Jurkat Cell Line Research Articles

Bioassay-Guided Fractionation of Endophytic Fungal Extract of Fusarium solani (Saccardo) against Cancer Cell Lines and Zebrafish Embryo

Abstract: Background: This study aimed to explore the toxicity and apoptotic potential of Fusarium solani extracts on cancer cell lines and zebrafish embryo. Materials and Methods: F. solani was initially extracted with ethyl acetate and methanol. Further purification was performed using column chromatography. Each fraction was assayed for its cytotoxic potential against four cancer cell lines using MTT assay. The apoptotic potential of the HepG2 cells was investigated using Hoechst 33342 dye and caspase 3/7. The in vivo developmental toxicity of the active fractions was assessed using zebrafish embryos. Results: The ethyl acetate extract and active fractions affected the cell viability of tested cell lines on dose dependent manner. The IC50 values of F1 fraction for Jurkat, HEK 293, MDA-MB-231 and HepG2 cell lines were 48.07, 60.09, 97. 82 and 69.7 μg/ml respectively whereas, the IC50 values of F7 fraction were 58.17μg/ml and 34.29 for HepG2 and HEK 293 cell lines respectively. Morphological changes of cell such as chromatin condensation as well as the expression of caspase 3/7 were observed. In agreement with cell line data, the active fraction obtained from F. solani were toxic for zebrafish embryos at ≥100 μg/ml and lower concentration (≤60μg/ml) induced various developmental defects. Conclusion: The F. solani ethyl acetate extract and its fractions showed anticancer activity in tested cancer cell lines and induced apoptosis via caspase 3/7 pathway and showed in vivo toxicity in zebrafish embryos. Key words: Cell lines, Endophyte, Fusarium solani, Toxicity, Zebrafish.

Hybrid molecules based on fullerene C60 and 5Z,9Z-dienoic acids: Synthesis and cytotoxic activity

The present research project details synthesis of new hybrid methanofullerenes based on acetylene and triazole esters of malonic acid containing 5Z,9Z-dienoic acids and fullerene C60 under Bingel-Hirsch conditions, including study of the cytotoxic activity with respect to Jurkat, K562, U937 and HL60 tumor cell lines. Hybrid methanofullerenes containing acetylenic fragments, unlike triazole substituents, were found to exhibit higher cytotoxicity, but are characterized by lower selectivity of action in relation to healthy cells.

Hydrazines in the Synthesis of Cytotoxic N-Aryl(alkyl)-N-(hexaoxazadispiroalkyl)amines

An efficient method has been developed for the synthesis of N-aryl(alkyl)-N-(hexaoxazadispiroalkanyl)amines by the reaction of 3,6-di(spiroalkanyl)-substituted heptaoxacycloundecanes with hydrazine derivatives (3-chlorophenylhydrazine, phenylhydrazine, 2,4-dinitrophenylhydrazine, and tert-butylhydrazine) with Sm-containing catalysts. It was found that amines with macrocyclic spiro-linked azatriperoxide substituents exhibit a high cytotoxic activity against Jurkat, K562, and U937 tumor cell line and normal fibroblast cell line.

Synthesis of new alkynyl containing 9-azabicyclo[4.2.1]nonatrienes from diynes and azepines

New 7-alkynyl-9-azabicyclo[4.2.1]nona-2,4,7-triene-9carboxylates were obtained upon the Co(acac)2(dppe)/Zn/ZnI2catalyzed [6p+2p]-cycloaddition of 1,3-diynes to ethyl/phenyl azepine-1-carboxylates. The structures of the obtained azacyclic compounds were established by 1D and 2D NMR spectroscopy. The compounds prepared were tested for antitumor activities in vitro against Jurkat, K562, U937 and HL60 cancer cell lines.

