Electroretinography in congenital nystagmus patients with a normal fundus examination.

PURPOSE: To identify the ophthalmic causes of congenital nystagmus with normal eye examination by electroretinography (ERG). STUDY DESIGN: Retrospective observational study. METHODS: We reviewed the medical records of patients younger than 6 months of age who presented between June 2008 and November 2011 with nystagmus and no other neurological signs following an otherwise normal eye examination. A complete ophthalmic examination and ERG (Nicolet Bravo system; Nicolet Biomedial & RETIscan; Roland Instruments), fundus photography, and Ishihara color test were performed to identify any ophthalmic causes of congenital nystagmus. RESULTS: Thirty-three patients met the criteria. Rod dysfunction was diagnosed in 4 patients (12.1%), cone dysfunction in 2 patients (6.1%), and cone-rod dysfunction in 1 patient (3.0%). The results of ERG were negative in 2 patients (6.1%). Idiopathic infantile nystagmus was diagnosed in the remaining 24 patients (72.7%) based on their normal ERG examination. CONCLUSIONS: In Korean congenital nystagmus patients with a normal fundus examination, achromatopsia and Leber's congenital amaurosis are uncommon causes. ERG is needed to make a definite diagnosis and provide prognostic information in congenital idiopathic nystagmus patients with a normal fundus examination.

Double-negative T cells utilize a TNFα-JAK1-ICAM-1 cytotoxic axis against acute myeloid leukemia.

ABSTRACT: Allogeneic double-negative T cells (DNTs) are a rare T-cell subset that effectively target acute myeloid leukemia (AML) without inducing graft-versus-host disease in an allogeneic setting. A phase 1 clinical trial demonstrated the feasibility, safety, and potential efficacy of allogeneic DNT therapy among patients with relapsed AML. However, the molecular mechanisms of DNT-mediated cytotoxicity against AML remain elusive. Thus, we used a flow cytometry-based high throughput screening to compare the surface molecule expression profile on DNTs during their interaction with DNT-susceptible or -resistant AML cells and identified a tumor necrosis factor α (TNFα)-dependent cytotoxic pathway in DNT-AML interaction. TNFα secreted by DNTs, upon encountering susceptible AML targets, sensitized AML cells to DNT-mediated killing, including those otherwise resistant to DNTs. Mechanistically, TNFα upregulated ICAM-1 on AML cells through a noncanonical JAK1-dependent pathway. DNTs then engaged with AML cells more effectively through an ICAM-1 receptor, lymphocyte function-associated antigen 1, leading to enhanced killing. These results reveal a TNFα-JAK1-ICAM-1 axis in DNT-mediated cytotoxicity against AML to improve therapeutic efficacy.

Evaluation of the Randox and Fuji Dri-Chem vcCRP-P assays of canine C-reactive protein.

In veterinary medicine, measurement of canine C-reactive protein (cCRP) is used widely to detect inflammatory diseases. We evaluated the precision of Randox and Fuji assays for cCRP, as well as accuracy, correlation, and agreement compared to a reference ELISA. Blood samples from 71 client-owned dogs (20 healthy, 51 diseased) were analyzed with the 3 assays. Inter-assay CVs were ~3.5% with both the Randox and Fuji assays. The mean biases were -1.90% for the Randox and -5.93% for the Fuji test; the targeted biases were ~8.5% for both assays. The CV, bias, and observed total error were acceptable for the 2 assays compared to ASVCP recommendations based on biological variation studies. The Spearman correlation coefficient for cCRP concentration compared with the reference ELISA was 0.83 for the Randox test and 0.92 for the Fuji test. Both assays measured cCRP precisely at intermediate and increased concentrations. Correlation with the reference ELISA was good, and both assays could be used to evaluate cCRP concentrations in veterinary practice. However, the assays did not reach analytical agreement; hence the results obtained by these assays are not interchangeable, and serial monitoring of cCRP requires the use of the same assay.

Allogeneic double-negative CAR-T cells inhibit tumor growth without off-tumor toxicities.

