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.

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.

Genetic diversity and different cross-neutralization capability of porcine circovirus type 2 isolates recently circulating in South Korea.

BACKGROUND: Porcine circovirus type 2 (PCV2) is a small single-stranded DNA virus and a primary cause of PCV-associated diseases (PCVAD) that result insubstantial economic loss for swine farms. Between 2016 and 2018, PCV2 field viruses were isolated from PCVAD-affected swine farms in South Korea and investigated for genetic and antigenic heterogeneity. RESULTS: The genetic analysis of ORF2 showed that the genotype of the Korean PCV2 field isolates has been rapidly shifted from PCV2a or 2b to mutant PCV2b known as PCV2d with 82.6 to 100% amino acid sequence similarity. PCV2-specific monoclonal antibodies (mAbs) demonstrated variable antigen-binding activity to four representative Korean PCV2 field isolates [QIA215 (PCV2a), QIA418 (PCV2b), QIA169 (PCV2d), and QIA244 (PCV2d)] without genotype specificity, and one mAb showed neutralization activity to QIA215. In a cross-virus neutralization assay using anti-PCV2 sera of pigs and guinea pigs injected with a commercial vaccine and the Korean PCV2 field isolates, the anti-porcine sera of a commercial vaccine had high neutralization activity against QIA215 and QIA418 with statistically lower activity against PCV2d viruses. Anti-guinea pig sera of QIA215, QIA418, QIA169, and a commercial vaccine had high neutralization activity against all of the viruses with significantly lower activity against QIA244. Importantly, anti-guinea pig sera of QIA244 had high neutralization activity against all of the viruses. CONCLUSIONS: This study confirmed genetic and antigenic diversity among recent PCV2 field isolates in Korean swine farms, and the strain-based difference in virus neutralization capability should be considered for more effective control by vaccination.

Geographic distribution and molecular analysis of porcine reproductive and respiratory syndrome viruses circulating in swine farms in the Republic of Korea between 2013 and 2016.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes devastating disease characterized by reproductive failure and respiratory problems in the swine industry. To understand the recent prevalence and genetic diversity of field PRRSVs in the Republic of Korea, open reading frames (ORFs) 5 and 7 of PRRSV field isolates from 631 PRRS-affected swine farms nationwide in 2013-2016 were analyzed along with 200 Korean field viruses isolated in 2003-2010, and 113 foreign field and vaccine strains. RESULTS: Korean swine farms were widely infected with PRRSVs of a single type (38.4 and 37.4% for Type 1 and Type 2 PRRSV, respectively) or both types (24.2%) with up to approximately 83% nucleotide sequence similarity to prototype PRRSVs (Lelystad or VR2332). Phylogenetic analysis based on the ORF5 nucleotide sequence revealed that Korean Type 1 field isolates were classified as subgroups A, B, and C under subtype 1, while Korean Type 2 field isolates were classified as lineages 1 and 5 as well as three Korean lineages (kor A, B, and C) with the highest infection prevalence in subgroup A (50.5%) and lineage 5 (15.3%) for Type 1 and Type 2 PRRSV, respectively, among ORF5-positive farms. In particular, the lineages kor B and C were identified as novel lineages in this study, and lineage kor B comprised only the field viruses isolated from Gyeongnam Province in 2014-2015, establishing regionally unique genetic characteristics. It has also recently been confirmed that commercialized vaccine-like viruses (subgroup C) of Type 1 PRRSV and NADC30-like viruses of Type 2 PRRSV (lineage 1) are spreading rapidly in Korean swine farms. The Korean field viruses were also expected to be antigenically variable as shown in the high diversity of neutralizing epitopes and N-glycosylation sites. CONCLUSIONS: This up-to-date information regarding recent field PRRSVs should be taken into consideration when creating strategies for the application of PRRS control measures, including vaccination in the field.

Covalent Functionalization of Multi-Walled Carbon Nanotubes Surface via Chemical Treatment.