Complete multi-chain adaptome amplification from whole blood and PBMC via novel dam-PCR technology

Abstract Profiling the immune repertoire through NGS allows detailed, sequence-specific insight into the adaptive immune response. One of the key challenges during immune receptor amplification is the formation of dimers, which can compete with the immune amplicons of interest during library preparation. Therefore, we developed a novel PCR technique, dimer-avoided multiplex PCR (dam-PCR), that effectively avoids primer dimer formation and allows for the all-in-one qualitative amplification of all BCR and TCR receptors in a single, quantitative reaction. Here, we present this technology’s capabilities of bias-free immune repertoire amplification, even for low frequency CDR3s. First, we demonstrate the ability of this method to accurately detect a Jurkat cell line that was spiked into pre-screened sorted donor CD4+ cells at 1%, 0.1%, 0.01%, 0.001%, and 0.0001%. RNA from each spike-in percentage was extracted and amplified in duplicate using our standard amplification protocol to assess repeatability of CDR3 discovery between replicates, as well as establish the limits of detection and quantitation. Then, we demonstrate the method’s ability to amplify a synthetic repertoire, created by spiking RNA from two individuals of known repertoire into each other at 75%, 50%, and 25%. The mixed RNA, as well as pure RNA from each individual, was then amplified to assess correlation between the predicted repertoire and amplified repertoire at each spike-in percentage. For both the Jurkat cell level spike-in test, as well as the RNA level spike-in test, the amplified repertoire was successfully predicted at an R2 value of > 0.99. This single reaction method will allow for a cost effective, all-inclusive, and quantitative analysis of immune repertoires.

A comparison of single cell capture methods and amplification strategies to pair alpha and beta

Abstract Immune repertoire amplification of T cell receptors coupled to NGS provides detailed, sequence-level insight into the immune system which can be beneficial for the development of personalized therapies. However, information related to the cognate pairing of alpha and beta chains is lost once RNA extraction is performed on the bulk sample. We have previously presented low through-put (iPair) and high throughput methods for single cell pairing of the alpha and beta receptor chain. Here, we compare different methods to amplify and pair alpha and beta from single cells, as well as track CDR3 rankings from the same sample when sorted for iPair, high throughput pairing, and bulk repertoire. Template switch oligo (TSO) with 5′ cell barcoding, 3′ cell barcoding, and iRepertoire’s multiplex primers were used independently to amplify cDNA from the same cells. The cells are sorted CD14-CD4+ with 1% Jurkat cell line spiked in. Eight thousand cells from the same population were loaded onto 2 different platforms to capture single cells: a droplet-based capture and a microwell array capture. The results demonstrate that the dominant CDR3 pairs in single cell data (regardless of capture method) match the dominant CDR3 clones in bulk repertoire. All single cell methods compared accurately called the 1% Jurkat spike-in and the top paired CDR3.

New 1Z,5Z-diene macrodiolides: Catalytic synthesis, anticancer activity, induction of mitochondrial apoptosis, and effect on the cell cycle.

An original scheme was developed for the synthesis of previously undescribed unsaturated macrodiolides containing a 1Z,5Z-diene moiety in 44-80% yields and with high stereoselectivity (>95%) based on the intermolecular esterification of α,ω-diols with α,ω-alka-nZ,(n+4)Z-dienedicarboxylic acids (1,12-dodeca-4Z,8Z-dienedicarboxylic acid, 1,14-tetradeca-5Z,9Z-dienedicarboxylic acid, 1,18-octadeca-7Z,11Z-dienedicarboxylic acid) catalyzed by hafnium triflate [Hf(OTf)4]. The unsaturated dicarboxylic acids were prepared via homo-cyclomagnesiation of tetrahydropyran ethers of O-containing 1,2-dienes with EtMgBr in the presence of Mg metal and the Cp2TiCl2 catalyst (10mol.%) and the subsequent Jones oxidation of pyran ethers formed after the acid hydrolysis of magnesacyclopentanes. The thus prepared macrodiolides exhibit high cytotoxic activity in vitro against Jurkat, K562, U937, Hek293 and HeLa cancer cell lines. It was found that induction of the programmed cell death in Jurkat cells by macrodiolides corresponds to the mitochondrial apoptosis pathway. Also, it was shown that the prepared macrodiolides efficiently suppress phosphorylation of Akt and p38 kinases and CREB transcription factor in cancer cells.

Effect of Pharmacological Inhibition of the Catalytic Activity of Phosphatases of Regenerating Liver in Early T Cell Receptor Signaling Dynamics and IL-2 Production.