The development of autologous chimeric antigen receptor T (CAR-T) cell therapies has revolutionized cancer treatment. Nevertheless, the delivery of CAR-T cell therapy faces challenges, including high costs, lengthy production times, and manufacturing failures. To overcome this, attempts have been made to develop allogeneic CAR-T cells using donor-derived conventional CD4+ or CD8+ T cells (Tconvs), but severe graft-versus-host disease (GvHD) and host immune rejection have made this challenging. CD3+CD4-CD8- double-negative T cells (DNTs) are a rare subset of mature T cells shown to fulfill the requirements of an off-the-shelf cellular therapy, including scalability, cryopreservability, donor-independent anticancer function, resistance to rejection, and no observed off-tumor toxicity including GvHD. To overcome the challenges faced with CAR-Tconvs, we evaluated the feasibility, safety, and efficacy of using healthy donor-derived allogeneic DNTs as a CAR-T cell therapy platform. We successfully transduced DNTs with a second-generation anti-CD19-CAR (CAR19) without hampering their endogenous characteristics or off-the-shelf properties. CAR19-DNTs induced antigen-specific cytotoxicity against B cell acute lymphoblastic leukemia (B-ALL). In addition, CAR19-DNTs showed effective infiltration and tumor control against lung cancer genetically modified to express CD19 in xenograft models. CAR19-DNT efficacy was comparable with that of CAR19-Tconvs. However, unlike CAR19-Tconvs, CAR19-DNTs did not cause alloreactivity or xenogeneic GvHD-related mortality in xenograft models. These studies demonstrate the potential of using allogeneic DNTs as a platform for CAR technology to provide a safe, effective, and patient-accessible CAR-T cell treatment option.

Adoptive Cell Therapy for T-Cell Malignancies.

T-cell malignancies are often aggressive and associated with poor prognoses. Adoptive cell therapy has recently shown promise as a new line of therapy for patients with hematological malignancies. However, there are currently challenges in applying adoptive cell therapy to T-cell malignancies. Various approaches have been examined in preclinical and clinical studies to overcome these obstacles. This review aims to provide an overview of the recent progress on adoptive cell therapy for T-cell malignancies. The benefits and drawbacks of different types of adoptive cell therapy are discussed. The potential advantages and current applications of innate immune cell-based adoptive cell therapy for T cell malignancies are emphasized.

Enhancing Therapeutic Efficacy of Double Negative T Cells against Acute Myeloid Leukemia Using Idelalisib.

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.

Venetoclax enhances T cell-mediated antileukemic activity by increasing ROS production.

Venetoclax, a Bcl-2 inhibitor, in combination with the hypomethylating agent azacytidine, achieves complete remission with or without count recovery in ∼70% of treatment-naive elderly patients unfit for conventional intensive chemotherapy. However, the mechanism of action of this drug combination is not fully understood. We discovered that venetoclax directly activated T cells to increase their cytotoxicity against acute myeloid leukemia (AML) in vitro and in vivo. Venetoclax enhanced T-cell effector function by increasing reactive oxygen species generation through inhibition of respiratory chain supercomplexes formation. In addition, azacytidine induced a viral mimicry response in AML cells by activating the STING/cGAS pathway, thereby rendering the AML cells more susceptible to T cell-mediated cytotoxicity. Similar findings were seen in patients treated with venetoclax, as this treatment increased reactive oxygen species generation and activated T cells. Collectively, this study presents a new immune-mediated mechanism of action for venetoclax and azacytidine in the treatment of AML and highlights a potential combination of venetoclax and adoptive cell therapy for patients with AML.

State-of-Art of Cellular Therapy for Acute Leukemia.

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.

Mitochondrial and Metabolic Pathways Regulate Nuclear Gene Expression to Control Differentiation, Stem Cell Function, and Immune Response in Leukemia.

Mitochondria are involved in many biological processes including cellular homeostasis, energy generation, and apoptosis. Moreover, mitochondrial and metabolic pathways are interconnected with gene expression to regulate cellular functions such as cell growth, survival, differentiation, and immune recognition. Metabolites and mitochondrial enzymes regulate chromatin-modifying enzymes, chromatin remodeling, and transcription regulators. Deregulation of mitochondrial pathways and metabolism leads to alterations in gene expression that promote cancer development, progression, and evasion of the immune system. This review highlights how mitochondrial and metabolic pathways function as a central mediator to control gene expression, specifically on stem cell functions, differentiation, and immune response in leukemia. SIGNIFICANCE: Emerging evidence demonstrates that mitochondrial and metabolic pathways influence gene expression to promote tumor development, progression, and immune evasion. These data highlight new areas of cancer biology and potential new therapeutic strategies.

CRISPR screen identifies genes that sensitize AML cells to double-negative T-cell therapy.

