Single-cell sequencing of tumour infiltrating T cells efficiently identifies tumour-specific T cell receptors based on the T cell activation score.

Adoptively transferred T cell receptor-engineered T cells are a promising cancer treatment strategy, and the identification of tumour-specific TCRs is essential. Previous studies reported that tumour-reactive T cells and TCRs could be isolated based on the expression of activation markers. However, since T cells with different cell states could not respond uniformly to activation but show a heterogeneous expression profile of activation and effector molecules, isolation of tumour-reactive T cells based on single activation or effector molecules could result in the absence of tumour-reactive T cells; thus, combinations of multiple activation and effector molecules could improve the efficiency of isolating tumour-specific TCRs. We enrolled two patients with lung adenocarcinoma and obtained their tumour infiltrating lymphocytes (TILs) and autologous tumour cells (ATCs). TILs were cocultured with the corresponding ATCs for 12 h and subjected to single-cell RNA sequencing. First, we identified three TCRs with the highest expression levels of IFNG and TNFRSF9 mRNA for each patient, yet only the top one or two recognized the corresponding ATCs in each patient. Next, we defined the activation score based on normalized expression levels of IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK, and PRF1 mRNA for each T cell and then identified three TCRs with the highest activation score for each patient. We found that all three TCRs in each patient could specifically identify corresponding ATCs. In conclusion, we established an efficient approach to isolate tumour-reactive TCRs based on combinations of multiple activation and effector molecules through single-cell RNA sequencing.

Theoretical charge plots as a tool for targeted and accelerated ion exchange chromatography method development of NANOBODYⓇ molecules.

NANOBODYⓇ molecules are an innovative class of biotherapeutics based on heavy chain only VHH immunoglobulins. Much like canonical antibodies, they are prone to the formation of charge variants and other post-translational modifications, which can potentially impact their critical quality attributes. Therefore, establishing high-resolution product-specific methods, such as IEX chromatography, is essential for evaluating the purity of these molecules. However, due to the lower surface charge of NANOBODYⓇ molecules, their charge-based elution behavior can differ considerably from that of classical antibodies, resulting in a more extensive method development set-up for these smaller molecules. Using an initial pH screening gradient based on theoretical protein charge plots, we investigated the IEX retention behavior of eight NANOBODYⓇ molecules with a wide range of pI values (pI 5.0 to 10.0). Our findings reveal that the charge-based chromatographic behavior of NANOBODYⓇ molecules cannot be solely attributed to the isoelectric point (pI) of the protein. Rather, a molecule-specific charge threshold was identified as a critical parameter for NANOBODYⓇ molecule retention. Furthermore, the protein charge plot also showed that NANOBODYⓇ molecule elution can be characterized by a charge plateau where the net charge of the protein remains constant over a certain pH range (∼ pH 5.5 to pH 8.0), further challenging the paradigm that elution pH and pI are fixed values. The application of this theoretical approach using protein charge plots to define NANOBODYⓇ molecule charge threshold and charge plateau parameters, can reduce overall IEX method development turnaround time by at least 2-fold.

T cells engineered with full-length NKG2D linked to signaling domains of 4-1BB and CD3ζ show enhanced antitumor activity.

Since NKG2D ligands (NKG2DLs) are primarily overexpressed on multiple types of solid tumors but absent on most normal tissues, NKG2DLs could be optimal antigens for CAR-T cells. To date, there have been two types of NKG2DL CARs: (i) the extracellular domain of NKG2D fused to the CD8a transmembrane domain, signaling domains of 4-1BB and CD3ζ (NKBz) and (ii) full-length NKG2D fused to the CD3ζ signaling domain (chNKz). Although NKBz- and chNKz-engineered T cells both showed antitumor activities, a comparison of their functions has not been reported. In addition, use of the 4-1BB signaling domain into the CAR construct could prolong the persistence and resistance to antitumor activities of CAR-T cells, we designed a new NKG2DL CAR, full-length NKG2D fused to the signaling domains of 4-1BB and CD3ζ (chNKBz). Among the two types of NKG2DL CAR-T cells reported in previous studies, we found that chNKz T cells had stronger antitumor ability than NKBz T cells in vitro, but their antitumor activity in vivo is similar. The chNKBz T cells showed antitumor activity superior to that of chNKz T cells and NKBz T cells in vitro and in vivo, providing a new option for the immunotherapy of NKG2DL-positive tumor patients.

TCR extracellular domain genetically linked to CD28, 2B4/41BB and DAP10/CD3ζ -engineered NK cells mediates antitumor effects.