We have previously shown the delivery of phosphatase of regenerating liver-1 (PRL-1) to the immunological synapse (IS) and proposed a regulatory role of the catalytic activity of PRLs (PRL-1, PRL-2 and PRL-3) in antigen-induced IL-2 production. Nonetheless, the expression in T cells and delivery to the IS of the highly homologous PRL-3, as well as the role of the catalytic activity of PRLs in antigen-induced early signaling, has not been investigated. Here, the expression of PRL-3 protein was detected in primary CD4 T cells and in the CD4 T cell line Jurkat (JK), in which an overexpressed GFP-PRL-3 fluorescent fusion protein trafficked through the endosomal recycling compartment and co-localized with PLCγ1 signaling sites at the IS. Pharmacological inhibition was used to compare the role of the catalytic activity of PRLs in antigen-induced early signaling and late IL-2 production. Although the phosphatase activity of PRLs was not critical for early signaling triggered by antigen, it seemed to regulate signaling dynamics and was necessary for proper IL-2 production. We propose that enzymatic activity of PRLs has a higher significance for cytokine production than for early signaling at the IS. However, further research will be necessary to deeply understand the regulatory role of PRLs during lymphocyte activation and effector function.

MPGES-1/PGE2 promotes the growth of T-ALL cells in vitro and in vivo by regulating the expression of MTDH via the EP3/cAMP/PKA/CREB pathway

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive haematological malignancy that is characterized by a high frequency of induction failure and by early relapse. Many studies have revealed that metadherin (MTDH) is highly expressed in a variety of malignant solid tumours and plays an important role in the occurrence and development of tumours. However, the relationship between the expression of MTDH and T-ALL has not yet been reported, and the regulatory factors of MTDH are still unknown. Our previous studies found that mPGES-1/PGE2 was important for promoting the growth of leukaemia cells. In the present study, we found that MTDH was highly expressed in primary T-ALL cells and in the Jurkat cell line. Our results showed that mPGES-1/PGE2 regulates the expression of MTDH through the EP3/cAMP/PKA-CREB pathway in T-ALL cells. Downregulation of MTDH inhibits the growth of Jurkat cells in vitro and in vivo. Our results suggest that MTDH could be a potential target for the treatment of T-ALL.

Effect of Polyvinyl Alcohol/Graphene Oxide Aerogel on the Biological Characteristics of Acute Leukemia Cell Lines

To investigate the effects of polyvinyl alcohol (PVA) + graphene oxide (GO, weight content 1 wt%) aerogel three-dimensional (3D) scaffolds culture system on the proliferation, phenotype and drug resistance of ALL cell line Jurkat and AML cell line HL-60. Jurkat cells and HL-60 cells were seeded in PVA+GO aerogel scaffolds for culture, and the structure of cells were observed by the scanning electron microscopy. Cell proliferation activity was measured by Cell Counting Kit-8 (CCK-8), cell phenotypes were analyzed by flow cytometry after fluorescent staining, then were compared with 2D cultured cells. Ara-C was used in drug resistance experiment, and CCK8 was used to detected cell proliferation activity. The proliferation activity of Jurkat cells grown in aerogel scaffolds was higher than that by 2D cultured in long-term culture. However, in HL-60 cells, the proliferation activity on 3D scaffold only at the 8th to 20th day was higher than that on the traditional 2D culture. Expression of CD4 in Jurkat cells increased after culture for 30 days, but the cell phenotypes in the 3D aerogel scaffolds were similar to 2D cultured cells. Phenotype of HL-60 cells was certainly changed after culture for 30 days, the cells can be divided into CD13+CD14-CD45+HLA-DR+,CD13-CD14--CD45+HLA-DR+ and CD13-CD14-CD45+HLA-DR- groups, and a new CD13+CD14-CD45-HLA-DR+ group of cells appeared in the cells cultured in 3D scaffolds, but not in 2D cultured cells. Drug resistance experiments showed that Jurkat cells in aerogel scaffolds have stronger drug resistance than those in 2D culture. PVA+GO (1 wt%) aerogel scaffolds can improve the proliferation and drug resistance of leukemia cells, and the phenotypes were the same as those in 2D culture, which can be used for cell amplification and biology characteristics studies and drug experiments. However, cell phenotypes should be analyzed before culture, and the effects of phenotypes changes on drug resistance should be eliminated.