Acute myeloid leukemia (AML) remains a devastating disease in need of new therapies to improve patient survival. Targeted adoptive T-cell therapies have achieved impressive clinical outcomes in some B-cell leukemias and lymphomas but not in AML. Double-negative T cells (DNTs) effectively kill blast cells from the majority of AML patients and are now being tested in clinical trials. However, AML blasts obtained from ∼30% of patients show resistance to DNT-mediated cytotoxicity; the markers or mechanisms underlying this resistance have not been elucidated. Here, we used a targeted clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) screen to identify genes that cause susceptibility of AML cells to DNT therapy. Inactivation of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitinating complex components sensitized AML cells to DNT-mediated cytotoxicity. In contrast, CD64 inactivation resulted in resistance to DNT-mediated cytotoxicity. Importantly, the level of CD64 expression correlated strongly with the sensitivity of AML cells to DNT treatment. Furthermore, the ectopic expression of CD64 overcame AML resistance to DNTs in vitro and in vivo. Altogether, our data demonstrate the utility of CRISPR/Cas9 screens to uncover mechanisms underlying the sensitivity to DNT therapy and suggest CD64 as a predictive marker for response in AML patients.

Efficacy and safety of dose escalation in male patients with overactive bladder showing poor efficacy after low-dose antimuscarinic treatment: A retrospective multicenter study.

PURPOSE: To analyze the efficacy and safety of standard-dose antimuscarinic treatment on male patients with overactive bladder (OAB) symptoms showing poor efficacy after low-dose antimuscarinics. MATERIALS AND METHODS: We retrospectively reviewed the data of 566 male patients aged ≥40 with OAB symptoms between January 2017 and June 2018. They were treated with low-dose antimuscarinics for at least 4 weeks and showed poor efficacy; therefore, they were switched to standard dose antimuscarinic treatment (5 mg of solifenacin) for ≥12 weeks. The international prostate symptom score (IPSS) and overactive bladder symptom score (OABSS) at baseline (V0), 4 weeks (V1), and 12 weeks (V2) were analyzed. Post void residual urine volume (PVR) was also recorded. RESULTS: The median age, body mass index, and prostate-specific antigen levels were 69.0 years, 24.2 kg/m², and 1.24 ng/dL, respectively. The mean value of the total IPSS and OABSS significantly decreased between V0 and V2 (from 16.73 to 13.69 and 7.33 to 5.34, respectively, all p<0.001). All component scores from each questionnaire demonstrated a significant decrease except for numbers three and six on the IPSS questionnaire. PVR was increased from V0 to V2 (36.40 to 68.90 mL, p=0.015). Four and nine patients experienced constipation and thirst, respectively, and all adverse effects were graded as ≤2. CONCLUSIONS: Standard dose antimuscarinic treatment using solifenacin (5 mg) may be a safe and effective treatment for patients with OAB symptoms refractory to low-dose antimuscarinic treatment.

Comparison of surgical outcomes between lateral rectus recession and medial rectus advancement for postoperative consecutive exotropia.

To compare the surgical outcomes of medial rectus advancement and lateral rectus recession in postoperative consecutive exotropia with single-stage adjustable suture surgery.Among 1003 patients who underwent bilateral medial rectus recession between November 1996 and March 2013, the patients who required surgery for consecutive exotopia were retrospectively reviewed. Nineteen patients underwent medial rectus advancement and 15 patients underwent lateral rectus recession. All patients underwent single-stage adjustable surgery under topical anesthesia and were followed up for at least 12 months.The mean follow-up duration was 2.4 years. At final follow-up, a successful surgical outcome was found in 12 patients (63.0%) in the medial rectus advancement group and 14 patients (93.3%) in the lateral rectus recession group (P = .039). The change in ocular deviation was correlated with the amount of recession (P = .008) and preoperative angle (P < .001) in the lateral rectus recession group.Lateral rectus recession showed a higher success rate with predictable and easily performed procedure than medial rectus advancement for the treatment of postoperative consecutive exotropia with adjustable suture.

Investigation on urinary and serum alpha klotho in dogs with chronic kidney disease.