NK cells, especially FDA-approved NK-92 cells, could be used for TCR engineering owing to their specialized cytotoxicity against tumors, safety profile and potential use as an off-the-shelf cellular therapy. The TCR complex requires assembly of TCR- α/ ß chains with CD3 molecules (CD3δ, CD3γ, CD3ε, CD3ζ) to be correctly expressed at the cell membrane, and yet NK cells lack expression of these CD3 subunits besides CD3ζ. Since transmembrane regions of TCR α and ß chains are involved in TCR complex assembly, transmembrane regions of TCR replaced by CD28 transmembrane domain could result in the expression of TCR independent of its companion CD3 subunits. However, since the absence of CD3 signaling components can influence the transmission of TCR signals to NK cells, it is necessary to add the signaling molecules of NK cells followed by CD28 transmembrane domain. Both CD3ζ and DAP10 play an important role in the activation and cytotoxicity of NK cells; moreover, 2B4 and 4-1BB are the main costimulatory molecules in NK cells. Therefore, we designed a chimeric TCR that consisted of the extracellular domains of the TCR α and ß chains specific for NYESO-1 fused to the CD28 transmembrane domain followed by the 41BB and CD3ζ signaling domains as well as the 2B4 and DAP10 signaling domain, respectively. The chimeric TCR genetically engineered NK-92 cells exhibit antigen-specific recognition and lysis of tumor cells both in vitro and in vivo. In addition, TCR-28-2B10/BBζ can be feasibly expressed in primary NK cells and exhibit antigen-reactive recognition and effect function. The overall encouraging data highlight the value of NK-92 cells and primary NK cells engineered to express therapeutic chimeric TCR for adoptive immunotherapies.

Autophagic flux restoration enhances the antitumor efficacy of tumor infiltrating lymphocytes.

BACKGROUND: Although adoptive cell therapy with tumor infiltrating lymphocytes (TILs) has mediated effective antitumor responses in several cancers, dysfunction and exhaustion of TILs significantly impair the therapeutic effect of TILs. Thus, it is essential to elucidate the exhausted characteristics of TILs and improve the antitumor effect of TILs by reversing their exhaustion. Here, we focused on the influence of autophagy on TILs in terms of T-cell activation, proliferation, and differentiation in vitro and in vivo. METHODS: We first evaluated autophagy level of TILs and influence of spermidine treatment on autophagy levels of TILs. Furthermore, we assessed the proliferative potential, phenotypical characteristics, T cell receptor (TCR) repertoire and antitumor activity of TILs with and without spermidine treatment. RESULTS: We found that autophagic flux of TILs, especially exhausted TILs that express inhibitory immunoreceptors and have impaired proliferative capacity and decreased production of cytotoxic effector molecules, was significantly impaired. The restoration of autophagic flux via spermidine treatment resulted in increased diversity of the TCR repertoire, reduced expression of inhibitory immunoreceptors (PD1, TIM3, or LAG3), enhanced proliferation and effector functions, which subsequently demonstrated the superior in vitro and in vivo antitumor activity of TILs. Our findings unveil that spermidine, as an autophagy inducer, reverses dysfunction and exhaustion of TILs and subsequently improves the antitumor activity of TILs. CONCLUSIONS: These data suggest that spermidine treatment presents an opportunity to improve adoptive TIL therapy for the treatment of solid tumors.

Autophagic flux restoration of senescent T cells improves antitumor activity of TCR-engineered T cells.

Objectives: Although adoptive cell therapy with T-cell receptor-engineered T cells (TCR-Ts) has mediated effective antitumor responses in several cancers, senescence of T cells could impair the therapeutic effect of TCR-Ts. Thus, it is essential to elucidate the characteristics of senescent TCR-Ts and how to subsequently improve their antitumor effect. Here, we focused on the influence of autophagy on TCR-Ts, since autophagy is tightly associated with the regulation of T-cell activation, proliferation and differentiation. Methods: We first evaluated autophagy level of senescent TCR-Ts, and then the senescent TCR-Ts were expanded in vitro for 7 days with and without spermidine treatment, respectively. Furthermore, the proliferative potential, phenotypical characteristics and functionality of the propagated senescent TCR-Ts were analysed in vitro and in vivo after 7-day ex vivo expansion. Results: We found that autophagic flux of senescent TCR-T cells was significantly impaired. The restoration of autophagic flux via spermidine treatment reduced the expression of inhibitory immunoreceptors (PD-1, TIM-3 or LAG-3), enhanced proliferation and effector functions and subsequently demonstrated the superior in vitro and in vivo antitumor activity of TCR-Ts. Conclusion: These data suggest that spermidine treatment presents an opportunity to improve the antitumor effect of TCR-Ts for the treatment of solid tumors.

Metabolic reprogramming by ex vivo glutamine inhibition endows CAR-T cells with less-differentiated phenotype and persistent antitumor activity.

