T cell receptor clonotype in tumor microenvironment contributes to intratumoral signaling network in patients with colorectal cancer.

Single-cell RNA sequencing (scRNA-seq) has contributed to understanding cellular heterogeneity and immune profiling in cancer. The aim of the study was to investigate gene expression and immune profiling in colorectal cancer (CRC) using scRNA-seq. We analyzed single-cell gene expression and T cell receptor (TCR) sequences in 30 pairs of CRC and matched normal tissue. Intratumoral lymphocytes were measured with digital image analysis. CRC had more T cells, epithelial cells, and myeloid cells than normal colorectal tissue. CRCs with microsatellite instability had more abundant T cells than those without microsatellite instability. Immune cell compositions of CRC and normal colorectal tissue were inversely correlated. CD4 + or CD8 + proliferating T cells, CD4 + effector memory T cells, CD8 + naïve T cells, and regulatory T cells of CRC showed higher TCR clonal expansion. Tumor epithelial cells interacted with immune cells more strongly than normal. T cells, myeloid cells, and fibroblasts from CRCs of expanded T cell clonotypes showed increased expression of genes related to TNF and NFKB signaling and T cell activation. CRCs of expanded T cell clonotypes also showed stronger cellular interactions among immune cells, fibroblasts, and endothelial cells. Pro-inflammatory CXCL and TNF signaling were activated in CRCs of expanded T cell clonotype. In conclusion, scRNA-seq analysis revealed different immune cell compositions, differential gene expression, and diverse TCR clonotype dynamics in CRC. TCR clonality expansion is associated with immune activation through T cell signaling and chemokine signaling. Patients with CRCs of expanded clonotype can be promising candidates for immunotherapy.

Myeloid-derived suppressor cells in pleural effusion as a diagnostic marker for early discrimination of pulmonary tuberculosis from pneumonia.

Introduction: Tuberculous pleural effusion (TPE) stands as one of the primary forms of extrapulmonary tuberculosis (TB) and frequently manifests in regions with a high prevalence of TB, consequently being a notable cause of pleural effusion in such areas. However, the differentiation between TPE and parapneumonic pleural effusion (PPE) presents diagnostic complexities. This study aimed to evaluate the potential of myeloid-derived suppressor cells (MDSCs) in the pleural fluid as a potential diagnostic marker for distinguishing between TPE and PPE. Methods: Adult patients, aged 18 years or older, who presented to the emergency room of a tertiary referral hospital and received a first-time diagnosis of pleural effusion, were prospectively enrolled in the study. Various immune cell populations, including T cells, B cells, natural killer (NK) cells, and MDSCs, were analyzed in both pleural fluid and peripheral blood samples. Results: In pleural fluid, the frequency of lymphocytes, including T, B, and NK cells, was notably higher in TPE compared to PPE. Conversely, the frequency of polymorphonuclear (PMN)-MDSCs was significantly higher in PPE. Notably, compared to traditional markers such as the neutrophil-to-lymphocyte ratio and adenosine deaminase level, the frequency of PMN-MDSCs emerged as a more effective discriminator between PPE and TPE. PMN-MDSCs demonstrated superior positive and negative predictive values and exhibited a higher area under the curve in the receiver operating characteristic curve analysis. PMN-MDSCs in pleural effusion increased the levels of reactive oxygen species and suppressed the production of interferon-gamma from T cells following nonspecific stimulation. These findings suggest that MDSC-mediated immune suppression may contribute to the pathology of both TPE and PPE. Discussion: The frequency of PMN-MDSCs in pleural fluid is a clinically useful indicator for distinguishing between TPE and PPE.

Degradation of dissolved sulfide in water using multi-hole dielectric barrier discharge.

