Carboxyfluorescein Succinimidyl Ester Research Articles

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.

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.

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).

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.

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.

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.

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.

Herpesvirus Entry Mediator as an Immune Checkpoint Target and a Potential Prognostic Biomarker in Myeloid and Lymphoid Leukemia.

Herpesvirus entry mediator (HVEM) is a molecular switch that can modulate immune responses against cancer. The significance of HVEM as an immune checkpoint target and a potential prognostic biomarker in malignancies is still controversial. This study aims to determine whether HVEM is an immune checkpoint target with inhibitory effects on anti-tumor CD4+ T cell responses in vitro and whether HVEM gene expression is dysregulated in patients with acute lymphocytic leukemia (ALL). HVEM gene expression in tumor cell lines and peripheral blood mononuclear cells (PBMCs) from ALL patients and healthy controls was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Tumor cells were left untreated (control) or were treated with an HVEM blocker before co-culturing with CD4+ T cells in vitro in a carboxyfluorescein succinimidyl ester (CFSE)-dependent proliferation assay. HVEM expression was upregulated in the chronic myelogenous leukemia cell line (K562) (FC = 376.3, p = 0.086) compared with normal embryonic kidney cells (Hek293). CD4+ T cell proliferation was significantly increased in the HVEM blocker-treated K562 cells (p = 0.0033). Significant HVEM differences were detected in ALL PBMCs compared with the controls, and these were associated with newly diagnosed ALL (p = 0.0011) and relapsed/refractory (p = 0.0051) B cell ALL (p = 0.0039) patients. A significant differentiation between malignant ALL and the controls was observed in a receiver operating characteristic (ROC) curve analysis with AUC = 0.78 ± 0.092 (p = 0.014). These results indicate that HVEM is an inhibitory molecule that may serve as a target for immunotherapy and a potential ALL biomarker.

Activated and Naïve Allogenic Human Placental Mesenchymal Stromal Cells Exert an Immunomodulatory Effect on Hidradenitis Suppurativa Patient Peripheral Blood Mononuclear Cells.

This pilot study aimed to evaluate the immunomodulatory effect of placental mesenchymal stem/stromal cells (MSCs) on peripheral blood mononuclear cells (PBMCs) from patients with hidradenitis suppurativa (HS). Blood samples were collected from 3 healthy and 3 patients with HS. Isolated PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin solution. The PBMCs of patients with HS were co-cultured with naïve MSCs (n-MSCs), activated with tumor necrosis factor (TNF)-α (10 ng/mL) and interferon (IFN)-γ (10 ng/mL) MSCs (a-MSCs), or adalimumab (30 μg/mL). The division index (proliferation inhibition) of PBMCs was analyzed by flow cytometry using the Proliferation Modeling tool after 5 days of coculture. The relative inflammatory gene expression dynamics and cytokine secretion were quantified in triplicate using real-time polymerase chain reaction (PCR) and Luminex assays. PBMCs from the HS control group showed statistically significant increases in interleukin (IL)-6 and IFN-γ cytokine concentrations and IL-17A gene expression when compared with healthy subjects. Statistically significant reduction of the division index was found in the a-MSCs group (P = 0.04). Also, the Luminex assay revealed significantly reduced proinflammatory cytokine concentrations of IL-9 (P = 0.022) and IL-17A (P = 0.022) in the a-MSCs group with the same trend of numerical lowering in n-MSCs group when compared to HS control. The results of real-time PCR revealed a numerical increase in the expression of the IL-1β, IL-36α, and TNF-α genes in both the a-MSCs and n-MSCs groups compared with the HS control. In conclusion, our findings suggest that MSCs can effectively curb PBMCs proliferation and suppress the production of inflammatory cytokines. Moreover, the preactivation of MSCs with IFN-γ and TNF-α before use can enhance their therapeutic effectiveness. Nevertheless, a larger sample size is imperative to validate these results.