The inadequate in vivo persistence of chimeric antigen receptor (CAR)-modified T cells has been shown to lead to poor therapeutic efficacy and disease recurrence. In vivo persistence is associated with the differentiation subsets infused, with less differentiated TN or TCM conferring superior renewal capacity and antitumor immunity compared to TEM or TEFF. However, ex vivo expanded CAR-T cells exhibit phenotypic heterogeneity with majority of TEM or TEFF subsets and very low populations of TN and TCM. The transition of differentiation subsets is closely correlated with T cell metabolism fitness. Effector T cell differentiation from TN or TCM requires glutamine uptake and metabolism. Using a CD19-specific CAR, we demonstrated that glutamine inhibition by adding the glutamine antagonist 6-Diazo-5-oxo-l-norleucine (DON) into the culture endows CAR-T cells with enhanced mitochondrial OXPHOS utilizing fatty acids and reduced glycolytic activity, and retains more TN or TCM subsets. DON- pretreated CAR-T cells exhibited stronger cytotoxic lysis in vitro and more robust elimination of tumor burdens in vivo. This study suggests that glutamine inhibition ex vivo would be a potential approach for modulating metabolism and differentiation state to improve the efficacy of CAR-T cell therapy.

[Quantitative analysis of fermented aerial part of Bupleurum chinense and prediction of their antimicrobial activity].

The aim of this study was to predict the anti-microbial components in the aerial part of Bupleurum chinense fermented by Lactobacillus plantarum through analyzing the correlation between contents of bioactive components and their inhibitory action for pathogenic bacteria. In this study, the UPLC-MS-MS detection method was established for eight flavonoids(kaempferol-3-O-ß-D-rutinoside, isoquercitrin, quercetin, isorhamnetin, rutin, iridin, quercetin-3-O-ß-L-arabinoside, kaempferol) and DL-3-phenyllactic acid, and the dynamic change of their contents at fermentation course were monitored. Meanwhile, the experiment employed five common no-naquatic pathogenic bacteria(Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Bacillus subtilis), and four common aquatic pathogenic bacteria(Aeruginosa hydrophila, Delayed Edwards, Vibrio alginolyticus, and Vibrio harveyi) to validate in vitro anti-microbial activity of the fermented aerial part of B. chinense at different fermentation time points. Finally, the Pearson correlation analysis was applied to predict the anti-microbial components of the fermented aerial part of B. chinense.The established UPLC-MS-MS method showed a good linearity and the widest linear range was from 0.19 µg·mL~(-1) to 50 µg·mL~(-1). The limit of quantitation and the limit of detection were 0.19-1.56 µg·mL~(-1) and 0.1-0.78 µg·mL~(-1) respectively. During the fermentation within 48 h, the contents of three flavonoids(rutin, quercetin-3-O-ß-L-arabinoside, isoquercitrin) and DL-3-phenyllactic acid from the fermented aerial part of B. chinense increased sharply. In the process of fermentation, the anti-microbial effect of the fermented aerial part of B. chinense on aquatic pathogens was significantly stronger than that on non-aquatic pathogens. Furthermore, Pearson correlation analysis predicted that isoquercitrin, rutin, quercetin-3-O-ß-L-arabinoside and DL-3-phenyllactic acid showed significant correlation with the four aquatic pathogens. This study revealed that the fermented aerial part of B. chinense had a high sensitivity to aquatic pathogens, which may be caused by the increased contents of isoquercitrin, rutin, quercetin-3-O-ß-L-arabinoside and DL-3-phenyllactic acid. In conclusion, this study provides a theoretical basis and new idea for the further development of the large amount of wasteful aerial part of Bupleurum chinense.

Progress of long non-coding RNA in esophageal squamous cell carcinoma / 肿瘤研究与临床

Esophageal cancer is one of the most frequent malignant tumors in the world, and esophageal squamous cell carcinoma is the most common one in China, and its molecular targeted therapy is the current research focus. More and more long non-coding RNAs (lncRNA) have been found to be involved in the occurrence, invasion and metastasis of various tumors with the deepening of researches, and they are likely to be potential targets for effective prevention and treatment of tumors. This article reviews the research progress of related lncRNA in esophageal squamous cell carcinoma in recent years.

Enzyme-free fluorescent detection of microcystin-LR using hairpin DNA-templated copper nanoclusters as signal indicator.