Biogas obtained from livestock manure is used as fuel for solid oxide fuel cells. Although H2S is a typical biogas, it is a fatal disadvantage for fuel-cell power generation and, thus, must be removed. In this study, we proposed an effective method for sulfide removal from water using a multi-hole dielectric barrier discharge (DBD) system. In this system, active species, such as ozone, ultraviolet rays, hydroxyl radicals, and hydrogen peroxide, were simultaneously generated. Under optimal conditions, dissolved sulfide (initial concentration: 120 mg/L) was completely degraded within 10 min in air plasma and 6 min in oxygen plasma. Changes in the physical properties of the sulfide-treated water were confirmed by measuring the pH, oxidation-reduction potential, and dissolved oxygen. Results of the by-product analysis showed that sulfide was converted into sulfate by reacting with a large amount of ozone, and the active species were emitted from the multi-hole DBD system. In summary, multi-hole DBD technology has demonstrated merit as a water-contaminant purification technology and for the removal of dissolved sulfide.

Discovery of GABA Aminotransferase Inhibitors via Molecular Docking, Molecular Dynamic Simulation, and Biological Evaluation.

γ-Aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that degrades γ-aminobutyric (GABA) in the brain. GABA is an important inhibitory neurotransmitter that plays important neurological roles in the brain. Therefore, GABA-AT is an important drug target that regulates GABA levels. Novel and potent drug development to inhibit GABA-AT is still a very challenging task. In this study, we aimed to devise novel and potent inhibitors against GABA-AT using computer-aided drug design (CADD) tools. Since the crystal structure of human GABA-AT was not yet available, we utilized a homologous structure derived from our previously published paper. To identify highly potent compounds relative to vigabatrin, an FDA-approved drug against human GABA-AT, we developed a pharmacophore analysis protocol for 530,000 Korea Chemical Bank (KCB) compounds and selected the top 50 compounds for further screening. Preliminary biological analysis was carried out for these 50 compounds and 16 compounds were further assessed. Subsequently, molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations were carried out. In the results, four predicted compounds, A07, B07, D08, and H08, were found to be highly potent and were further evaluated by a biological activity assay to confirm the results of the GABA-AT activity inhibition assay.

Machine Learning-Based Drug Repositioning of Novel Janus Kinase 2 Inhibitors Utilizing Molecular Docking and Molecular Dynamic Simulation.

Machine learning algorithms have been increasingly applied in drug development due to their efficiency and effectiveness. Machine learning-based drug repurposing can contribute to the identification of novel therapeutic applications for drugs with other indications. The current study used a trained machine learning model to screen a vast chemical library for new JAK2 inhibitors, the biological activities of which were reported. Reference JAK2 inhibitors, comprising 1911 compounds, have experimentally determined IC50 values. To generate the input to the machine learning model, reference compounds were subjected to RDKit, a cheminformatic toolkit, to extract molecular descriptors. A Random Forest Regression model from the Scikit-learn machine learning library was applied to obtain a predictive regression model and to analyze each molecular descriptor's role in determining IC50 values in the reference data set. Then, IC50 values of the library compounds, comprised of 1,576,903 compounds, were predicted using the generated regression model. Interestingly, some compounds that exhibit high IC50 values from the prediction were reported to possess JAK inhibition activity, which indicates the limitations of the prediction model. To confirm the JAK2 inhibition activity of predicted compounds, molecular docking and molecular dynamics simulation were carried out with the JAK inhibitor reference compound, tofacitinib. The binding affinity of docked compounds in the active region of JAK2 was also analyzed by the gmxMMPBSA approach. Furthermore, experimental validation confirmed the results from the computational analysis. Results showed highly comparable outcomes concerning tofacitinib. Conclusively, the machine learning model can efficiently improve the virtual screening of drugs and drug development.

Vismodegib Identified as a Novel COX-2 Inhibitor via Deep-Learning-Based Drug Repositioning and Molecular Docking Analysis.