Due to the strong hydrophobic and van der Waals interactions between individual carbon nanotubes (CNTs), these particles easily aggregate with themselves. When CNTs were introduced into a polymer matrix as a filler, aggregations formed that can adversely affect the mechanical and thermal properties of polymer/CNTs composites. To prevent aggregation, covalent functionalizations via chemical treatments using H2SO4/HNO3, H2O2/H2O and a silane coupling agent(STX)-glycidoxypropyltrimethoxysilane, GPTMS) on the CNTs were chosen in this study. Moreover, the effect of the functional groups on the solubility of CNTs in tetrahydrofuran (THF) was investigated. The surface-modified multi-walled carbon nanotubes (MWCNTs) were also characterized and compared with pristine MWCNTs using several techniques. Morphology changes in surfacemodified MWCNTs were observed by Raman spectroscopy and Field-Emission Scanning Electron Microscopy (FE-SEM) images. Qualitative analyses of the functional groups on the surface-modified MWCNTs were performed by Fourier Transform Infrared Spectroscopy (FT-IR). Additionally, quantitative analyses were performed by X-Ray Photoelectron Spectroscopy (XPS), Energy Dispersive Spectroscopy (EDS), a titration method and Thermogravimetric analysis (TGA).

Impact of Postprocedural TIMI Flow on Long-Term Clinical Outcomes in Patients with Acute Myocardial Infarction.

This study aimed to evaluate the clinical prognostic implications of postprocedural Thrombolysis in Myocardial Infarction (TIMI) flow in acute myocardial infarction patients. A total of 2796 ST-elevation myocardial infarction (STEMI) and 1720 non ST-elevation myocardial infarction (NSTEMI) patients treated in 8 hospitals affiliated with the Catholic University of Korea and Chonnam National University Hospital were analyzed. The study populations were divided according to the final TIMI flow. The primary outcome were the major adverse cardiac events (MACE), defined as a composite of cardiac deaths (CD), nonfatal myocardial infarctions (MI), and target lesion revascularization (TLR). Over a median follow-up of 3.3 years (minimum 2 to maximum 5 years), MACE and CD occurred more frequently in STEMI patients with TIMI ≤ 2 group than those with TIMI 3 (MACE: adjusted hazard ratio [aHR], 1.962; 95% confidence interval [CI] 1.513 to 2.546, P < 0.001, CD: aHR, 3.154, CI 2.308 to 4.309, P < 0.001). However, there was no significant difference between the two subgroups in NSTEMI (aHR, 0.932; 95% CI 0.586 to 1.484, P = 0.087). In STEMI patients, good postprocedural TIMI flow after PCI was associated with favorable clinical outcomes. And the effect of poor TIMI flow in STEMI was on death, not the components of MACE. Meanwhile, postprocedural TIMI flow had no effect on long-term outcomes in NSTEMI patients.

Sasa quelpaertensis Nakai extract suppresses porcine reproductive and respiratory syndrome virus replication and modulates virus-induced cytokine production.

Although Sasa quelpaertensis Nakai, a dwarf bamboo, is known to exert a variety of beneficial effects on health, its antiviral effect remains to be elucidated. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating viral pathogens of swine and has a substantial economic impact on the global pork industry. Therefore, the present study was conducted to determine whether Sasa quelpaertensis Nakai extract (SQE) inhibits PRRSV infection in cultured porcine alveolar macrophages (PAMs). Our results demonstrated that SQE treatment suppressed the replication of PRRSV in a dose-dependent manner. The antiviral activity of SQE on PRRSV replication was found to be primarily exerted at early times postinfection. Treatment with SQE resulted in marked reduction of viral genomic and subgenomic RNA synthesis, viral protein expression, and progeny virus production. Notably, pro-inflammatory cytokine production in PAM cells infected with PRRSV was shown to be modulated in the presence of SQE. Taken together, our data indicate that SQE has potential as a therapeutic agent against PRRSV.

Re-emergence of foot-and-mouth disease in the Republic of Korea caused by the O/ME-SA/Ind-2001e lineage.

The O/ME-SA/Ind-2001e foot-and-mouth disease virus (FMDV) lineage is a pandemic strain that has recently become dominant within East and Southeast Asia. During May 2023, this viral lineage spread to the Republic of Korea, where 11 outbreaks were detected on cattle and goat farms located in Cheongju and Jeungpyeong. Infected animals displayed typical FMD signs including vesicular lesions with drooling and anorexia. Molecular diagnostic testing and genetic analysis (VP1 sequencing) showed that the causative FMDVs belonged to the O/ME-SA/Ind-2001e lineage and shared the closest nucleotide identity (97.95-99.21%) to viruses that have been collected from Mongolia and South-East Asian countries. Phylogenetic analyses showed that these sequences were distinct to those collected from the previous Korean O/ME-SA/Ind-2001e lineage outbreaks in 2019, demonstrating that these cases are due to a new incursion of the virus into the country. Prompt implementation of emergency vaccination using antigenically matched serotype O vaccines (r1 value: 0.74-0.93), together with intensive active surveillance on farms surrounding the infected premises has successfully prevented further spread of FMD. These recent FMD outbreaks reinforce the importance of research to understand the risks associated with transboundary pathways in the region, in order to reduce the possibility of a further reintroduction of FMD into the Republic of Korea.

