Succinimidyl Ester Research Articles

Overcoming GABA-induced Treg suppression of immunity by ABAT to augment CD8+T cell anti-tumor immune response in liver cancer

Background: ABAT, a key enzyme in GABA catabolism, modulates anti-tumor immune activity across various cancers. However, the molecular mechanisms by which the ABAT/GABA axis exerts immune regulation in the liver cancer microenvironment remain unclear. Methods: ABAT expression in liver cancer tissues was scrutinized via the TCGA-LIHC database, and the 5-year survival rates of liver cancer patients were appraised through Kaplan-Meier survival analyses. The mRNA levels of ABAT in liver cancer cell lines were quantified by qRT-PCR. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), colony formation, and Transwell assays were employed to gauge the influence of ABAT overexpression on liver cancer cell growth, proliferation, migration, and invasion, respectively. Western blot evaluated EMT-related protein expression. ELISA quantified GABA, IL-10, TGF-β1, Granzyme B, and IFN-γ in the culture medium. Flow cytometry was used to measure the frequency of CD25+FOXP3+ cells and the expression of CD25, CD69, and PD-1 on CD8+T cells in co-culture. Carboxifluorescein diacetate succinimidyl ester (CFSE) dilution assays were performed to assess the proliferative activity of CD8+T cells. Results: ABAT was found to be underexpressed in liver cancer tissues and cells, and such underexpression is indicative of a poorer prognosis for patients, while its overexpression was shown to curb the malignancy of liver cancer cells. Upon overexpression of ABAT, there is a decrease in GABA levels in the cell supernatant, coupled with an increase in IL-10 and TGF-β1 cell number, and an upsurge in the CD25+FOXP3+ cell ratios, all of which were restored by the addition of exogenous GABA. Furthermore, the depletion of ABAT led to a reduction in the proliferation and tumor-killing ability of CD8+T cells, effects that were reversed by the application of GABAA receptor inhibitors. Conclusion: ABAT functions to inhibit Treg differentiation in liver cancer through downregulation of GABA, thus promoting the antitumor activity of CD8+T cells.

TMIC-21. PYK2/MAPK/ERK SIGNALING PATHWAY REGULATES EXTRACELLULAR VESICLE RELEASE IN GLIOBLASTOMA

Abstract Glioblastoma (GBM), the deadliest brain cancer, typically proves fatal within a year despite treatment. Tumor-associated myeloid cells (TAMs) support GBM growth, treatment resistance, and immune surveillance. Extracellular vesicles (EVs) facilitate communication between tumor and TAMs, exchanging proteins, lipids, RNA, and genetic material. As EVs can travel through the bloodstream to distant tissues, they trigger signaling pathways and metabolic changes in remote organs while also recruiting bone marrow-derived cells and modulating immune responses. Therefore, EVs serve as a crucial mechanism for the distant regulation of the tumor’s immune microenvironment. We hypothesize that Pyk2/MAPK/Erk signaling regulates EV release by modulating the actin cytoskeleton, impacting TAM infiltration and activation in tumors. This study aims to investigate the role of Pyk2/MAPK/Erk in specific EV release in GBM cells, potentially offering insights into therapeutic targets. Using confocal imaging and flow cytometric analysis of EVs, purified from medium conditioned from human primary GBM cell lines with and without Pyk2 CRISPR/Cas9 knock-out (Pyk2KO), the study identified that Pyk2KO cells do not release the population of EVs with diameter bigger then 600nm and up-regulate release of smaller EVs. Additionally, flow cytometric analysis of EVs, released from cells, fluorescently labeled with membrane dye carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), detected 14% of CFSE+ events in EV’s population, purified from Pyk2WT, vs. to 2.5% from Pyk2KO cells. Membrane labeling of cells enables the tracking of microvesicles (MV) shedding and was used to differentiate between MV and ES based on the process of their biogenesis. Considering that larger size and the transition of fluorescence from the plasma membrane are indicative of MVs, our data indicate that Pyk2 is involved in the regulation of MV release. FUNDING: PRSTRT2022 and NIH Grant 1R15CA287203

Poly (ADP-Ribose) Polymerase 1 Induces Cyclic GMP-AMP Synthase-stimulator of Interferon Genes Pathway Dysregulation to Promote Immune Escape of Colorectal Cancer Cells