Development and validation of a flow cytometry method to examine circulating leukocyte subpopulations in barramundi (Lates calcarifer)

Bacterial infections are a major contributor to losses in aquaculture production. Diagnosis of these bacterial diseases is based on sacrificing fish and isolating the infectious agent from the internal organs. Infections lead to an inflammatory response in the host that is characterized by changes in leukocyte profile and activity. We hypothesized that the profile of circulating peripheral blood leukocytes and markers of their activity, such as reactive oxygen species production, would change following an event of bacterial infection in fish. We therefore developed a flow cytometry-based methodology for analyzing blood leukocytes in barramundi (Lates calcarifer), then used it to compare healthy fish to those infected with a common pathogenic bacterium, Vibrio harveyi. The initial step of the analysis relied on the effective separation of erythrocytes and leukocytes, and two methods were tested: one based on hypotonic lysis of red blood cells, and the other involving stain-based differentiation using carboxyfluorescein succinimidyl ester (CFSE). Fluorescence-activated cell sorting analysis revealed significant differences in leukocyte parameters of infected and control fish using the stain-based approach vs. hypotonic lysis. Changes in blood leukocytes included an increase in the proportion of monocytes and decrease in that of lymphocytes in infected fish. Furthermore, cell size and granularity of the different leukocyte populations changed over time in infected fish as compared to controls. Monitoring peripheral blood leukocytes provides a non-lethal analysis of inflammation that allows repeated sampling of individual fish over time. This is beneficial given the high variability between individual fish. The use of CFSE-staining for leukocyte isolation proved to be advantageous over a method based on lysis of erythrocytes.

Analysis and Development of Antigen-specific T Cells Derived from Peripheral Blood Mononuclear Cells of Healthy Donors.

Adoptive cell therapy using antigen-specific T cells is a promising treatment modality for cancer patients. Various methods to isolate specific T cells and identify corresponding T cell receptor (TCR) sequences are known. This study aimed to identify antigen-specific TCR from T cells isolated using carboxyfluorescein succinimidyl ester (CFSE), which marks proliferating activated T cells. CFSE stained healthy donor peripheral blood mononuclear cells (PBMCs) were treated with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) peptides for seven days. Then, proliferating T cells with decreased CFSE staining were isolated and single cell VDJ sequencing was performed on isolated T cells to identify antigen-specific TCRs. As antigen-specific TCR candidates, ten TCR clones were selected for the CMV antigen and five for the EBV antigen. The reactivity of ten CMV TCR-transduced T cells and one EBV TCR-transduced T cell toward T2 cells pulsed with CMV or EBV peptide was confirmed via NFAT-luciferase, IFN-γ ELISA, and cytotoxicity assays. Identification of antigen-specific TCRs with CFSE staining is a valid method for the development of effective immunotherapy. The identified CMV- or EBV-specific TCRs can be used for adoptive cell therapy to treat cancer.

Investigation into Cardiac Myhc-α 334-352-Specific TCR Transgenic Mice Reveals a Role for Cytotoxic CD4 T Cells in the Development of Cardiac Autoimmunity.

Myocarditis is one of the major causes of heart failure in children and young adults and can lead to dilated cardiomyopathy. Lymphocytic myocarditis could result from autoreactive CD4+ and CD8+ T cells, but defining antigen specificity in disease pathogenesis is challenging. To address this issue, we generated T cell receptor (TCR) transgenic (Tg) C57BL/6J mice specific to cardiac myosin heavy chain (Myhc)-α 334-352 and found that Myhc-α-specific TCRs were expressed in both CD4+ and CD8+ T cells. To investigate if the phenotype is more pronounced in a myocarditis-susceptible genetic background, we backcrossed with A/J mice. At the fourth generation of backcrossing, we observed that Tg T cells from naïve mice responded to Myhc-α 334-352, as evaluated by proliferation assay and carboxyfluorescein succinimidyl ester staining. The T cell responses included significant production of mainly pro-inflammatory cytokines, namely interferon (IFN)-γ, interleukin-17, and granulocyte macrophage-colony stimulating factor. While the naïve Tg mice had isolated myocardial lesions, immunization with Myhc-α 334-352 led to mild myocarditis, suggesting that further backcrossing to increase the percentage of A/J genome close to 99.99% might show a more severe disease phenotype. Further investigations led us to note that CD4+ T cells displayed the phenotype of cytotoxic T cells (CTLs) akin to those of conventional CD8+ CTLs, as determined by the expression of CD107a, IFN-γ, granzyme B natural killer cell receptor (NKG)2A, NKG2D, cytotoxic and regulatory T cell molecules, and eomesodermin. Taken together, the transgenic system described in this report may be a helpful tool to distinguish the roles of cytotoxic cardiac antigen-specific CD4+ T cells vs. those of CD8+ T cells in the pathogenesis of myocarditis.