The Role of the Actin‐Binding Protein Cortactin in the Activation of T Cells: Implications for T‐Cell Acute Lymphoblastic Leukemia

T‐cell acute lymphoblastic leukemia (T‐ALL) is an aggressive neoplasm due to high rates of organ infiltration and relapse, which are major obstacles for curing the disease. Infiltration of T‐ALL cells requires extravasation, a migratory process highly dependent on actin cytoskeleton dynamics. Cortactin is an actin‐binding protein that regulates actin remodeling and thus cell migration. Recently, we showed that cortactin overexpression in B‐cell ALL was correlated with infiltration, steroid treatment failure and relapse. However, it is unknown whether cortactin is expressed in leukemic T cells and what role it plays in T‐ALL biology. We analyzed cortactin expression in murine and human normal T cells and T‐ALL cell lines, and cortactin functions using different in vitro and in vivo methods such as transendothelial migration and xenotransplantation assays. We observed that the human leukemic T cell lines Jurkat, CEM and Molt‐3, as well as the leukemic murine T cell line 6645/4 expressed the 80 kDa WT isoform of cortactin. Importantly, all cell lines had significantly higher levels of cortactin than normal resting CD3+ cells. In addition, leukemic T cells transmigrated, infiltrated and colonized bone marrow organoids significantly more than normal T cells. Of note, in vivo xenotransplantation assays revealed that infiltrated leukemic T cells diminished cortactin expression suggesting that cortactin is needed for initial organ invasion, but not for subsequent functions within the organ. As leukemic cells display hyperactive TCR signaling, we examined cortactin and HS1 levels after activation of T cells. To this end, we used α‐CD3 and α‐CD28 to stimulate resting normal CD3+ cells and leukemic T cells, and found that in CD3+, Jurkat and CEM cells, cortactin was up‐regulated during activation (~1000 fold‐over resting) in a time‐dependent manner, reaching a peak after full activation at 72 hours. Cortactin upregulation was also seen after phorbol 12‐myristate 13‐acetate and ionomycin stimulation in all cells. Importantly, levels of the hematopoietic homologue of cortactin, HS1, diminished as cortactin expression increased in CD3+ cells. Taken together, our results show that cortactin is overexpressed in leukemic T cells to endow them with enhanced migratory abilities. Cortactin is upregulated in both leukemic and normal T cells upon activation suggesting that cortactin might be an important protein for effector functions of activated T cells such as cytokine secretion.

Histone Deacetylase Inhibitors Enhance CRISPR‐Cas9 Cutting Efficiency

The CRISPR/Cas9 technology is a prominent genome‐editing tool as an adaptable nuclease capable of producing a double strand break at almost any genomic loci. However, the modification of hematopoietic stem and progenitor cells (HSPC) via the homology directed repair (HDR) pathway is still inefficient. We hypothesize that histone deacetylase inhibitors (HDACi), such as Valproic Acid (VPA) and Sodium Butyrate (NaB), could enhance HDR efficiency by increasing the accessibility of the genome‐editing machinery. To address the potential utilization of HDACi therapeutically, we began by assessing the effect of VPA and NaB on cell growth and viability. No statistically significant effect on cell growth or viability was observed at concentrations as high as 50mM in a myeloid‐erythroid leukemic cell line (K562 cells). However, K562 cells only represent one lineage of the HSPC progeny. Therefore, we also treated an immortalized T lymphocyte cell line (Jurkat cells), representative of the lymphoid lineage, with either VPA or NaB. No effect on the growth pattern or viability of Jurkat cells was evident at concentrations as high as 5mM VPA and NaB. However, Jurkat cells did appear to be more susceptible to HDACi. Therefore, we ascertained that concentrations of HDACi lower than 5mM would be tolerated in HSPCs. At concentrations as low as 0.0005mM HDACi an enhancement in CRISPR cutting efficiency was evidenced as assessed in both K562 cells and Jurkat cells. This enhancement did not appear to be locus specific, in that two sets of CRISPR guide RNAs were analyzed revealing similar trends of enhancement. If the same trend follows, treatment with HDACi can be expanded to virtually any genome editing experiment utilizing the CRISPR‐Cas9 system to enhance cutting and thus therapeutic gene editing.Support or Funding InformationCSUPERBEffect of Valproic Acid and Butyrate on the Growth Curve of Hematopoietic Cell Lines A) Schematic representation of the experimental design utilized to test the effect of HDACi on the division of two hematopoietic cell lines. B) K562 cells treated with 300mM and 100mM VPA were not observed to divide. C) K562 cells treated with 500mM NaB were not observed to divide. D) Jurkat cells appear to be more susceptible to VPA. E) Jurkat cells treated with 50mM NaB proliferated at a much slower rate.Figure 1

Expansion and CD2/CD3/CD28 stimulation enhance Th2 cytokine secretion of human invariant NKT cells with retained anti-tumor cytotoxicity