Colorectal cancer (CRC) ranks third globally in cancer incidence and mortality, posing a significant human concern. Recent advancements in immunotherapy are noteworthy. This study explores immune modulation for CRC treatment. Initially targeting poly (ADP-ribose) polymerase 1 (PARP-1), a gene overexpressed in CRC tissues per The Cancer Genome Atlas, we examined its correlation with immune cell infiltration using the Tumor Immune Estimation Resource tool. Quantitative reverse transcription polymerase chain reaction assessed PARP-1 mRNA and inflammation-related gene expression in tumor tissues and cells. Assessing CD8+ T-cell proliferation and cytotoxicity towards HCT116 cells involved carboxyfluorescein diacetate succinimidyl ester and lactate dehydrogenase kits. Chemotaxis was gauged using a Transwell system in a CD8+ T-cell coculture setup, with immunofluorescence revealing cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) levels in HCT116 cells. Enzyme-linked immunosorbent assay kits measured CD8+ T-cell cytokine secretion. The findings suggested that PARP-1 was overexpressed in CRC tissues and cells and this overexpression was positively correlated with Treg cell infiltration. Overexpression of PARP-1 could significantly reduce the proportion of cGAS and STING-positive cells in HCT116 cells, dampen the proliferation, tumor-killing capacity, and chemotaxis of CD8+ T cells, and inhibit the secretion of related cytokines. The introduction of STING agonists could reverse the effects caused by overexpressed PARP-1. In vivo experiments affirmed the independent anti-tumor effects of PARP-1 inhibitors and STING agonists, synergistically inhibiting tumor growth. Silencing PARP-1 in HCT116 cells potentially boosts CD8+ T-cell activity against these cells through the cGAS-STING pathway.

CircDDX21 alleviates trophoblast dysfunction and Treg differentiation in recurrent spontaneous abortion via miR-520a-5p/ FOXP3/PD-L1 axis.

Recurrent spontaneous abortion (RSA) is a common complication during pregnancy, which is a burden to patients both physically and mentally. Circular RNAs (circRNAs) play important roles in RSA. However, the roles of circDDX21 in RSA development remain unknown. Decidual samples were harvested from healthy pregnant women and RSA patients. In HTR-8/SVneo and Bewo trophoblast cells, proliferation and migration were analyzed by cell counting kit-8 (CCK-8)/5-ethynyl-2'-deoxyuridine (EdU) staining and transwell/wound healing assays, respectively. CD4+ T cells from peripheral blood mononuclear cells of patients were incubated with trophoblast-conditioned medium. Regulatory T cells (Treg) proliferation was detected by carboxyfluorescein succinimidyl ester (CFSE) assay. Treg proportion, Treg/T helper 17 cells (Th17) ratio, and cytokines were measured using flow cytometry. The association among genes was validated using dual-luciferase assay, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP). CircDDX21 and Forkhead box P3 (FOXP3) decreased, while miR-520a-5p increased in the decidual tissues of RSA patients. CircDDX21 overexpression promoted trophoblast proliferation and migration, and facilitated CD4+ T cell differentiation into Treg. CircDDX21 targeted miR-520a-5p to elevate FOXP3. MiR-520a-5p overexpression reversed the promoted trophoblast cell function of circDDX21 overexpression in HTR-8/SVneo cells. FOXP3 overexpression reversed the repressed trophoblast cell function elicited by miR-520a-5p overexpression in HTR-8/SVneo cells. FOXP3 promoted Treg differentiation by transcriptionally upregulating programmed cell death ligand 1 (PD-L1). CircDDX21 ameliorated trophoblast dysfunction and Treg differentiation in RSA via miR-520a-5p/FOXP3/PD-L1 axis.

Chemically Self-Assembled Monolayer Semiconducting Single-Walled Carbon Nanotube-Based Biosensor Platform for Amyloid-β Detection.

This paper presents a platform for amyloid-β (Aβ) biosensors, employing nearly monolayer semiconducting single-walled carbon nanotubes (sc-SWNTs) via click reaction. A high-purity sc-SWNT ink was obtained by employing a conjugated polymer wrapping method with the addition of silica gel. Aβ detection involved monitoring the electrical resistances of the sc-SWNT layers. Electrical resistances increased rapidly corresponding to the concentration of amyloid-β 1-42 (Aβ1-42) peptides. Furthermore, we introduced Aβ peptides onto the 1-pyrenebutanoic acid succinimidyl ester (PBASE) linker, confirming that only the chemical adsorption of the peptide by the antibody-antigen reaction yielded a significant change in electrical resistance. The optimized sensor exhibited a high sensitivity of 29% for Aβ at a concentration of 10 pM. Notably, the biosensor platform featuring chemically immobilized sc-SWNT networks can be customized by incorporating various bioreceptors beyond Aβ antibodies.

Setting a Successful Sorting for Extracellular Vesicle Isolation.