P1223 Lactobacillus rhamnosus GG/p40 encapsuled cationic host defense peptide play protective roles on immune-related colitis by inducing differentiation and immune suppression of regulatory T cells

Abstract Background The immune-related colitis (IRC) is the most prominent in immune-related adverse events (irAE). The association between gut microbiota and IRC development has been revealed. Our study aimed to explore the protective effect and mechanism of the probiotic Lactobacillus rhamnosus GG (LGG) and its secretory protein p40 on IRC. The self-assembled cationic host defense peptide (CHDP) hydrogel was applied to protect oral p40 protein from digestive damage and target enriched the site of colitis. Methods The IRC model mice were intervened with LGG by gavage (i.g.), p40 by enema (en.), or p40 encapsuled by CHDP (i.g.). The T cell phenotype of colonic lamina propria mononuclear cells (LPMC) was measured by flow cytometry. The indocyanine green (ICG) encapsuled by CHDP was gavage in IRC model mice, the distribution of CHDP was monitored by fluorescence signals imaging via IVIS, to assess the adhesive effect of CHDP in mice colitis. In vitro, the proportion of regulatory T cells (Treg) differentiated was measured by LGG supernatant co-cultured with CD4+ naïve T cells, and the division index of CD4+T cells was measured by LGG supernatant co-cultured with CD4+T cells and Treg (CD4+T cells: Treg=1:1) by carboxyfluorescein succinimidyl ester (CFSE) staining test. Results Compared with DSS+anti-PD-1+anti-CTLA-4 IRC model group, LGG (i.g.) group had lower weight loss, DAI score, histopathological score, relative IL-6 and TNF-α mRNA level, IL-6 and TNF-α concentration and higher colon length (P&amp;lt;0.05) (Fig.1A-L). P40 (en.) group had lower weight loss, DAI score, histopathological score and higher colon length (P&amp;lt;0.05) (Fig.1M-T). CHDP+p40 (i.g.) group had lower weight loss, DAI score, histopathological score, relative IL-6 and TNF-α mRNA level, IL-6 concentration and higher colon length (P&amp;lt;0.05) (Fig.1M-W). The percentages of CD25+FoxP3+Treg/CD4+T cells of colonic LPMC in LGG, p40 (en.), and CHDP+p40 (i.g.) group were significantly higher than IRC model group (P&amp;lt;0.05) (Fig.1X-Z, α). The average 28hr-residual rate of fluorescence imaging radiance in IRC-CHDP+ICG group was higher than both NC-CHDP+ICG and IRC-ICG group (Fig.2β-δ), and the average radiance of colon in IRC-CHDP+ICG group was also the highest (P&amp;lt;0.05) (Fig.2ε-ζ). In vitro, LGG 107 CFU/mL, 108 CFU/mL and 5 × 108 CFU/mL (P&amp;lt;0.05) could significantly promote Treg differentiation with concentration dependent manner (Fig.2η-ι), and the suppression of Treg on CD4+T cells with lower division index compared with control group (Fig.2κ). Conclusion LGG/p40 played protective role by promoting the differentiation and immune suppression of Treg cells in IRC. CHDP hydrogel could protect p40 protein from the digestive damage, and target enriched the inflamed lesion to improve the efficacy.