Background aimsKey obstacles in human iNKT cell translational research and immunotherapy include the lack of robust protocols for dependable expansion of human iNKT cells and the paucity of data on phenotypes in post-expanded cells. MethodsWe delineate expansion methods using interleukin (IL)-2, IL-7 and allogeneic feeder cells and anti-CD2/CD3/CD28 stimulation by which to dependably augment Th2 polarization and direct cytotoxicity of human peripheral blood CD3+Vα24+Vβ11+ iNKT cells. ResultsGene and protein expression profiling demonstrated augmented Th2 cytokine secretion (IL-4, IL-5, IL-13) in expanded iNKT cells stimulated with anti-CD2/CD3/CD28 antibodies. Cytotoxic effector molecules including granzyme B were increased in expanded iNKT cells after CD2/CD3/CD28 stimulation. Direct cytotoxicity assays using unstimulated expanded iNKT cell effectors revealed α-galactosyl ceramide (α-GalCer)-dependent killing of the T-ALL cell line Jurkat. Moreover, CD2/CD3/CD28 stimulation of expanded iNKT cells augmented their (α-GalCer-independent) killing of Jurkat cells. Co-culture of expanded iNKT cells with stimulated responder cells confirmed contact-dependent inhibition of activated CD4+ and CD8+ responder T cells. DiscussionThese data establish a robust protocol to expand and novel pathways to enhance Th2 cytokine secretion and direct cytotoxicity in human iNKT cells, findings with direct implications for autoimmunity, vaccine augmentation and anti-infective immunity, cancer immunotherapy and transplantation.

In vitro mechanistic investigation of polycyclic cage-like heterocyclic hybrid possessing diverse pharmacophoric units

A novel polycyclic cage-like heterocyclic hybrid comprising several privileged structures such as pyrroloisoquinoline, 4-pyridinone and a α,β -unsaturated ketone moiety has been synthesized in good yield employing a microwave mediated [3 + 2]-cycloaddition/annulation strategy in 1-butyl-3-methylimidazolium bromide ([bmim]Br). The compound thus synthesized was analyzed for in vitro anticancer activity employing MCF-7, Jurkat, HCT 116 and NCI-H460 cell lines. The effect of polycyclic cage-like heterocyclic hybrid on cells viability was analyzed by MTT assay and its DNA damage efficiency was confirmed by several examinations like apoptosis study, cell cycle analysis, caspase-3 expression analysis and TUNEL assay. Mitochondrial membrane potential was determined by JC-1 staining. The polycyclic cage-like heterocyclic hybrid bearing diverse functionalities played a major role in DNA damage of cancerous cells and in consequence inhibited the DNA cells proliferation.

Comparative in vitro Studies of the Topoisomerase I Inhibition and Anticancer Activities of Metallated N‐Confused Porphyrins and Metallated Porphyrins

Using the original approach, a series of metallated N-confused porphyrins and metallated porphyrins have been synthesized and characterized. For all the synthesized porphyrins, in vitro studies of cytotoxic activity against K562, U937, HL-60, Jurkat, A549 and HeLa cancer cell lines, the ability to induce apoptosis and effects on the cell cycle as well as the kinetics of proliferative activity of porphyrins and their respective metallated complexes in real time have been developed. The inhibitory activity of metallated porphyrins against human topoisomerase I and the possible mechanism of inhibition have been carried out by modelling using molecular docking.

Statistical significance of cluster membership for unsupervised evaluation of cell identities.

MotivationSingle-cell RNA-sequencing (scRNA-seq) allows us to dissect transcriptional heterogeneity arising from cellular types, spatio-temporal contexts and environmental stimuli. Transcriptional heterogeneity may reflect phenotypes and molecular signatures that are often unmeasured or unknown a priori. Cell identities of samples derived from heterogeneous subpopulations are then determined by clustering of scRNA-seq data. These cell identities are used in downstream analyses. How can we examine if cell identities are accurately inferred? Unlike external measurements or labels for single cells, using clustering-based cell identities result in spurious signals and false discoveries.ResultsWe introduce non-parametric methods to evaluate cell identities by testing cluster memberships in an unsupervised manner. Diverse simulation studies demonstrate accuracy of the jackstraw test for cluster membership. We propose a posterior probability that a cell should be included in that clustering-based subpopulation. Posterior inclusion probabilities (PIPs) for cluster memberships can be used to select and visualize samples relevant to subpopulations. The proposed methods are applied on three scRNA-seq datasets. First, a mixture of Jurkat and 293T cell lines provides two distinct cellular populations. Second, Cell Hashing yields cell identities corresponding to eight donors which are independently analyzed by the jackstraw. Third, peripheral blood mononuclear cells are used to explore heterogeneous immune populations. The proposed P-values and PIPs lead to probabilistic feature selection of single cells that can be visualized using principal component analysis (PCA), t-distributed stochastic neighbor embedding (t-SNE) and others. By learning uncertainty in clustering high-dimensional data, the proposed methods enable unsupervised evaluation of cluster membership.Availability and implementation https://cran.r-project.org/package=jackstraw.Supplementary information Supplementary data are available at Bioinformatics online.