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) contain a set of microRNAs with regenerative and anti-inflammatory roles. Therefore, purified MSC-EVs are envisioned as a next-generation therapeutic option for a wide array of diseases. In this protocol, we report the strategy for successfully sorting EVs from the supernatant of adipose-derived MSCs (ASCs), often used in orthopedics regenerative medicine applications. First, we described the sample preparation, focusing on EV isolation and labeling steps with carboxyfluorescein succinimidyl ester (CFSE) for fluorescence detection; subsequently, we detailed the sorting process, which constitutes the main part of the protocol. In addition to the rules defined by MISEV 2023 and MIFlowCyt EV guidelines, we applied specific experimental conditions concerning nozzle size, frequency, and sheath pressure. Morphological parameters are established using beads of diameters selected to cover the theoretical range of EV size. After ASC-EVs sorting, we performed a purity check of the sorted fraction by re-analyzing it with the sorter and verifying the EV size distribution with the nanoparticle tracking analysis technique. Due to the increasing importance of EVs, having a pure population to study and characterize is becoming crucial. Here, we demonstrate a winner strategy to set up sorting to achieve this goal.

The PreS-Based Recombinant Vaccine VVX001 Induces Hepatitis B Virus Neutralizing Antibodies in a Low-Responder to HBsAg-Based HBV Vaccines

Background: Approximately 10–20% of subjects vaccinated with HBsAg-based hepatitis B virus (HBV) vaccines are non-responders. BM32 is a recombinant grass pollen allergy vaccine containing the HBV-derived preS surface antigen as an immunological carrier protein. PreS includes the binding site of HBV to its receptor on hepatocytes. We investigated whether immunological non-responsiveness to HBV after repeated HBsAg-based vaccinations could be overcome by immunization with VVX001 (i.e., alum-adsorbed BM325, a component of BM32). Methods: A subject failing to develop protective HBV-specific immunity after HBsAg-based vaccination received five monthly injections of 20 µg VVX001. PreS-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA) and micro-array technology. Serum reactivity to subviral particles of different HBV genotypes was determined by sandwich ELISA. PreS-specific T cell responses were monitored by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and subsequent flow cytometry. HBV neutralization was assessed using cultured HBV-infected HepG2 cells. Results: Vaccination with VVX001 induced a strong and sustained preS-specific antibody response composed mainly of the IgG1 subclass. PreS-specific IgG antibodies were primarily directed to the N-terminal part of preS containing the sodium taurocholate co-transporting polypeptide (NTCP) attachment site. IgG reactivity to subviral particles as well as to the N-terminal preS-derived peptides was comparable for HBV genotypes A–H. A pronounced reactivity of CD3+CD4+ lymphocytes specific for preS after the complete injection course remaining up to one year after the last injection was found. Maximal HBV neutralization (98.4%) in vitro was achieved 1 month after the last injection, which correlated with the maximal IgG reactivity to the N-terminal part of preS. Conclusions: Our data suggest that VVX001 may be used as a preventive vaccination against HBV even in non-responders to HBsAg-based HBV vaccines.

In-utero transfer of decidualized endometrial stromal cells increases the frequency of regulatory T cells and normalizes the abortion rate in the CBA/J × DBA/2 abortion model.

Failure to adequate decidualization leads to adverse pregnancy outcomes including pregnancy loss. Although there are plenty of reports underscoring immune dysfunction as the main cause of abortion in CBA/J females mated with DBA/2 males (CBA/J × DBA/2), little is known about the potential role of impaired endometrial decidualization. Endometrial stromal cells (ESCs) from CBA/J mice were in-vitro decidualized, and the proteome profile of the secretome was investigated by membrane-based array. CBA/J mice were perfused In-utero with either decidualized ESCs (C×D/D), undecidualized ESCs (C×D/ND), or PBS (C×D/P) 12 days before mating with DBA/2 males. Control mice were not manipulated and were mated with male DBA/2 (C×D) or Balb/c (C×B) mice. On day 13.5 of pregnancy, reproductive parameters were measured. In-vivo tracking of EdU-labeled ESCs was performed using fluorescence microscopy. The frequency of regulatory T cells (Tregs) in paraaortic/renal and inguinal lymph nodes was measured by flow cytometry. The proliferation of pregnant CBA/J splenocytes in response to stimulation with DBA/2 splenocytes was assessed by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) flow cytometry. In C×D/D mice, the resorption rate was reduced to match that seen in the C×B group. Intrauterine perfused ESCs appeared in uterine stroma after 2 days, which remained there for at least 12 days. There was no difference in the number of implantation sites and embryo weight across all groups. The frequency of Tregs in the inguinal lymph nodes was similar across all groups, but it increased in the paraaortic/renal lymph nodes of C×D/D mice to the level found in C×B mice. No significant changes were observed in the proliferation of splenocytes from pregnant C×D/D compared to those of the C×D group in response to stimulation with DBA/2 splenocytes. Decidualization of ESCs was associated with a profound alteration in ESC secretome exemplified by alteration in proteins involved in extracellular matrix (ECM) remodeling, response to inflammation, senescence, and immune cell trafficking. Our results showed that the deficiency of Tregs is not the primary driver of abortion in the CBA/J × DBA/2 model and provided evidence that impaired endometrial decidualization probably triggers endometrial immune dysfunction and abortion in this model.