Peripheral blood regulatory B and T cells are decreased in patients with focal epilepsy

Patients with focal epilepsy of unknown cause (FEoUC) may display T cell infiltration in post-surgery brain specimens and increased serum levels of pro-inflammatory cytokines produced by B and T cells, indicating potential involvement of adaptive immunity. Our study aimed to investigate the peripheral blood distribution of B and T cell subgroups to find clues supporting the distinct organization of adaptive immunity in FEoUC. Twenty-two patients with FEoUC and 25 age and sex matched healthy individuals were included. Peripheral blood mononuclear cells were immunophenotyped by flow cytometry. Expression levels of anti-inflammatory cytokines and FOXP3 were measured by real-time PCR. Carboxyfluorescein succinimidyl ester (CFSE) proliferation assay was conducted using CD4+ T cells. Patients with FEoUC showed significantly decreased regulatory B (Breg), B1a, plasmablast and regulatory T (Treg) cell percentages, and increased switched memory B and Th17 cell ratios. Moreover, CD4+CD25+CD49d− Tregs of FEoUC patients displayed significantly reduced TGFB1 and FOXP3, but increased IL10 gene expression levels. CD4+ helper T cells of patients with FEoUC gave more exaggerated proliferation responses to phytohemagglutinin, anti-CD3 and anti-CD28 stimulation. Patients with FEoUC display increased effector lymphocyte, decreased regulatory lymphocyte ratios, and impaired Treg function and enhanced lymphocyte proliferation capacity. Overall, this pro-inflammatory phenotype lends support to the involvement of adaptive immunity in FEoUC.

Identification and analysis of alloreactive T lymphocytes from peripheral blood mononuclear cells.

Alloreactive T-cell responses against mismatched MHC or minor histocompatibility antigens may result in deleterious graft-versus-host disease (GVHD) and increased morbidity and mortality in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nevertheless, these T-cell responses may be directed against residual tumor cells (the graft-versus-tumor effect, GVT), thus preventing relapse of the disease. Recent findings have shown that CD45RA+ naïve T cells, but not CD45RA- memory T cells are the major contributors to GVHD, thus leading to clinical trials where CD45RA+-depleted, memory-enriched T-cell products are adoptively transferred following allo-HSCT to prevent GVHD and enhance immune reconstitution. However, residual alloreactivity may still be present in the memory T-cell compartment, thus contributing to prevent disease relapse by GVT. Here, we describe a simple cell-based protocol to identify alloreactive naïve and memory T cells by co-culturing T-cell subsets and third-party antigen-presenting cells. The responding cells are identified following dilution of carboxyfluorescein succinimidyl ester (CFSE) and upregulation of the activation marker CD25. These CFSE-diluting cells can be further phenotyped by high-dimensional flow cytometry, or purified with a cell sorter for downstream genomic and functional assays.

A Robust Capillary Electrophoresis with Laser-Induced Fluorescence Detection (CE-LIF) Method for Quantitative Compositional Analysis of Trace Amino Acids in Hypersaline Samples.

The search for life in our solar system can be enabled by the characterization of extreme environments representing conditions expected on other planets within our solar system. Molecular abundances observed in these environments help establish instrument design requirements, including limits of detection and pH/salt tolerance, and may be used for validation of proposed planetary science instrumentation. Here, we optimize capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) separations for low limit of detection quantitative compositional analysis of amino acids in hypersaline samples using carboxyfluorescein succinimidyl ester (CFSE) as the amine-reactive fluorescent probe. Two methods were optimized for identification and quantification of proteinogenic amino acids, those with and those without acidic side chains, with limits of detection as low as 250 pM, improving on previous CFSE-amino acid CE-LIF methods by an order of magnitude. The resilience of the method to samples with high concentrations of Mg2+ (>4 M diluted to >0.4 M for analysis) is demonstrated on a sample collected from the salt harvesting facility South Bay Salt Works in San Diego, CA, demonstrating the highest Mg2+ tolerance for CE-LIF methods used in amino acid analyses to date. This advancement enables the rapid and robust analysis of trace amino acids and the search for biosignatures in hypersaline systems.

Effect of streptococcal arginine deiminase on the function of CD4&lt;sup&gt;+&lt;/sup&gt; and CD8&lt;sup&gt;+&lt;/sup&gt;T lymphocytes

