Activation Of Jurkat Research Articles

A convenient single-cell newly synthesized transcriptome assay reveals gene expression dynamics during early-stage T-cell activation.

Sequencing newly synthesized transcriptome alongside regular transcriptome in single cells enables the study of gene expression temporal dynamics during rapid chromatin and gene regulation processes. However, existing assays to profile single-cell newly synthesized transcriptome require in-house technical expertise to achieve high cellular throughput, limiting their widespread application. Here, we developed NOTE-seq, a method that simultaneously profiles regular and newly synthesized transcriptomes in single cells. NOTE-seq integrates 4-thiouridine labeling of newly synthesized RNA, thiol-alkylation-based chemical conversion, and a streamlined workflow on the 10X Genomics platform, offering high cellular throughput that is accessible and convenient for regular biology laboratories without specialized single-cell expertise. Using NOTE-seq, we characterized the temporal dynamics of gene expression during early-stage T-cell activation in Jurkat and naïve T cells, identified transcription factors and regulons, and discovered Fli-1 as a master transcription factor for gene regulation upon T-cell stimulation. Interestingly, chemotherapeutic topoisomerase inhibitor affects Fli-1 level in T cells, indicating potential complications for the immune system.

Abstract 4998: Pralsetinib induces opportunistic infection in RET fusion-positive NSCLC patients via inhibition of IL-2 production by blocking Jak3-Stat5 activation

Abstract Background: Oncogenic alteration in RET, representing 1-2% of non-small cell lung cancer (NSCLC), is one of the important targets. Pralsetinib is a selective RET inhibitor that targets RET fusions. We present a case series on opportunistic infections and the mechanism of immunosuppression. Methods: From October 2021 to March 2022, we administered pralsetinib to a total of 15 patients with NSCLC harboring RET fusion. We retrospectively analyzed the clinical efficacy and adverse event related to pralsetinib. To investigate the potential impact of pralsetinib on T-cells, we examined cytokine release from Jurkat T (JT) cells after pralsetinib treatment. To test if Stat5 bind to IL-2 promotor, we conducted Chromatin Immunoprecipitation (ChIP). Results: Out of a total of 18 patients with measurable disease, 14 patients (93%) achieved a partial response. With the median follow-up duration of 8.7 months, four cases of opportunistic infections were occurred. Notably, three patients (16.6%) experienced invasive pulmonary aspergillosis, and one patient (5.5%) experienced cytomegalovirus pneumonia. To mimic conditions akin to activated T cells, we treated JT cells with PMA and ionomycin, inducing IL-2 release through the binding of NFAT, AP-1, and NF-κB to the IL-2 promoter. Following approximately two weeks of pralsetinib treatment, a decrease of IL-2 releases, the inhibition of Jak3 and the reduced-expression of JunB and c-Jun was observed. Additionally, to investigate the direct correlation between Jak3 and IL-2 release, JT cells were treated with ritlecitinib, the Jak3-selective inhibitor, for approximately two weeks. Like pralsetinib, a reduction in IL-2 release was observed. Furthermore, based on the evidence that Stat5 inhibition leads to reduced IL-2 release, we also showed that the transcription factor Stat5, a direct downstream signaling component of Jak3, binds to the IL-2 promoter during the activation of Jurkat T cells. Conclusion: Pralsetinib inhibits additional IL-2 production by blocking Jak3/Stat5 activation triggered by IL-2 released during early T cell activation. As a result, the Jak3 inhibition induced by pralsetinib leads to a decrease in JunB/c-Jun expression and blocking Stat5 activation by reducing IL-2 release. Consequently, Pralsetinib-related opportunistic infections may be caused by inhibition of the JAK3 pathway and IL-2 release. Citation Format: Shinkyo Yoon, Hyun-Min Ryu, Sang-We Kim, Kang-Seo Park, Dae Ho Lee. Pralsetinib induces opportunistic infection in RET fusion-positive NSCLC patients via inhibition of IL-2 production by blocking Jak3-Stat5 activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4998.

Immunotherapy Study on Non-small-Cell Lung Cancer (NSCLC) Combined with Cytotoxic T Cells and miRNA34a.