Approbation of a Homologous Model of the Antitumor Vaccine Based on Mature Mouse Dendritic Cells to Study the Biodistribution of the Cell Product.

Homologous animal cell product was obtained in protocol developed for female BALB/c mice. Dendritic cell (DC) migration from the injection site into the draining lymph nodes was evaluated. The number of DC labeled with carboxyfluorescein succinimidyl ester (CFSE) in draining lymph nodes increased from 5.3% (16 h) to 13.3% (48 h) (p=0.028) with a maximum at 72 h (15.4%, p=0.003). The immunophenotype of CFSE-DC detected in murine lymph nodes corresponded to the immunophenotype of mature vaccine DCs: they expressed differentiation markers CD11c, CD80, CD83, and CD86 (p>0.05 vs initial DC).

Multi-sensor integration on one microfluidics chip for single-stranded DNA detection

PurposeThis study aims to improve detection efficiency of fluorescence biosensor or a graphene field-effect transistor biosensor. Graphene field-effect transistor biosensing and fluorescent biosensing were integrated and combined with magnetic nanoparticles to construct a multi-sensor integrated microfluidic biochip for detecting single-stranded DNA. Multi-sensor integrated biochip demonstrated higher detection reliability for a single target and could simultaneously detect different targets.Design/methodology/approachIn this study, the authors integrated graphene field-effect transistor biosensing and fluorescent biosensing, combined with magnetic nanoparticles, to fabricate a multi-sensor integrated microfluidic biochip for the detection of single-stranded deoxyribonucleic acid (DNA). Graphene films synthesized through chemical vapor deposition were transferred onto a glass substrate featuring two indium tin oxide electrodes, thus establishing conductive channels for the graphene field-effect transistor. Using π-π stacking, 1-pyrenebutanoic acid succinimidyl ester was immobilized onto the graphene film to serve as a medium for anchoring the probe aptamer. The fluorophore-labeled target DNA subsequently underwent hybridization with the probe aptamer, thereby forming a fluorescence detection channel.FindingsThis paper presents a novel approach using three channels of light, electricity and magnetism for the detection of single-stranded DNA, accompanied by the design of a microfluidic detection platform integrating biosensor chips. Remarkably, the detection limit achieved is 10 pm, with an impressively low relative standard deviation of 1.007%.Originality/valueBy detecting target DNA, the photo-electro-magnetic multi-sensor graphene field-effect transistor biosensor not only enhances the reliability and efficiency of detection but also exhibits additional advantages such as compact size, affordability, portability and straightforward automation. Real-time display of detection outcomes on the host facilitates a deeper comprehension of biochemical reaction dynamics. Moreover, besides detecting the same target, the sensor can also identify diverse targets, primarily leveraging the penetrative and noninvasive nature of light.

Evaluating Nanoparticulate Vaccine Formulations for Effective Antigen Presentation and T-Cell Proliferation Using an In Vitro Overlay Assay.

Inducing T lymphocyte (T-cell) activation and proliferation with specificity against a pathogen is crucial in vaccine formulation. Assessing vaccine candidates' ability to induce T-cell proliferation helps optimize formulation for its safety, immunogenicity, and efficacy. Our in-house vaccine candidates use microparticles (MPs) and nanoparticles (NPs) to enhance antigen stability and target delivery to antigen-presenting cells (APCs), providing improved immunogenicity. Typically, vaccine formulations are screened for safety and immunostimulatory effects using in vitro methods, but extensive animal testing is often required to assess immunogenic responses. We identified the need for a rapid, intermediate screening process to select promising candidates before advancing to expensive and time-consuming in vivo evaluations. In this study, an in vitro overlay assay system was demonstrated as an effective high-throughput preclinical testing method to evaluate the immunogenic properties of early-stage vaccine formulations. The overlay assay's effectiveness in testing particulate vaccine candidates for immunogenic responses has been evaluated by optimizing the carboxyfluorescein succinimidyl ester (CFSE) T-cell proliferation assay. DCs were overlaid with T-cells, allowing vaccine-stimulated DCs to present antigens to CFSE-stained T-cells. T-cell proliferation was quantified using flow cytometry on days 0, 1, 2, 4, and 6 upon successful antigen presentation. The assay was tested with nanoparticulate vaccine formulations targeting Neisseria gonorrhoeae (CDC F62, FA19, FA1090), measles, H1N1 flu prototype, canine coronavirus, and Zika, with adjuvants including Alhydrogel® (Alum) and AddaVax™. The assay revealed robust T-cell proliferation in the vaccine treatment groups, with variations between bacterial and viral vaccine candidates. A dose-dependent study indicated immune stimulation varied with antigen dose. These findings highlight the assay's potential to differentiate and quantify effective antigen presentation, providing valuable insights for developing and optimizing vaccine formulations.