BACKGROUND: As a co-receptor for fibroblast growth factor 23, klotho plays a pivotal role in phosphate metabolism. The kidney is known to be the main source of soluble alpha-klotho and the principal regulator of its concentration. Previous studies in human participants showed that the concentration of soluble alpha-klotho in serum and urine decreased in chronic kidney disease (CKD) patients. However, no previous study has assessed soluble alpha-klotho levels in dogs. This study aimed to measure serum and urinary alpha-klotho levels in CKD dogs and identify their associations with International Renal Interest Society (IRIS) CKD stages and other parameters known to be associated with CKD. RESULTS: Serum and urinary alpha klotho concentrations were measured by a commercially available canine-specific sandwich enzyme-linked immunosorbent assay kit and compared between groups by a nonparametric Kruskal-Wallis test. Spearman's correlation coefficient was used to evaluate the relationships between variables. A stepwise multiple regression analysis was performed to estimate the effects of independent predictors on klotho concentrations. The urine klotho-to-creatinine ratio (UrKl/Cr) was significantly lower in stage 3 dogs than the control group and was significantly lower in dogs with stage 3 and 4 CKD than in those with stage 1 and 2 disease. UrKl/Cr was negatively correlated with serum symmetric dimethylarginine (sSDMA), blood urea nitrogen (BUN), creatinine, and phosphorus concentration. Serum alpha-klotho concentration in dogs with stages 2 and 3 CKD was significantly lower than those in the control group. There was no significant correlation between serum alpha-klotho and BUN, creatinine, and phosphorus concentrations. No statistically significant differences were observed in UrKl/Cr and serum alpha-klotho concentration between groups based on sex, age, urine protein-to-creatinine ratio (UPC), or blood pressure. CONCLUSIONS: UrKl/Cr decreased in dogs with advanced CKD, and it was negatively correlated with sSDMA, BUN, creatinine, and phosphorus concentrations. Thus, klotho is associated with CKD and its clinical consequences, including CKD-mineral bone disorder, in dogs. Although serum klotho concentration was negatively correlated with sSDMA levels, it was not apparently related to IRIS CKD stage or other parameters known to be associated with CKD.

Targeting late-stage non-small cell lung cancer with a combination of DNT cellular therapy and PD-1 checkpoint blockade.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: This study supports the use of DNT cells for adoptive cellular therapy against lung cancer either alone or in combination with anti-PD-1.

Cellular immunotherapy for acute myeloid leukemia: How specific should it be?

Significant improvements in the survival of patients with hematological cancers following hematopoietic stem cell transplantation provide evidence supporting the potency of immune cell-mediated anti-leukemic effects. Studies focusing on immune cell-based cancer therapies have made significant breakthroughs in the last few years. Adoptive cellular therapy (ACT), and chimeric antigen receptor (CAR) T cell therapy, in particular, has significantly increased the survival of patients with B cell acute lymphoblastic leukemia and aggressive B cell lymphoma. Despite antigen-negative relapses and severe toxicities such as cytokine release syndrome after treatment, CAR-T cell therapies have been approved by the FDA in some conditions. Although a number of studies have tried to achieve similar results for acute myeloid leukemia (AML), clinical outcomes have not been as promising. In this review, we summarize recent and ongoing studies on cellular therapies for AML patients, with a focus on antigen-specific versus -nonspecific approaches.

Human double negative T cells target lung cancer via ligand-dependent mechanisms that can be enhanced by IL-15.

BACKGROUND: The advents of novel immunotherapies have revolutionized the treatment of cancer. Adoptive cellular therapies using chimeric antigen receptor T (CAR-T) cells have achieved remarkable clinical responses in B cell leukemia and lymphoma but the effect on solid tumors including lung cancer is limited. Here we present data on the therapeutic potential of allogeneic CD3+CD4-CD8- double negative T (DNT) cells as a new cellular therapy for the treatment of lung cancer and underlying mechanisms. METHODS: DNTs were enriched and expanded ex vivo from healthy donors and phenotyped by flow cytometry. Functionally, their cytotoxicity was determined against primary and established non-small-cell lung cancer (NSCLC) cell lines in vitro or through in vivo adoptive transfer into xenograft models. Mechanistic analysis was performed using blocking antibodies against various cell surface and soluble markers. Furthermore, the role of IL-15 on DNT function was determined. RESULTS: We demonstrated that ex vivo expanded DNTs can effectively lyse various human NSCLC cells in vitro and inhibit tumor growth in xenograft models. Expanded DNTs have a cytotoxic phenotype, as they express NKp30, NKG2D, DNAM-1, membrane TRAIL (mTRAIL), perforin and granzyme B, and secrete IFNγ and soluble TRAIL (sTRAIL). DNT-mediated cytotoxicity was dependent on a combination of tumor-expressed ligands for NKG2D, DNAM-1, NKp30 and/or receptors for TRAIL, which differ among different NSCLC cell lines. Furthermore, stimulation of DNTs with IL-15 increased expression of effector molecules on DNTs, their TRAIL production and cytotoxicity against NSCLC in vitro and in vivo. CONCLUSION: Healthy donor-derived DNTs can target NSCLC in vitro and in vivo. DNTs recognize tumors via innate receptors which can be up-regulated by IL-15. DNTs have the potential to be used as a novel adoptive cell therapy for lung cancer either alone or in combination with IL-15.