Investigating the inhibitory effect of miR-34a, miR-449a, miR-1827, and miR-106b on target genes including NOTCH1, c-Myc, and CCND1 in human T cell acute lymphoblastic leukemia clinical samples and cell line.

Objective(s):microRNAs are small non-coding molecules that regulate gene expression in various biological processes. T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy accompanied with genetic aberrations and accounts for 20% of children’s and adult’s ALL. Notch signaling pathway dysregulation occurs in 60% of T-ALL cases. In the present study, we aimed to determine the relationship between miRNAs and genes involved in Notch signaling pathway. Materials and Methods:Considering the role of the pathway and its down-stream genes in proliferation, differentiation, cell cycle, and apoptosis, NOTCH1, c-Myc, and CCND1 genes were selected as target genes. Using bioinformatics studies, miR-34a, miR-449a, miR-1827, and miR-106b were selected as miRNAs targeting the above-mentioned genes. We evaluated these genes and miRNAs in T-ALL clinical samples as well as Jurkat cell line, in which NOTCH1 is overexpressed. Results:Quantitative Real-Time PCR indicated that NOTCH1, c-Myc, and CCND1 were overexpressed in samples with decreased expression of miR-34a. In addition, we observed that samples with decreased expression of miR-449a showed increased expression of NOTCH1 and CCND1. Furthermore, we analyzed the expression of miR-1827 and miR-106b, which target c-Myc and CCND1, respectively. We found out that the expression of miR-1827, miR-106b, and their respective target genes were inversely correlated in 80% and 75% of the cases (r=0.8), respectively. Furthermore, in Jurkat cell line, the expression of target genes was increased while the candidate miRNAs except miR-34a were decreased. Conclusion:These miRNAs can be proposed as biomarkers and new therapeutic targets in T-ALL patients who have NOTCH1 overexpression.

Construction of a new anti-CD123 chimeric antigen receptor T cells and effect of anti-acute myeloid leukemia

目的构建一种新的靶向CD123的嵌合抗原受体T细胞(CAR-T细胞)，为CD123阳性白血病的免疫治疗提供实验参考。方法通过单克隆筛选技术获得能稳定分泌CD123抗体的杂交瘤细胞株6E11，将杂交瘤细胞扩增后腹腔注射至Balb/c小鼠腹腔内，收集腹水并处理、纯化得到单克隆抗体，测定抗体效价并对其特异性进行验证；RT-PCR法获得轻链和重链可变区序列，并以此为基础利用分子克隆技术构建一种新的靶向CD123嵌合抗原受体，包装病毒后感染T细胞制备CD123CAR-T细胞，通过功能实验初步探讨6E11CAR-T细胞体外抗白血病的能力。结果①获得1株稳定分泌抗人CD123抗体的杂交瘤细胞株6E11并获得其可变区序列。②6E11单克隆抗体对CD123蛋白亲和性高，解离常数(Kd值)为2.10nmol/L，特异性识别CD123阳性细胞且与CD123阴性细胞无交叉反应。 ③成功构建了CD123CAR慢病毒载体，感染T细胞后获得了靶向CD123的CAR-T细胞(6E11CAR-T)，感染效率大于60％。 ④6E11CAR-T能明显杀伤CD123阳性靶细胞MV4-11，效靶比1∶1时6E11CAR-T细胞对MV4-11细胞的杀伤比例明显高于 Vecor-T细胞[(98.60±1.20)％对(20.28±6.74)％，P<0.001]，但对CD123阴性靶细胞K562没有明显杀伤作用。 ⑤MV4-11细胞可以显著激活6E11CAR-T，但对Vecor-T细胞无明显激活作用[(26.33±3.30)％对(1.17±0.06)％，P<0.001]。⑥6E11CAR-T与MV4-11细胞共培养上清中细胞因子水平均显著高于Vecor-T组[IL-2：(92.90±1.51)pg/ml对(6.05±3.41)pg/ml，P<0.001；TNF-α：(1 407.20±91.95)pg/ml对(7.86±0.85)pg/ml，P<0.001；IFN-γ：(5 614.60±170.17)pg/ml对(8.42±2.70)pg/ml，P<0.001]但与 K562细胞共培养后，两组各细胞因子水平差异无统计学意义。 ⑦6E11CAR-T在与CD123阳性髓系白血病(AML)原代细胞共培养过程中被显著激活，且能有效杀伤原代 AML细胞。结论杂交瘤细胞株6E11能稳定分泌高效特异的抗人CD123单克隆抗体，可用于检测表达人CD123的细胞，也能应用在靶向人CD123蛋白的肿瘤免疫治疗中，以 6E11Ig可变区序列为抗原识别区的CD123CAR-T细胞，具有明确的体外抗白血病活性，为进一步的临床研究奠定了基础。