Xuebijing enhances antitumor efficacy of anti-CD19 CAR-T cells

ObjectiveTo investigate the effects and mechanisms of Xuebijing injection (XBJ) on Chimeric antigen receptor-T (CAR-T) cell function and its therapeutic potential against CAR-T therapy-associated cytokine storms (CRS). MethodsAnti-CD19 CAR-T cells were established based on FMC63 antibodies. Different doses of XBJ (1 and 10 mg/mL) were added to the culture system. Untreated anti-CD19 CAR-T cells served as negative controls. After 48-h co-culture, the effects of XBJ on CAR-T cell function were assessed. Carboxyfluorescein diacetate succinimidyl ester staining was used to assess the effect of XBJ on CAR-T cell proliferation. Flow cytometry, luciferase reporter gene assays, and real time cellular analysis were employed to evaluate the effects of XBJ on CAR-T cell cytotoxicity in vitro. RNA-sequencing was performed to analyze the effects of XBJ on CAR-T cell gene expression. Network pharmacology predicted potential XBJ therapeutic targets for CRS, which were verified in a THP-1 macrophage inflammation model. ResultsXBJ enhanced both the proliferation and tumor killing capacities of CAR-T cells. Transcriptome analysis showed that XBJ treatment affects multiple genes and pathways in CAR-T cells, with differential gene enrichment in multiple cell proliferation and growth factor pathways. Potential targets for CRS control by XBJ were predicted using network pharmacology, and the inhibitory effect of XBJ on the expression of relevant genes was verified using a macrophage model. ConclusionThe results of this study indicate that XBJ can enhance the killing effect of CAR-T cells on tumor cells and that the mechanism is related to the regulation of T cell proliferation and activation. Moreover, XBJ inhibited excessive inflammation associated with CAR-T therapy. However, the current findings remain to be further validated through in vivo experiments.

Increased circulating LOX-1+ neutrophils activate T cells and demonstrate a pro-inflammatory role in allergic rhinitis

BackgroundLow-density neutrophils are heterogeneous immune cells with immunosuppressive (such as polymorphonuclear myeloid-derived suppressor cells [PMN-MDSC]) or pro-inflammatory (such as low-density granulocytes [LDG]) properties that have been well described in multiple cancers and immune diseases. However, its role in allergic rhinitis (AR) is still unclear. MethodsIn the present study, we defined low-density neutrophils as CD14−CD11B+CD15+LOX-1+ (LOX-1+ neutrophils), and their levels in the peripheral blood (PB) were evaluated and compared between patients with AR and healthy donors using flow cytometric analysis. LOX-1 expression on polymorphonuclear neutrophils was identified. Carboxyfluorescein succinimidyl ester (CFSE)-stained CD3+ T cells were cultured alone or with LOX-1+ neutrophils, T cell proliferation was assessed using flow cytometry, and pro-inflammatory cytokines in the supernatants were detected using enzyme-linked immunosorbent assay (ELISA). Clinicopathological analyses were performed to gain a thorough understanding of LOX-1+ neutrophils. ResultsWe determined that LOX-1+ neutrophils were significantly increased in the PB of patients with AR, and LOX-1 expression in neutrophils from patients with AR was elevated. Interestingly, LOX-1+ neutrophils derived from patients with AR, unlike PMN-MDSC, promoted T cell proliferation and pro-inflammatory cytokine production. Moreover, clinicopathological analysis revealed that there was no any relation between circulating LOX-1+ neutrophil levels and the levels of IgE, age and sex. ConclusionThese findings indicate that elevated circulating LOX-1+ neutrophils play a pro-inflammatory role in AR.

Succinimide-Functionalized Reduced Graphene Oxide Nanosheets: A High-throughput Resistive Sensing Platform for Age-Related Macular Degeneration Biomarker Determination Using Human Tears.