Artificial intelligence algorithms have been increasingly applied in drug development due to their efficiency and effectiveness. Deep-learning-based drug repurposing can contribute to the identification of novel therapeutic applications for drugs with other indications. The current study used a trained deep-learning model to screen an FDA-approved drug library for novel COX-2 inhibitors. Reference COX-2 data sets, composed of active and decoy compounds, were obtained from the DUD-E database. To extract molecular features, compounds were subjected to RDKit, a cheminformatic toolkit. GraphConvMol, a graph convolutional network model from DeepChem, was applied to obtain a predictive model from the DUD-E data sets. Then, the COX-2 inhibitory potential of the FDA-approved drugs was predicted using the trained deep-learning model. Vismodegib, an anticancer agent that inhibits the hedgehog signaling pathway by binding to smoothened, was predicted to inhibit COX-2. Noticeably, some compounds that exhibit high potential from the prediction were known to be COX-2 inhibitors, indicating the prediction model's liability. To confirm the COX-2 inhibition activity of vismodegib, molecular docking was carried out with the reference compounds of the COX-2 inhibitor, celecoxib, and ibuprofen. Furthermore, the experimental examination of COX-2 inhibition was also carried out using a cell culture study. Results showed that vismodegib exhibited a highly comparable COX-2 inhibitory activity compared to celecoxib and ibuprofen. In conclusion, the deep-learning model can efficiently improve the virtual screening of drugs, and vismodegib can be used as a novel COX-2 inhibitor.

Computational Exploration of the Effects of Mutations on GABA Aminotransferase in GABA Aminotransferase Deficiency.

Gamma-aminobutyric acid (GABA) transaminase-also called GABA aminotransferase (GABA-AT)-deficiency is a rare autosomal recessive disorder characterized by a severe neonatal-infantile epileptic encephalopathy with symptoms such as seizures, hypotonia, hyperreflexia, developmental delay, and growth acceleration. GABA transaminase deficiency is caused by mutations in GABA-AT, the enzyme responsible for the catabolism of GABA. Mutations in multiple locations on GABA-AT have been reported and their locations have been shown to influence the onset of the disease and the severity of symptoms. We examined how GABA-AT mutations influence the structural stability of the enzyme and GABA-binding affinity using computational methodologies such as molecular dynamics simulation and binding free energy calculation to understand the underlying mechanism through which GABA-AT mutations cause GABA-AT deficiency. GABA-AT 3D model depiction was carried out together with seven individual mutated models of GABA-AT. The structural stability of all the predicted models was analyzed using several tools and web servers. All models were evaluated based on their phytochemical values. Additionally, 100 ns MD simulation was carried out and the mutated models were evaluated using RMSD, RMSF, Rg, and SASA. gmxMMPBSA free energy calculation was carried out. Moreover, RMSD and free energy calculations were also compared with those obtained using online web servers. Our study demonstrates that P152S, Q296H, and R92Q play a more critical role in the structural instability of GABA-AT compared with the other mutated models: G465R, L211F, L478P, and R220K.

Virtual screening of flavonoids against Plasmodium vivax Duffy binding protein utilizing molecular docking and molecular dynamic simulation.

BACKGROUND: Plasmodium vivax (P. vivax) is one of the highly prevalent human malaria parasites. Due to the presence of extravascular reservoirs, P. vivax is extremely challenging to manage and eradicate. Traditionally, flavonoids have been widely used to combat various diseases. Recently, biflavonoids were discovered to be effective against Plasmodium falciparum. METHOD: In this study, in silico approaches were utilized to inhibit Duffy binding protein (DBP), responsible for Plasmodium invasion into red blood cells (RBC). The interaction of flavonoid molecules with the Duffy antigen receptor for chemokines (DARC) binding site of DBP was investigated using a molecular docking approach. Furthermore, molecular dynamic simulation studies were carried out to study the stability of top-docked complexes. RESULTS: The results showed the effectiveness of flavonoids, such as daidzein, genistein, kaempferol, and quercetin, in the DBP binding site. These flavonoids were found to bind in the active region of DBP. Furthermore, the stability of these four ligands was maintained throughout the 50 ns simulation, maintaining stable hydrogen bond formation with the active site residues of DBP. CONCLUSION: The present study suggests that flavonoids might be good candidates and novel agents against DBP-mediated RBC invasion of P. vivax and can be further analyzed in in vitro studies.

Investigation of Flavonoid Scaffolds as DAX1 Inhibitors against Ewing Sarcoma through Pharmacoinformatic and Dynamic Simulation Studies.

Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX1) is an orphan nuclear receptor encoded by the NR0B1 gene. The functional study showed that DAX1 is a physiologically significant target for EWS/FLI1-mediated oncogenesis, particularly Ewing Sarcoma (ES). In this study, a three-dimensional DAX1 structure was modeled by employing a homology modeling approach. Furthermore, the network analysis of genes involved in Ewing Sarcoma was also carried out to evaluate the association of DAX1 and other genes with ES. Moreover, a molecular docking study was carried out to check the binding profile of screened flavonoid compounds against DAX1. Therefore, 132 flavonoids were docked in the predicted active binding pocket of DAX1. Moreover, the pharmacogenomics analysis was performed for the top ten docked compounds to evaluate the ES-related gene clusters. As a result, the five best flavonoid-docked complexes were selected and further evaluated by Molecular Dynamics (MD) simulation studies at 100 ns. The MD simulation trajectories were evaluated by generating RMSD, hydrogen bond plot analysis, and interaction energy graphs. Our results demonstrate that flavonoids showed interactive profiles in the active region of DAX1 and can be used as potential therapeutic agents against DAX1-mediated augmentation of ES after in-vitro and in-vivo evaluations.

Computational Exploration of Licorice for Lead Compounds against Plasmodium vivax Duffy Binding Protein Utilizing Molecular Docking and Molecular Dynamic Simulation.

Plasmodium vivax (P. vivax) is one of the human's most common malaria parasites. P. vivax is exceedingly difficult to control and eliminate due to the existence of extravascular reservoirs and recurring infections from latent liver stages. Traditionally, licorice compounds have been widely investigated against viral and infectious diseases and exhibit some promising results to combat these diseases. In the present study, computational approaches are utilized to study the effect of licorice compounds against P. vivax Duffy binding protein (DBP) to inhibit the malarial invasion to human red blood cells (RBCs). The main focus is to block the DBP binding site to Duffy antigen receptor chemokines (DARC) of RBC to restrict the formation of the DBP-DARC complex. A molecular docking study was performed to analyze the interaction of licorice compounds with the DARC binding site of DBP. Furthermore, the triplicates of molecular dynamic simulation studies for 100 ns were carried out to study the stability of representative docked complexes. The leading compounds such as licochalcone A, echinatin, and licochalcone B manifest competitive results against DBP. The blockage of the active region of DBP resulting from these compounds was maintained throughout the triplicates of 100 ns molecular dynamic (MD) simulation, maintaining stable hydrogen bond formation with the active site residues of DBP. Therefore, the present study suggests that licorice compounds might be good candidates for novel agents against DBP-mediated RBC invasion of P. vivax.

Exploration of Flavonoids as Lead Compounds against Ewing Sarcoma through Molecular Docking, Pharmacogenomics Analysis, and Molecular Dynamics Simulations.

Ewing sarcoma (ES) is a highly malignant carcinoma prevalent in children and most frequent in the second decade of life. It mostly occurs due to t(11;22) (q24;q12) translocation. This translocation encodes the oncogenic fusion protein EWS/FLI (Friend leukemia integration 1 transcription factor), which acts as an aberrant transcription factor to deregulate target genes essential for cancer. Traditionally, flavonoids from plants have been investigated against viral and cancerous diseases and have shown some promising results to combat these disorders. In the current study, representative flavonoid compounds from various subclasses are selected and used to disrupt the RNA-binding motif of EWS, which is required for EWS/FLI fusion. By blocking the RNA-binding motif of EWS, it might be possible to combat ES. Therefore, molecular docking experiments validated the binding interaction patterns and structural behaviors of screened flavonoid compounds within the active region of the Ewing sarcoma protein (EWS). Furthermore, pharmacogenomics analysis was used to investigate potential drug interactions with Ewing sarcoma-associated genes. Finally, molecular dynamics simulations were used to investigate the stability of the best selected docked complexes. Taken together, daidzein, kaempferol, and genistein exhibited a result comparable to ifosfamide in the proposed in silico study and can be further analyzed as possible candidate compounds in biological in vitro studies against ES.