Immunotherapy has emerged as a promising approach for cancer treatment, and the use of microRNAs (miRNAs) as therapeutic agents has gained significant attention. In this study, we investigated the effectiveness of immunotherapy utilizing miRNA34a and Jurkat T cells in inducing cell death in non-small-cell lung cancer cells, specifically A549 cells. Moreover, we explored the impact of Jurkat T cell activation and miRNA34a delivery using iron oxide nanorods (IONRs) on the killing of cancer cells. A549 cells were cocultured with both activated and inactivated Jurkat T cells, both before and after the delivery of miRNA34a. Surprisingly, our results revealed that even inactive Jurkat T cells were capable of inducing cell death in cancer cells. This unexpected observation suggested the presence of alternative mechanisms by which Jurkat T cells can exert cytotoxic effects on cancer cells. We stimulated Jurkat T cells using anti-CD3/CD28 and analyzed their efficacy in killing A549 compared to that of the inactive Jurkat T cells in conjunction with miRNA34a. Our findings indicated that the activation of Jurkat T cells significantly enhanced their cytotoxic potential against cancer cells compared to their inactive counterparts. The combined treatment of A549 cells with activated Jurkat T cells and miRNA34a demonstrated the highest level of cancer cell death, suggesting a synergistic effect between Jurkat T cell activation and miRNA therapy. Besides the apoptosis mechanism for the Jurkat T cells' cytotoxic effects on A549 cells, we furthermore investigated the ferroptosis pathway, which was found to have an impact on the cancer cell killing due to the presence of miRNA34a and IONRs as the delivery agent inside the cancer cells.

Pharmacological activities of extracts from different parts of <i>Cirsium japonicum</i> var <i>spinossimum</i>

Purpose: To evaluate the antioxidant, anti-inflammatory, inhibitory and cell proliferation activities of Cirsium japonicum (C. japonicum) var. spinossimum.
 Methods: Phytochemicals (polyphenols, flavonoids, and silymarin) in leaves, roots, and seeds of C. japonicum var. spinossimum were quantified using high-performance liquid chromatography (HPLC). Antioxidant activity was investigated on 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. Anti-inflammatory activity was investigated on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 cells. Immunostimulant and hair loss prevention was confirmed through 3'-{1-((phenylamino)-carbonyl)-3, 4-tetrazolium}bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate (XTT)-based cell proliferation assays using Jurkat cells and human follicle dermal papilla cells (HFDPC).
 Results: Extract from seeds exhibited the highest total polyphenol content (610.08 ± 32.40 mg GAE/g), as well as the highest total flavonoid content, while the total polyphenol content of leaves and root extracts were similar. The RC50 values for seed extract against ABTS and DPPH radicals were 8.20 ± 0.02 µg/mL and 40.13 ± 2.47 µg/mL, respectively. In particular, all six Silymarin derivatives were found to be highest in the seed extract. The C. japonicum var. spinossimum seed extract (CSE) showed significant (p < 0.05) nitric oxide (NO) inhibitory activity in RAW264.7 cells induced by LPS at all concentrations (12.5, 25, 50, 100 and 200 µg/mL) and exhibited significant (p < 0.05) cell proliferation activity in Jurkat and hair follicle dermal papilla cells (HFDPC) at 25 µg/mL.
 Conclusion: Extract of C. japonicum var. spinossimum seeds contain polyphenols and flavonoids and show significant NO inhibitory and cell proliferation activity in Jurkat and HFDPC. Therefore, Cirsium japonicum is a promising source of anti-inflammatory and anticancer agents.

Dual Role of Necroptosis in Cervical Cancer: Promoting Tumor Aggression and Modulating the Immune Microenvironment via the JAK2-STAT3 Pathway.

In the dynamic landscape of cervical cancer (CC) pathophysiology, this study aimed to elucidate the role of necroptosis in modulating tumor proliferation, invasion, and the immune microenvironment in CC. In this study, the impact of necroptosis on CC was evaluated through a series of bioinformatical analyses and experimental approaches. The impact of necroptosis on CC was illustrated by analyzing its effects on tumor aggression, immune responses, and the JAK2-STAT3 signaling pathway. Bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor (VEGF), was also evaluated for its potential induction of necroptosis in CC cells and its interaction with necroptosis inhibitors. Additionally, the study assessed the influence of necroptosis on the immune microenvironment, particularly in T-cell-related pathways and the expression of tumor suppressor genes in CC. Necroptosis was found to enhance VEGFA expression through the activation of the JAK2-STAT3 pathway, promoting tumor proliferative and invasive capabilities in CC. Bevacizumab induced necroptosis in CC cells, potentially leading to resistance to therapy. The combination of bevacizumab with necroptosis inhibitors attenuated VEGFA expression, suggesting a novel therapeutic strategy. Additionally, necroptosis activated T-cell-related pathways and promoted the infiltration and activation of Jurkat T cells. CD3D-a tumor suppressor gene in CC-was identified as a critical marker and its expression could be upregulated by necroptosis via the JAK2-STAT3 pathway in Jurkat T cells. Treatment of CC cells with supernatants from necroptosis-induced Jurkat cells resulted in reduced tumor cell proliferation and invasion. This study reveals a complex interaction between necroptosis, tumor progression, and the immune response in CC. The findings propose a nuanced approach to leveraging necroptosis for therapeutic interventions, highlighting the potential of combining necroptosis inhibitors with existing therapies to improve treatment outcomes in CC.

Mapping genetic effects on cell type-specific chromatin accessibility and annotating complex immune trait variants using single nucleus ATAC-seq in peripheral blood.

