy and durability of DNT-mediated activity with phosphoinositide 3-kinase delta (PI3Kδ) inhibition. Mechanistically, DNTs treated with the PI3Kδ inhibitor, Idelalisib (Ide), exhibited early memory phenotype with superior viability and proliferative capacity but less cell exhaustion. Collectively, the findings from this study support the use of Ide-treated DNTs to improve its therapeutic outcome.