Synthesis of UV/blue light-emitting aluminum hydroxide with oxygen vacancy and their application to electrically driven light-emitting diodes.

Aluminum hydroxide nanoparticles, one of the essential luminescent materials for display technology, bio-imaging, and sensors due to their non-toxicity, affordable pricing, and rare-earth-free phosphors, are synthesized via a simple method at a reaction time of 10 min at a low temperature of 200 °C. By controlling the precursor's ratio of aluminum acetylacetonate to oleic acid, UV or blue light-emitting aluminum hydroxides with oxygen defects and carbonyl radicals can be synthesized. As a result, aluminum hydroxide (Al(OH)3-x ) nanoparticles overwhelmingly emit UVA light (390 nm) because of the oxygen defects in nanoparticles, and carbon-related radicals on the nanoparticles are responsible for the blue-light emission at 465 nm. Electrically driven light-emitting devices are applied using luminescent aluminum hydroxide as an emissive layer, that consists of a cost-efficient inverted bottom-emission structure as [ITO (cathode)/ZnO/emissive layers/2,2'-bis(4-(carbazol-9-yl)phenyl)-biphenyl (BCBP)/MoO3/Al (anode)]. The device with aluminum hydroxide as an emissive layer shows a maximum luminance of 215.48 cd m-2 and external quantum efficiency (EQE) of 0.12%. The new method for synthesizing UV-blue emitting aluminum hydroxides and their application to LEDs will contribute to developing the field of non-toxic optoelectronic material or UV-blue emitting devices.

Phenotypic Differences of CD103+ Tissue-Resident Memory T Cells Associated with Various Cancers.

BACKGROUND/AIMS: The presence and clinical importance of tissue-resident memory T (TRM) cells have been recently described in association with various cancer types. However, the frequency and the traditional naïve-effector-memory phenotypic characteristics of TRM cells are largely unknown. METHODS: We analyzed single-cell populations of colorectal cancer (CC, n = 18), stomach cancer (SC, n = 13), renal cell carcinoma (RCC, n = 19), and breast cancer (BC, n = 16) by dissociation of tumor tissue with collagenase/hyaluronidase. We investigated populations of naïve, effector, and memory T and TRM cells by flow cytometry. RESULTS: Among CD8- cells, CC was associated with a significantly higher proportion of CD103+ T cells than other tumor types (p < 0.001). Among CD8+ cells, CC and SC were associated with higher CD103+ T-cell proportions than RCC and BC (p < 0.001). Significantly more CD8+ than CD8- cells expressed CD103 (p < 0.001). In association with SC, RCC, and BC, CD8+ T cells had a similar T-cell phenotype composition pattern: fewer effector T cells and more memory-type T cells among CD103+ cells compared with CD103- cells (p < 0.05). Tumors with higher proportion of CD103+ cells had no specific clinicopathologic characteristics than those with lower proportion of CD103+ cells. CONCLUSION: TRM cell abundance and phenotypes varied among CC, SC, RCC, and BC. Further studies regarding the functional differences of TRM associated with various tumors are warranted.

Two putative probiotic strains improve diet-induced hypercholesterolemia through modulating intestinal cholesterol uptake and hepatic cholesterol efflux.

AIMS: Two putative probiotic strains, Lacticaseibacillus (Lc.) rhamnosus BFE5264 and Lactiplantibacillus (Lp.) plantarum NR74, have been shown to suppress cholesterol uptake and promote cholesterol efflux in Caco-2 cells. However, an in vivo beneficial effect of these strains on plasma cholesterol levels has not been verified yet; neither have the underlying mechanisms of regulating cholesterol metabolism clarified thus far. This study has focused on these two aspects. METHODS AND RESULTS: A murine model has been used, and the animals receiving a high-fat/high-cholesterol diet showed elevated plasma cholesterol levels. However, supplementation of Lc. rhamnosus BFE5264 and Lp. plantarum NR74 resulted in the down regulation of Niemann-Pick C1-like 1 (NPC1L1) in the intestine in addition to counteracting the diet-induced suppression of low-density lipoprotein receptor expression in the liver. ATP Binding Cassette Subfamily A Member 1 (ABCA1) was only significantly increased upon administration of Lc. rhamnosus BFE5264. CONCLUSIONS: The present findings demonstrate that supplementation with Lc. rhamnosus BFE5264 and Lp. plantarum NR74 may improve diet-induced hypercholesterolemia by suppression of cholesterol absorption in the small intestine and by supporting the regulation of cholesterol metabolism in the liver. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to understanding the beneficial effects of probiotics on host cholesterol metabolism and underlying mechanisms related to hypercholesterolemia.