Microcystin-LR (MC-LR) is a potent hepatotoxin that is often associated with blooms of cyanobacteria. The analysis of trace MC-LR plays important role in environmental and health fields. Herein, we developed a low-cost and enzyme-free detection method of MC-LR by using hairpin DNA-templated copper nanoclusters (hpDNA-CuNCs) as fluorescent probe. The hpDNA-template was designed and fabricated by a MC-LR aptamer loop and a double strand stem, which can specifically recognize target MC-LR with strong affinity. The AT-rich and complementary double strand stem serves as a template for the formation of CuNCs. The formed fluorescent sensing probe of hpDNA-CuNCs exhibits maximum emission wavelength at 575 nm. Upon the addition of target MC-LR into the hpDNA-CuNCs, we observed fluorescence was quenched considerably due to the high affinity between MC-LR and hpDNA aptamer strand loop, which indicated a conformational change of hairpin probe from the stem-loop DNA structure to single-stranded DNA. Then, the change of fluorescence intensity can be used to monitor the concentration of MC-LR from 0.005 to 1200 µg L-1 with a detection limit of 0.003 ng L-1. Compared with the previous reports, this method does not require complex DNA sequence design, fluorescence dye label and sophisticated experimental techniques. Moreover, the target MC-LR in real water samples has been detected.

[Identification and Characterization of a Hypothermic Alkaliphilic Aerobic Denitrifying Bacterium Pseudomonas monteilii Strain H97].

Low temperatures and high pH generally inhibit bio-denitrification. Thus, it is important to explore psychrotrophic and alkali-resistant microorganisms for nitrogen degradation. This study mainly focused on the identification of an alkaliphilic strain and preliminary exploration of its denitrification characteristics. Based on morphological observations, phospholipid fatty acids and 16S rRNA gene sequence analysis, strain H97, which was isolated from the winter paddy field in Guizhou province, was identified as Pseudomonas monteilii. Till date, there were few reports about the denitrification characteristics of Pseudomonas monteilii. The effects of environmental factors such as temperature, inoculation quantity, C/N ratio, initial pH, and carbon source were investigated using simulated wastewater. The optimum conditions for nitrate and total nitrogen removal by H97 were: inoculum size 1.5×106 CFU·(100 mL)-1; initial pH 9.0; C/N=15; 15℃; and sodium succinate as the carbon source. The nitrate and total nitrogen removal efficiencies were 97.69% and 96.32%, respectively, at optimum conditions with an initial nitrate nitrogen concentration of 50.0 mg·L-1. The temperature experiments indicated that the optimal temperature for highest nitrogen removal efficiency was 15℃, and that the strain H97 could survive in a wide range of 15-40℃. Additionally, the nitrate and total nitrogen efficiencies at the initial pH value of 7.0-11.0 were 91.21% and 79.10%, respectively, and the denitrification capacity then decreased to 64.75% at the initial pH 12.0. These results indicated that strain H97 showed cold and alkali resistance, which suggests an application potential for the treatment of alkaline nitrogen polluted water in the southern winter.

[Heterotrophic Nitrification and Aerobic Denitrification of the Hypothermia Aerobic Denitrification Bacterium: Arthrobacter arilaitensis].

High concentrations of ammonium, nitrate and nitrite nitrogen were employed to clarify the abilities of heterotrophic nitrification and aerobic denitrification of Arthrobacter arilaitensis strain Y-10. Meanwhile, by means of inoculating the strain suspension into the mixed ammonium and nitrate, ammonium and nitrite nitrogen simulated wastewater, we studied the simultaneous nitrification and denitrification ability of Arthrobacter arilaitensis strain Y-10. In addition, cell optical density was assayed in each nitrogen removal process to analyze the relationship of cell growth and nitrogen removal efficiency. The results showed that the hypothermia denitrification strain Arthrobacter arilaitensis Y-10 exhibited high nitrogen removal efficiency during heterotrophic nitrification and aerobic denitrification. The ammonium, nitrate and nitrite removal rates were 65.0%, 100% and 61.2% respectively when strain Y-10 was cultivated for 4 d at 15°C with initial ammonium, nitrate and nitrite nitrogen concentrations of 208.43 mg · L⁻¹, 201.16 mg · L⁻¹ and 194.33 mg · L⁻¹ and initial pH of 7.2. Nitrite nitrogen could only be accumulated in the medium containing nitrate nitrogen during heterotrophic nitrification and aerobic denitrification process. Additionally, the ammonium nitrogen was mainly removed in the inorganic nitrogen mixed synthetic wastewater. In short, Arthrobacter arilaitensis Y-10 could conduct nitrification and denitrification effectively under aerobic condition and the ammonium nitrogen removal rate was more than 80.0% in the inorganic nitrogen mixed synthetic wastewater.

New technologies for DNA analysis--a review of the READNA Project.

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.

Setting the scene: taxonomy of personal health monitoring.

In order to provide a systematic basis for communication in trans-disciplinary research projects, there is a need for taxonomies and ontologies. Our developed taxonomy of personal health monitoring (PHM) is based on a systematic literature review and an iterative adaption process with trans-disciplinary partners. The construction method of the taxonomy is an ongoing process and need regularly updates.