Age-related macular degeneration (AMD) is a well-recognized affliction among the elderly, causing vision impairment ranging from blurred vision to complete blindness. This underscores the critical need for accurate, precise, and early detection methods. Herein, we developed a noninvasive, label-free electrical biosensor, constructed on an economical printed circuit board (PCB) substrate, designed specifically for the precise quantification of AMD biomarker: complement component III (C3). The hydrothermally reduced graphene oxide (rGO) was deposited between gold-interdigitated microelectrodes, forming a conductive channel. The fabricated C3 biosensor exhibits a low detection limit of 0.4342 ng/mL and an impressive sensitivity of 9.238 ((ΔR/R)/ng.mL-1)/cm2 with a regression coefficient of 0.9815 calibrated within the clinical C3 range of 10-30 ng/mL. This excellent performance is ascribed to the synergistic effects of 1-pyrenebutanoic acid succinimidyl ester (PBASE) linker and conducting properties of rGO as they generate large active sites for higher anti-C3 antibody immobilization, thereby enhancing sensitivity and specificity. Furthermore, the performance of this proposed C3 sensor chip was validated with enzyme-linked immunosorbent assay (ELISA) using five human tear samples exhibiting an outstanding correlation of a regression value of 0.9774. The unparalleled merits of this newly crafted C3 biosensor transcend those of preceding platforms, boasting superior accuracy and precision in quantifying C3 levels in human tears, accelerated operational speed with results attainable within a mere 15 min, cost-effectiveness, and excellent sensitivity.

(Invited) Developing a Graphene Field-Effect Biosensor for Genetic Biomarkers: Simulations Unveil How the Device Sensitivity Is Related to DNA-Graphene Interactions at the Nanoscale

Graphene field-effect transistor biosensors (GFETs) offer a promising platform to detect cancer DNA biomarkers at low concentration [1]. At their core, they have an electronic circuit containing a graphene sheet – a two-dimensional nanomaterial made of a single layer of carbon atoms – whose electronic properties are very sensitive to nearby electric charges. Leveraging that feature, the electrical current generated by a GFET can thus be made very sensitive to the concentration of a specific DNA sequence in a biological sample. To achieve this, the complementary DNA sequence is linked to the graphene to bind specifically the target DNA. In most GFETs, the link is done using 1-pyrenebutanoic acid succinimidyl ester (PBASE) because its pyrene group stacks on the graphene through pi-pi interactions, without disrupting the electronic properties of the graphene. However, recent experiments in the Bouilly group have shown variations in GFET sensitivity depending on the incubation time of graphene with PBASE.Here, we use a computational approach developed in our previous work [2] to investigate at the nanoscale the role of PBASE linker molecules on the sensitivity of the GFET. First, we use molecular dynamics simulations to characterize the conformational ensemble of a 10-nt single-stranded DNA sequence linked to a graphene sheet using PBASE. Second, we estimate the electrical response of the GFET from the electrostatic potential generated by these DNA conformations on the graphene, as determined using electrostatic Poisson-Boltzmann simulations. Our results show that the conformational ensemble of DNA is strongly affected by the presence of other PBASE adsorbed on the graphene, thus changing the electrostatic potential generated on the graphene. The predicted change of the electrostatic potential should impact measurably the detection signal of the GFET.We are now using these simulations to explain the experimental characterizations of a GFET designed to detect a genetic biomarker important in breast cancer profiling. Our simulations have the potential to support the development of GFETs with better sensitivity for DNA detection.[1] Béraud, A., Sauvage, M., Bazán, C. M., Tie, M., Bencherif, A., and Bouilly, D. (2021) Graphene field-effect transistors as bioanalytical sensors: design, operation and performance. Analyst. 146:403-428.[2] Côté, S., Mousseau, N., and Bouilly, D. (2022) The molecular origin of the electrostatic gating of single-molecule field-effect biosensors investigated by molecular dynamics simulations. Phys. Chem. Chem. Phys. 24:4174-4186.

Adapting the protocol for studying the functional capacity of T lymphocytes thawed from cryopreservation

Background. Immunological studies are impossible without long-term storage of cryopreserved biomaterial. There are no standard procedures for working with cryopreserved mononuclear leukocytes.The aim of the study. To optimize the protocol for culturing T lymphocytes thawed after cryopreservation by assessing their viability and proliferative capacity.Methods. Mononuclear leukocytes were isolated from the peripheral blood of relatively healthy volunteers (n = 18). Cells were subjected to controlled freezing down to –80 °C and were transferred to liquid nitrogen. First step: after thawing, the cells were stained with CFSE (carboxyfluorescein succinimidyl ester), were divided into two parts and cultured in the presence/absence of interleukin 2 (IL-2). Cell proliferation was stimulated with phytohemagglutinin (type P). Cells were incubated for 7 days. Sample analysis was performed using flow cytometry. Second stage: thawed cells were divided into three parts. Two parts were resuspended in a full growth medium with IL-2 and were placed in a thermostat (+37 °C) to “rest” for one hour or overnight. After “resting”, the cells were stained with CFSE. One third of the thawed leukocytes were stained with CFSE immediately after thawing. Cells were stimulated, cultured and analyzed the same way at both stages of the study.Results. It has been established that adding IL-2 to the culture medium contributes to a better cell survival. In the presence of IL-2, stimulated CD4+ and CD8+ T lymphocytes produced more daughter cell generations. At the end of the 7-day incubation “rested” samples had reduced leukocyte counts compared to the samples that were cultured immediately after thawing. The number of daughter cell generations formed by stimulated CD4+ and CD8+ T cells decreased when the “rest” stage was included into the study protocol.Conclusion. Adding IL-2 into culture medium can increase the viability and mitotic capacity of thawed T cells, making their state more similar to that of freshly isolated lymphocytes. Cell “rest” after thawing negatively affects the viability and proliferative activity of T lymphocytes during their weekly incubation.