The Association of Estrogen Receptor Activity, Interferon Signaling, and MHC Class I Expression in Breast Cancer.

PURPOSE: The expression of major histocompatibility complex class I (MHC I) has previously been reported to be negatively associated with estrogen receptor (ER) expression. Furthermore, MHC I expression, level of tumor-infiltrating lymphocytes (TILs), and expression of interferon (IFN) mediator MxA are positively associated with one another in human breast cancers. This study aimed to investigate the mechanisms of association of MHC I with ER and IFN signaling. MATERIALS AND METHODS: The human leukocyte antigen (HLA)-ABC protein expression was analyzed in breast cancer cell lines. The expressions of HLA-A and MxA mRNAs were analyzed in MCF-7 cells in Gene Expression Omnibus (GEO) data. ER and HLA-ABC expressions, Ki-67 labeling index and TIL levels in tumor tissue were also analyzed in ER+/ human epidermal growth factor receptor 2 (HER2)- breast cancer patients who randomly received either neoadjuvant chemotherapy or estrogen modulator treatment followed by resection. RESULTS: HLA-ABC protein expression was decreased after ß-estradiol treatment or hESR-GFP transfection and increased after fulvestrant or IFN-γ treatment in cell lines. In GEO data, HLA-A and MxA expression was increased after ESR1 shRNA transfection. In patients, ER Allred score was significantly lower and the HLA-ABC expression, TIL levels, and Ki-67 were significantly higher in the estrogen modulator treated group than the chemotherapy treated group. CONCLUSION: MHC I expression and TIL levels might be affected by ER pathway modulation and IFN treatment. Further studies elucidating the mechanism of MHC I regulation could suggest a way to boost TIL influx in cancer in a clinical setting.

A DNA Topoisomerase II Inhibitor Results in Ex Vivo Differentiation of THP-1 Cells and Activation of Dendritic Cells.

BACKGROUND/AIM: Effective ex vivo maturation of dendritic cells (DCs) can increase the efficiency of cancer immunotherapy. We aimed to identify novel chemicals with the potential to differentiate and activate immature DCs (iDCs) to mature DCs (mDCs). MATERIALS AND METHODS: The expression of surface markers on THP-1 monocytes treated with the screened compounds was analyzed using FACS. Subsequent DC subset analysis and secreted cytokine profiling were also performed. RESULTS: FACS analysis showed that THP-1 cells treated with amsacrine hydrochloride, a DNA topoisomerase II inhibitor, exhibited the typical phenotype of conventional DCs (cDCs). The expression of DC activation markers was also increased after amsacrine treatment. The profile of cytokines produced by THP-1 cells treated with amsacrine was similar to that of mDCs. CONCLUSION: Amsacrine has an ex vivo capability of differentiating THP-1 monocytes into cDCs. As amsacrine has been used as a stable chemotherapeutic agent in humans, it can be useful for producing mDCs for cancer immunotherapy.

Efficient and stable blue quantum dot light-emitting diode.