Multiple incursions of foot-and-mouth disease virus serotype O into the Republic of Korea between 2010 and 2019.

This study characterised type O foot-and-mouth disease (FMD) viruses recovered from outbreaks that were reported between 2010 and 2019 in the Republic of Korea. We used 96 newly generated whole-genome sequences (WGS) along with 131 already published WGSs from samples collected from countries in East and Southeast Asia. We identified at least eight independent introductions of O/SEA/Mya-98 and O/ME-SA/Ind-2001e FMDV strains into the Republic of Korea during the study period, which were closely related to the sequences of viruses circulating in the East and Southeast Asia neighbourhood with over 97 % nucleotide identity. Spatial-temporal transitions of O/SEA/Mya-98 lineage viruses recovered from the largest outbreak (2014-16) showed that after initial cases were detected within a 15-day period in July 2014, a single introduction of the same virus during December 2014 generated extensive forward virus transmission between farms that lasted until March 2016. We estimated that secondary transmissions were responsible for infection on 44 % FMD affected farms, over a total of 14 generations of infection. We eastimated a median evolutionry rate of 2.51 × 10-5 nt/site/day, which is similar for other FMD epidemic scenarios. These findings suggest that regular incursions of different FMDV lineages into the Republic of Korea have posed a continuous threat from endemic countries of East and Southeast Asia. These data highlight the importance of active cooperation and information exchange on FMD situation within Asian countries and assessment about the likely risk routes of virus movement is highly necessary to prevent further incursion and virus spread of FMDV in the Republic of Korea.

Big data-based risk assessment of poultry farms during the 2020/2021 highly pathogenic avian influenza epidemic in Korea.

Outbreaks of H5-type highly pathogenic avian influenza (HPAI) in poultry have been reported in various parts of the world. To respond to these continuous threats, numerous surveillance programs have been applied to poultry raising facilities as well as wild birds. In Korea, a surveillance program was developed aimed at providing a preemptive response to possible outbreaks at poultry farms. The purpose of this study is to comprehensively present the risks of HPAI evaluated by this program in relation to actual outbreak farms during the epidemic of 2020/2021. A deep learning-based risk assessment program was trained based on the pattern of livestock vehicles visiting poultry farms and HPAI outbreaks to calculate the risk of HPAI for farms linked by the movement of livestock vehicles (such farms are termed "epidemiologically linked farms"). A total of 7,984 risk assessments were conducted, and the results were categorized into four groups. The proportion of the highest risk level was greater in duck farms (13.6%) than in chicken farms (8.8%). Among the duck farms, the proportion of the highest risk level was much greater in farms where breeder ducks were raised (accounting for 26.4% of the risk) than in farms where ducks were raised to obtain meat (12.8% of the risk). A higher risk level was also found in cases where the species of the outbreak farm and epidemiologically linked farms were the same (proportion of the highest risk level = 13.2%) compared to that when the species between the two farms were different (7.9%). The overall proportion of farms with HPAI outbreaks among epidemiologically linked farms (attack rate, AR) was 1.7% as HPAI was confirmed on 67 of the 3,883 epidemiologically linked farms. The AR was highest for breeder ducks (15.3%) among duck farms and laying hens (4.8%) among chicken farms. The AR of the pairs where livestock vehicles entered the inner farm area was 1.3 times (95% confidence interval: 1.4-2.9) higher than that of all pairs. With the risk information provided, customized preventive measures can be implemented for each epidemiologically linked farm. The use of this risk assessment program would be a good example of information-based surveillance and support decision-making for controlling animal diseases.

Enhanced detection and serotyping of foot-and-mouth disease virus serotype O, A, and Asia1 using a novel multiplex real-time RT-PCR.