Tankyrase 2 promotes lung cancer cell malignancy.

Tankyrase 2 (TNKS2) is a potential candidate molecular target for the prognosis and treatment of non-small cell lung cancer (NSCLC), but its biological functions are unclear. To investigate the biological functions of TNKS2 in NSCLC. Using a lentiviral vector, we generated H647 model cells with TNKS2 knockdown by RNA interference and A549 model cells with TNKS2 overexpression by transfection with a TNKS2 overexpressing plasmid. Increased and decreased expression levels of TNKS2 in the two cell lines were verified using real-time reverse transcriptase-polymerase chain reaction and Western blot analyses. Cell apoptosis, proliferation, and migration were determined using flow cytometry, carboxyfluorescein succinimidyl ester staining, and scratch assay, respectively. Immunofluorescence staining was conducted to examine TNKS2 and β-catenin expression levels in the two transfected cell lines and the non-transfected cells. TNKS2 mRNA and protein expression was significantly higher in the highly malignant NCI-H647 cells, while it remained at a low level in the less malignant A549 cells. Lentivirus-mediated overexpression of TNKS2 in A549 cells resulted in a 3-fold increase in gene expression and a 1.7-fold increase in protein expression (P < 0.01). Conversely, shRNA interference targeting TNKS2 Led to an 8-fold decrease in gene expression and a 3-fold decrease in protein expression (P < 0.01) in NCI-H647 cells. Furthermore, the cell apoptosis rate was significantly reduced (50%) and cell migration rate was increased (35%) in the TNKS2 overexpression group than in the control group (P < 0.05). In contrast, shTNKS2 promoted apoptosis by more than one fold and reduced migration by 60% (P < 0.05). Immunofluorescence analysis revealed enhanced nuclear localization of β-catenin fluorescence signal associated with high TNKS2 expression levels. Western blot analysis investigating TNKS2/β-catenin-related proteins indicated consistent changes between TNKS2 and β-catenin expression in lung cancer cells, whereas Axin displayed an opposite trend (P < 0.05). The obtained results revealed that TNKS2 may serve as an adverse prognostic factor and a potential therapeutic target in NSCLC.

18q Deletion Syndrome Presenting with Late-Onset Combined Immunodeficiency

Patients with chromosome 18q deletion syndrome generally experience hypogammaglobulinemia. Herein, we describe two patients with chromosome 18q deletion syndrome who presented with late-onset combined immune deficiency (LOCID), which has not been previously reported. Patient 1 was a 29-year-old male with 18q deletion syndrome, who was being managed for severe motor and intellectual disabilities at the Yamabiko Medical Welfare Center for 26 years. Although the patient had few infections, he developed Pneumocystis pneumonia at the age of 28. Patient 2, a 48-year-old female with intellectual disability and congenital malformations, was referred to Tokyo Medical and Dental University Hospital with abnormal bilateral lung shadows detected on her chest radiography. Computed tomography showed multiple lymphadenopathies and pneumonia. A lymph node biopsy of the inguinal region revealed granulomatous lymphadenitis, and a chromosomal examination revealed 18q deletion. Array-based genomic hybridization analysis revealed deletion at 18q21.32-q22.3 for patient 1 and at 18q21.33-qter for patient 2. Immune status work-up of the two patients revealed panhypogammaglobulinemia, decreased number of memory B cells and naïve CD4+ and/or CD8+ cells, reduced response on the carboxyfluorescein diacetate succinimidyl ester T-cell division test, and low levels of T-cell receptor recombination excision circles and Ig κ-deleting recombination excision circles. Consequently, both patients were diagnosed with LOCID. Although patients with 18q deletion syndrome generally experience humoral immunodeficiency, the disease can be further complicated by cell-mediated immunodeficiency, causing combined immunodeficiency. Therefore, patients with 18q deletion syndrome should be regularly tested for cellular/humoral immunocompetence.

Beyond the Bile: Exploring the Microbiome and Metabolites in Cholangiocarcinoma.