The visualization of accurate colour information using quantum dots has been explored for decades, and commercial products employing environmentally friendly materials are currently available as backlights1. However, next-generation electroluminescent displays based on quantum dots require the development of an efficient and stable cadmium-free blue-light-emitting device, which has remained a challenge because of the inferior photophysical properties of blue-light-emitting materials2,3. Here we present the synthesis of ZnSe-based blue-light-emitting quantum dots with a quantum yield of unity. We found that hydrofluoric acid and zinc chloride additives are effective at enhancing luminescence efficiency by eliminating stacking faults in the ZnSe crystalline structure. In addition, chloride passivation through liquid or solid ligand exchange leads to slow radiative recombination, high thermal stability and efficient charge-transport properties. We constructed double quantum dot emitting layers with gradient chloride content in a light-emitting diode to facilitate hole transport, and the resulting device showed an efficiency at the theoretical limit, high brightness and long operational lifetime. We anticipate that our efficient and stable blue quantum dot light-emitting devices can facilitate the development of electroluminescent full-colour displays using quantum dots.

T cell receptor repertoires of ex vivo-expanded tumor-infiltrating lymphocytes from breast cancer patients.

A higher level of tumor-infiltrating lymphocytes (TILs) is associated with better prognosis in breast cancer patients. Adoptive transfer of lymphocytes coupled with conventional therapies has appealed to many clinicians and investigators as an effective treatment strategy for cancer patients, which necessitates efficient activation and expansion of cytotoxic T lymphocytes precisely targeting cancer cells. To comprehensively understand composition of TILs and to provide a grounding in adoptive T cell therapy, we analyzed the T cell receptor (TCR) repertoires in ex vivo-expanded TILs from nine breast cancer patients via next-generation sequencing. For the three of them, TCR repertoires of TILs gathered after the initial culture during 2 weeks were additionally analyzed and compared to those of TILs that underwent ex vivo rapid expansion procedure (REP). Diversity of TCR repertoire was variable among the patients. V/J segment usage in the clonotypes was similar among patients, with variable distribution of read counts for each V/J segment. The top 50% of most frequently observed VJ combinations was present in > 80% of the total clonotypes. Compared with TCGA data, the samples contained a similar amount of recurrent CDR3 sequences, but clonotype expansion was variable among the samples. In terms of clinicopathologic factor, presence of in vitro reactivity among triple-negative breast cancer cases seemed to be related to lower Shannon's index, but p value was not statistically significant. In addition, the proportion of CD45RO+ cells out of CD8+ T cells were negatively correlated with Shannon's diversity index for both TCRα and TCRß chains (p = 0.010) via Spearman test. In this study, we identified a heterogeneous pattern of expanded T cell clones and stable usage of V/J segments in ex vivo-expanded TILs from breast cancer patients. Further large-scale studies are requisite to elucidate the clinical significance of TCR repertoires.

Peripheral natural killer cells and myeloid-derived suppressor cells correlate with anti-PD-1 responses in non-small cell lung cancer.

Inhibition of immune checkpoint proteins like programmed death 1 (PD-1) is a promising therapeutic approach for several cancers, including non-small cell lung cancer (NSCLC). Although PD-1 ligand (PD-L1) expression is used to predict anti-PD-1 therapy responses in NSCLC, its accuracy is relatively less. Therefore, we sought to identify a more accurate predictive blood biomarker for evaluating anti-PD-1 response. We evaluated the frequencies of T cells, B cells, natural killer (NK) cells, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), mononuclear myeloid-derived suppressor cells (M-MDSCs), and Lox-1+ PMN-MDSCs in peripheral blood samples of 62 NSCLC patients before and after nivolumab treatment. Correlation of immune-cell population frequencies with treatment response, progression-free survival, and overall survival was also determined. After the first treatment, the median NK cell percentage was significantly higher in responders than in non-responders, while the median Lox-1+ PMN-MDSC percentage showed the opposite trend. NK cell frequencies significantly increased in responders but not in non-responders. NK cell frequency inversely correlated with that of Lox-1+ PMN-MDSCs after the first treatment cycle. The NK cell-to-Lox-1+ PMN-MDSC ratio (NMR) was significantly higher in responders than in non-responders. Patients with NMRs ≥ 5.75 after the first cycle had significantly higher objective response rates and longer progression-free and overall survival than those with NMRs <5.75. NMR shows promise as an early predictor of response to further anti-PD-1 therapy.