Rapid and accurate detection and serotyping of foot-and-mouth disease (FMD) virus (FMDV) is essential for implementing control policies against emergent FMD outbreaks. Current serotyping assays, such as VP1 reverse transcription-polymerase chain reaction (RT-PCR)/sequencing (VP1 RT-PCR/sequencing) and antigen detection enzyme-linked immunosorbent assay (ELISA), have problems with increasing serotyping failure of FMDVs from FMD outbreaks. This study was conducted to develop a multiplex real-time RT-PCR for specific detection and differential serotyping of FMDV serotype O, A, and Asia 1 directly from field clinical samples. Primers and probes were designed based on 571 VP1 coding region sequences originated from seven pools. Multiplex real-time RT-PCR using these primers and probes demonstrated serotype-specific detection with enhanced sensitivity compared to VP1 RT-PCR/sequencing for reference FMDV (n = 24). Complete serotyping conformity between the developed multiplex real-time RT-PCR and previous VP1 RT-PCR/sequencing was demonstrated using FMDV field viruses (n = 113) prepared in cell culture. For FMDV field clinical samples (n = 55), the serotyping rates of multiplex real-time RT-PCR and VP1 RT-PCR/sequencing were 92.7% (51/55) and 72.7% (40/55), respectively. Moreover, the developed multiplex real-time RT-PCR demonstrated improved FMDV detection (up to 33.3%) and serotyping (up to 67.7%) capabilities for saliva samples when compared with 3D real-time RT-PCR and VP1 RT-PCR/sequencing, during 10 days of challenge infection with FMDV serotype O, A, and Asia 1. Collectively, this study suggests that the newly developed multiplex real-time RT-PCR assay may be useful for the detection and differential serotyping of FMDV serotype O, A, and Asia 1 in the field.

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.

In Vitro Propagation of Gastrochilus matsuran (Makino) Schltr., an Endangered Epiphytic Orchid.

Gastrochilus matsuran (Makino) Schltr. (Orchidaceae) populations are declining quickly because of overexploitation, climatic changes, and deforestation; therefore, mass-production protocols are required for this orchid. Natural propagation of this species is often hampered by meager seed germination and slow growth. Thus, our aim was to establish an effective protocol for the in vitro propagation of G. matsuran and reduce the risk of its extinction. We investigated the impacts of culture media, coconut water (CW), and plant hormones (gibberellic acid (GA3), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA), and thidiazuron (TDZ)) on asymbiotic germination, multiplication and conversion of protocorms, and plantlet development. Maximal seed germination (93.3%) was achieved on ½ MS medium without vitamins plus 5% CW, 1 µM NAA, and 1.5 µM GA3. Secondary protocorm formation was best achieved on ½ MS medium without vitamins plus 2 µM TDZ. The conversion of protocorms into seedlings was maximized by supplementation with 2 µM IBA or 1 µM NAA. Acclimatized plantlets that exhibited exuberant growth on sphagnum moss were reintroduced to tree trunks in a natural habitat, with a 67% survival rate. This in vitro propagation procedure would be helpful for the mass production and conservation of this rare epiphytic orchid.

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.

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.

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.

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.

An enhanced immunochromatographic strip test using colloidal gold nanoparticle-labeled dual-type N proteins for detection of antibodies to PRRS virus.

Porcine reproductive and respiratory syndrome (PRRS) is recognized as one of the most important infectious diseases causing serious economic loss in the swine industry worldwide. Due to its increasing genetic diversity, a rapid and accurate diagnosis is critical for PRRS control. The immunochromatographic strip test (ICST) is a rapid and convenient type of immunoassay. In this study, an on-site immunochromatographic assay-based diagnostic method was developed for detection of PRRS virus (PRRSV)-specific antibodies. The method utilized colloidal gold nanoparticle-labeled dual-type nucleocapsid proteins encoded by open reading frame 7. We evaluated 991 field samples from pig farms and 66 serum samples from experimentally PRRSV-inoculated pigs. Based on true PRRSV-specific antibody-positive or -negative sera determined by immunofluorescence assay and IgM enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of the ICST were 97.5% and 91.1%, respectively, similar to those of a commercial ELISA (IDEXX PRRS X3 Ab). More importantly, the ICST was completed within 15 min and could detect the PRRSV-specific antibody at an earlier stage of infection (3-7 days) than that of ELISA (7+ days). The results demonstrate that the developed ICST has great potential as an on-farm diagnostic method, providing excellent diagnostic performance in a quick and convenient manner.

Allogeneic Human Double Negative T Cells as a Novel Immunotherapy for Acute Myeloid Leukemia and Its Underlying Mechanisms.

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.