Selective anticancer activity of synthetic peptides derived from the host defence peptide tritrpticin

Antimicrobial peptides (AMPs) constitute a diverse family of peptides with the ability to protect their host against microbial infections. In addition to their ability to kill microorganisms, several AMPs also exhibit selective cytotoxicity towards cancer cells and are collectively referred to as anticancer peptides (ACPs). Here a large library of AMPs, mainly derived from the porcine cathelicidin peptide, tritrpticin (VRRFPWWWPFLRR), were assessed for their anticancer activity against the Jurkat T cell leukemia line. These anticancer potencies were compared to the cytotoxicity of the peptides towards normal cells isolated from healthy donors, namely peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs; where hemolytic activity was assessed). Among the active tritrpticin derivatives, substitution of Arg by Lys enhanced the selectivity of the peptides towards Jurkat cells when compared to PBMCs. Additionally, the side chain length of the Lys residues was also optimized to further enhance the tritrpticin ACP selectivity at low concentrations. The mechanism of action of the peptides with high selectivity involved the permeabilization of the cytoplasmic membrane of Jurkat cells, without formation of apoptotic bodies. The incorporation of non-natural Lys-based cationic amino acids could provide a new strategy to improve the selectivity of other synthetic ACPs to enhance their potential for therapeutic use against leukemia cells.

Cordycepin exhibits a suppressive effect on T cells through inhibiting TCR signaling cascade in CFA-induced inflammation mice model

Objective: Cordycepin has been shown to exhibit multiple pharmacological activities, such as antitumor, antifungi, antivirus, and immune-regulation activities, and is involved in the regulation of T cells. However, cordycepin that affects T cell activity is still not clear, and the molecular mechanism of cordycepin in regulation of TCR signaling has not yet been elucidated. In this study, the potential effect of cordycepin on T cells was observed in CFA-induced inflammation mice model, and the function of cordycepin in regulating TCR signaling cascade was investigated.Methods: A CFA-induced inflammation mice model was established for observing the effect of cordycepin on the thymus and spleen swellings, and T cell infiltration in paw tissue was detected by immunohistochemistry. The protein expression or phosphorilation was detected by western blotting, and the NFAT1 nuclear translocation was determined by fluorescence imaging. The cell proliferation, apoptosis, and IL-2 production were analyzed by CCK-8 method, flow cytometry, and ELISA.Results: In the mice model, the thymus and spleen swellings were suppressed and the T cell infiltration in paw tissue was inhibited by cordycepin at a concentration of 10 mg/kg. Although the expressions of ZAP70 and PLCγ1 were not significantly changed in the human T cell line Jurkat with cordycepin pretreatment, the CD3-antibody-induced phosphorylations of ZAP70 and PLCγ1 were markedly blocked. The protein level of p85 decreased when Jurkat cells were pretreated with cordycepin, and cordycepin blocked TCR downstream molecule Erk phosphorylation and NFAT1 nuclear translocation. Further investigation revealed that cordycepin inhibited T cell proliferation, reduced IL-2 production, and induced T cell apoptosis. Conclusions: These findings suggest that cordycepin regulates TCR signaling to inhibit excessive T cell activation in inflammation. Thus, cordycepin may be a potential therapeutic application in inflammation-associated diseases.