Cholangiocarcinoma (CCC) still has a high mortality rate despite improvements in diagnostic and therapeutic techniques. The role of the human microbiome in CCC is poorly understood, and a recent metagenomic analysis demonstrated a significant correlation between microbiome-associated carcinogenesis and CCC. This study aimed to investigate changes in microbiome composition associated with CCC and its metabolic signature by integrating taxonomic and functional information with metabolomics data and in vitro experimental results. From February 2019 to January 2021, this study included patients who underwent endoscopic retrograde cholangiopancreatography (ERCP), both with and without a diagnosis of CCC. Bile samples were collected via endoscopic nasobiliary drainages (ENBD) and subjected to DNA extraction, PCR amplification of the bacterial 16S rRNA gene V3-V4 region, and data analysis using QIIME2. In vitro Carboxyfluorescein succinimidyl ester (CFSE) proliferation and Annexin V/PI apoptosis assays were performed to investigate the effects of metabolites on CCC cells. A total of 24 patients were included in the study. Bile fluid analysis revealed a significantly higher abundance of Escherichia coli in the CCC group. Alpha diversity analyses exhibited significant differences between the CCC and non-CCC groups, and Nuclear Magnetic Resonance (NMR) spectroscopy metabolic profiling identified 15 metabolites with significant concentration differences; isoleucine showed the most notable difference. In vitro experiments demonstrated that isoleucine suppressed CCC cell proliferation but did not induce apoptosis. This research underlines the significance of biliary dysbiosis and specific bile metabolites, such as isoleucine, in influencing the development and progression of CCC.

Abstract PO1-25-06: Integrated analysis of single-cell RNA and VDJ-sequencing data to identify T cell marker expressed on antigen-specific T cells

Abstract Background In recent years, the advancement of adoptive cell transfer-based immunotherapies has significantly contributed to the evolution of cancer treatment. The crucial step in achieving successful treatment lies in the identification of T cells that exhibit reactivity towards tumor-specific antigens. However, the heterogeneous composition of tumor antigen-specific T cells poses a challenge as there are no standardized biomarkers available for their identification. To overcome this limitation, we investigated alternative approaches, employing single cell RNA and VDJ sequencing to identify antigen-specific T cells. Methods First, peripheral blood mononuclear cells (PBMCs) obtained from healthy human donors were stained with carboxyfluorescein succinimidyl ester (CFSE) to facilitate the identification of proliferating antigen-specific T cells. Then the PBMCs were exposed to 1 ug/ml of CMV pp65 peptide. After a 2-day incubation period with the CMV peptide, interleukin-2 (IL-2) was administered to stimulate T cell expansion. At various time points (0, 1, 2, 3, 5, and 7 days) following the CMV peptide treatment, CD8+ T cells were isolated through fluorescence-activated cell sorting (FACS) analysis. On day 7, the CD8+ T cells were further segregated into CFSE- and CFSE+ cell populations. The single-cell transcriptomes and T cell receptor (TCR) repertoire of the CD8+ T cells were subsequently examined using the Chromium Single Cell Reagent Kit. Results The TCR repertoire analysis of CD8+ CFSE- T cells revealed the dominance of 10 major clones, accounting for 93.9% of the total cells. These clones were confirmed to be CMV-specific through various in vitro assays, including NFAT-luciferase, IFN-γ ELISA, and cytotoxicity assay. In contrast, CD8+ CFSE+ T cells did not exhibit significant overlap with the major TCR clones found in CFSE- CD8+ T cells, comprising only 0.02% of the total cells. Gene set enrichment analysis demonstrated that the upregulated genes in CFSE- CD8+ T cells, when compared to CFSE+ T cells, were related to cell cycle proliferation and T cell cytotoxicity. Furthermore, these genes exhibited a matching upregulation pattern with CFSE- T cells from another batch of the same donor, as well as from two other healthy donors. In addition, there was a moderate increase in the expression of the upregulated genes in CFSE- CD8+ T cells at day 3 compared to day 0-2. In this end, we selected a set of nine genes that were consistently upregulated in CFSE- CD8+ T to serve as markers for antigen-specific T cells, employing a logistic regression model. This gene set showed high sensitivity (97.7%) and specificity (95.6%) in identifying T cell clones that respond to CMV antigen at day 3. Conclusions We have discovered potential markers for antigen-specific CD8+ T cells. These results can offer the promising opportunity for earlier isolation of T cells that exhibit reactivity towards tumor-specific antigens. This breakthrough has significant implications, as it can potentially contribute to cost savings in adoptive cell transfer-based immunotherapies. Citation Format: Byung-Kwan Jeong, Young-Ae Kim, Jungwook Park, Baknoon Ham, Jiheyong Kim, Chae Lyul Lim, Hee Jin Lee. Integrated analysis of single-cell RNA and VDJ-sequencing data to identify T cell marker expressed on antigen-specific T cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-06.