Arginine metabolism plays an important role in regulating the functions of immune cells in mammals. Pathogenic microbes use the mechanism of arginine depletion to suppress the immune response during infection. Arginine deiminase is a microbial arginine-hydrolyzing enzyme important for survival at low pH in the focus of infection, or in phagolysosomes, as well as under low-glucose conditions. The effect of bacterial arginine deiminase on the functions of adaptive immune cells remains poorly understood. The aim of our study was to evaluate the effect of streptococcal arginine deiminase on the proliferation and autophagy of CD4+ and CD8+ human peripheral blood T lymphocytes.The enzyme effects were studied with supernates of ultrasonic lysates from parental Streptococcus pyogenes M49-16, and its isogenic mutant with inactivated arcA gene (Streptococcus pyogenes M49-16delarcA). The study was performed with blood samples of healthy donors. The fraction of mononuclear leukocytes was isolated by centrifugation in a Ficoll density gradient. To evaluate proliferation levels, a method based on the staining of intracellular proteins with vital fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) was used. The level of autophagy was studied using the fluorescent Lysotracker Green DND-26 dye. To analyze the proliferation and autophagy of T helper cells (CD3+CD4+) and cytotoxic T lymphocytes (CD3+CD4-), the obtained cell suspensions were stained with antibodies against CD4, CD45RA, and CD3. The proportion of necrotic cells was determined by staining with a fluorescent DNA-binding DAPI dye. The normality of the distribution was assessed by Shapiro–Wilk test. The data were analyzed using Kruskal–Wallis criterion, followed by Mann–Whitney criterion for pairwise comparisons and expressed as median and interquartile ranges (Q0.25-Q0.75).When comparing the effects of supernatants from the parental and mutant streptococcal strains, which differed in expression of arginine deiminase gene, we have shown that the bacterial enzyme had no effect on the functions of inactive lymphocytes. However, streptococcal arginine deiminase completely suppressed proliferation of CD4+ and CD8+T lymphocytes stimulated with anti-CD2/CD3/CD28 antibodies. These effects were accompanied by a decrease in the autophagy levels. At the same time, arginine deiminase did not exert cytotoxic effects upon lymphocytes. L-arginine applied at the doses exceeding physiological levels caused restoration of the cellular functions. There were no differences between the studied parameters of CD4+ and CD8+T lymphocyte subsets.The obtained data show that the antiproliferative effect of arginine demimnase may be associated with ability of the enzyme to inhibit autophagy and confirm an opportunity of the bacterial enzyme to suppress host adaptive immune responses.

High BCR::ABL1 Expression Defines CD34+ Cells with Significant Alterations in Signal Transduction, Short-Proliferative Potential and Self-Renewal Ability.

Chronic Myeloid Leukemia (CML) is a clonal disorder of the hematopoietic stem cell caused by expression of the BCR::ABL1 oncoprotein. High BCR::ABL1 levels have been associated to proliferative advantage of leukemic cells, blast crisis progression and tyrosine kinase inhibitors (TKIs) inefficacy. We have previously shown that high BCR::ABL1/GUSIS transcripts measured at diagnosis are associated with inferior responses to standard dose Imatinib (IM). However, the mechanisms underlying the higher rates of disease progression and development of TKIs resistance dependent on elevated BCR::ABL1 levels remain unclear. Leukemic cells were collected from CML patients showing, at diagnosis, high or low BCR::ABL1/GUSIS. BCR::ABL1 expression levels were measured using real-time PCR. Short-term culture and long-term culture-initiating cells assays were employed to investigate the role of BCR::ABL1 gene-expression levels on proliferation, clonogenicity, signal transduction, TKIs responsiveness and self-renewal ability. Cell division was performed by carboxyfluorescein-succinimidyl ester (CFSE) assay. We found that BCR::ABL1 oncogene expression levels correlate in both PMNs and CD34+ cells. Furthermore, high oncogene levels increased both proliferation and anti-apoptotic signaling via ERK and AKT phosphorylation. Moreover, high BCR::ABL1 expression reduced the clonogenicity of leukemic CD34+ cells and increased their sensitivity to high doses IM but not to those of dasatinib. Furthermore, we observed that high BCR::ABL1 levels are associated with a reduced self-renewal of primitive leukemic cells and, also, that these cells showed comparable TKIs responsiveness with cells expressing lower BCR::ABL1 levels. Interestingly, we found a direct correlation between high BCR::ABL1 levels and reduced number of quiescent leukemic cells caused by increasing their cycling. Higher BCR::ABL1 levels improving the proliferation, anti-apoptotic signaling and reducing self-renewal properties cause an increased expansion of leukemic clone.