Developing Allogeneic Double-Negative T Cells as a Novel Off-the-Shelf Adoptive Cellular Therapy for Cancer.

PURPOSE: To expand clinical-grade healthy donor-derived double-negative T cells (DNT) to a therapeutically relevant number and characterize their potential to be used as an "off-the-shelf" adoptive cellular therapy (ACT) against cancers. EXPERIMENTAL DESIGN: We developed methods to expand DNTs under GMP conditions and characterized their surface molecule expression pattern using flow cytometry-based high-throughput screening. We investigated the off-the-shelf potential of clinical-grade DNTs by assessing their cytotoxicity against various cancer types and their off-tumor toxicity in vitro and in xenograft models and determining the effect of cryopreservation under GMP conditions on cell viability and cytotoxicity. Further, we determined the susceptibility of DNTs to conventional allogeneic T cells in vitro and in vivo. RESULTS: Clinical-grade DNTs expanded 1,558 ± 795.5-fold in 17 days with >90% purity. Expanded DNTs showed potent in vitro cytotoxic activity against various cancer types in a donor-unrestricted manner. DNTs enhanced the survival of mice infused with a lethal dose of EBV-LCL and significantly reduced leukemia engraftment in xenograft models. Expanded DNTs cryopreserved using GMP-compliant reagents maintained viability and anticancer functions for at least 600 days. Live allogeneic DNTs did not induce cytotoxicity of alloreactive CD8+ T cells in vitro, and coinfusion of DNTs with peripheral blood mononuclear cells (PBMC) from a different donor into mice resulted in coengraftment of DNTs and PBMC-derived allogeneic conventional T cells in the absence of cytotoxicity toward DNTs, suggesting the lack of host-versus-graft reaction. CONCLUSIONS: We have established a method to generate therapeutic numbers of clinical-grade DNTs that fulfill the requirements of an off-the-shelf ACT.

Pharmacokinetics and pharmacodynamics of intravenous esomeprazole at 2 different dosages in dogs.

BACKGROUND: Although the demand for esomeprazole is increasing in veterinary medicine, the pharmacokinetics (PK) and pharmacodynamics of esomeprazole have been described in only a few studies. OBJECTIVE: To determine the PK of 0.5 and 1 mg/kg esomeprazole administered IV q12h and to investigate its effects on intragastric pH in healthy dogs. ANIMALS: Six adult Beagles. METHODS: Open-label, randomized, and crossover design. The dogs received 0.5 or 1 mg/kg esomeprazole IV q12h for 48 hours. Plasma concentrations of esomeprazole were measured by high-performance liquid chromatography-tandem mass spectrometry. Intragastric pH was determined using the Bravo pH monitoring system and recorded as mean percentage time (MPT) for which pH was ≥3 and ≥4 for 24 hours in each group. RESULTS: The peak plasma concentration and area under the curve from the time of dosing to the last measurable concentration in the 1 mg/kg group were higher than those in the 0.5 mg/kg group. However, when the dosage normalized, intergroup differences were not significant. The MPTs for which intragastric pH was ≥3 and ≥4 for 48 hours were 88% ± 7% and 81% ± 9% for the 0.5 mg/kg group and 90% ± 9% and 85% ± 11% for the 1 mg/kg group, respectively, with no significant intergroup differences. CONCLUSIONS AND CLINICAL IMPORTANCE: The pharmacokinetic parameters and acid suppressant effect for 0.5 and 1 mg/kg esomeprazole were not significantly different. Furthermore, the efficacy of esomeprazole 0.5 mg/kg IV q12h was sufficient to increase intragastric pH in Beagles.

Targeting chemotherapy-resistant leukemia by combining DNT cellular therapy with conventional chemotherapy.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Our results demonstrate the feasibility and benefit of using DNTs as an immunotherapy after the administration of conventional chemotherapy.