Gene regulation is highly cell type-specific and understanding the function of non-coding genetic variants associated with complex traits requires molecular phenotyping at cell type resolution. In this study we performed single nucleus ATAC-seq (snATAC-seq) and genotyping in peripheral blood mononuclear cells from 13 individuals. Clustering chromatin accessibility profiles of 96,002 total nuclei identified 17 immune cell types and sub-types. We mapped chromatin accessibility QTLs (caQTLs) in each immune cell type and sub-type using individuals of European ancestry which identified 6,901 caQTLs at FDR < .10 and 4,220 caQTLs at FDR < .05, including those obscured from assays of bulk tissue such as with divergent effects on different cell types. For 3,941 caQTLs we further annotated putative target genes of variant activity using single cell co-accessibility, and caQTL variants were significantly correlated with the accessibility level of linked gene promoters. We fine-mapped loci associated with 16 complex immune traits and identified immune cell caQTLs at 622 candidate causal variants, including those with cell type-specific effects. At the 6q15 locus associated with type 1 diabetes, in line with previous reports, variant rs72928038 was a naïve CD4+ T cell caQTL linked to BACH2 and we validated the allelic effects of this variant on regulatory activity in Jurkat T cells. These results highlight the utility of snATAC-seq for mapping genetic effects on accessible chromatin in specific cell types.

Novel scFv against Notch Ligand JAG1 Suitable for Development of Cell Therapies toward JAG1-Positive Tumors

The Notch signaling ligand JAG1 is overexpressed in various aggressive tumors and is associated with poor clinical prognosis. Hence, therapies targeting oncogenic JAG1 hold great potential for the treatment of certain tumors. Here, we report the identification of specific anti-JAG1 single-chain variable fragments (scFvs), one of them endowing chimeric antigen receptor (CAR) T cells with cytotoxicity against JAG1-positive cells. Anti-JAG1 scFvs were identified from human phage display libraries, reformatted into full-length monoclonal antibodies (Abs), and produced in mammalian cells. The characterization of these Abs identified two specific anti-JAG1 Abs (J1.B5 and J1.F1) with nanomolar affinities. Cloning the respective scFv sequences in our second- and third-generation CAR backbones resulted in six anti-JAG1 CAR constructs, which were screened for JAG1-mediated T-cell activation in Jurkat T cells in coculture assays with JAG1-positive cell lines. Studies in primary T cells demonstrated that one CAR harboring the J1.B5 scFv significantly induced effective T-cell activation in the presence of JAG1-positive, but not in JAG1-knockout, cancer cells, and enabled specific killing of JAG1-positive cells. Thus, this new anti-JAG1 scFv represents a promising candidate for the development of cell therapies against JAG1-positive tumors.

Abstract 3433: Novel dual antagonist anti-LILRB1/LILRB2 antibodies reprogram myeloid cells and potentiate T cells

Abstract Background: Genome-wide association studies have identified HLA-G and LILRB1/2 loci associated with cancer risk. LILRB1 and LILRB2 are distinct inhibitory immune checkpoint receptors that recognize MHC-I ligands, particularly immunosuppressive HLA-G. LILRB1-mediated inhibition can lead to impairment of T-cell cytotoxicity and preventing the efficient engulfment of tumor cells by macrophages as a “don’t eat me” signal. LILRB2 is expressed primarily by myeloid cells, especially in the tumor microenvironment, as an important immunosuppressive signal to inhibit the stimulation of an immune response. A dual antagonist antibody targeting both LILRB1 and LILRB2 could be more effective for cancer immunotherapy than targeting either LILRB1 or LILRB2 individually. Methods: Mice were immunized with recombinant D1-D2 domain of LILRB1 and LILRB2 proteins. Resulted heavy chain and light chain variable regions of IgG were PCR amplified from splenocytes and bone marrow plasma cells. PCR amplicons were built into yeast libraries. Yeast displayed scFv were screened for LILRB1 and LILRB2 binding. The dual binders were converted to effector silent human IgG1 and evaluated for ability to block HLA-G binding to LILRB1/RB2. The dual binder and dual blocker were further assessed in series of assays using human T cells and various primary myeloid cell subsets, including human monocyte-derived macrophages (hMDMs), tumor-associated macrophages (TAMs), dendritic cells (DCs) and monocytes. Results: We have discovered a panel of anti-LILRB1/2 antibodies that bind to both LILRB1 and LILRB2 with high affinity and block the interactions to HLA-G efficiently. The anti-LILRB1/2 antibodies inhibit both LILRB1 and LILRB2 signaling and modulate the function of immune cells. Anti-LILRB1/LILRB2-mediatted LILRB1 blockade: 1) enhances phagocytosis of tumor cells by macrophages; 2) releases HLA-G mediated inhibition on TCR activation in Jurkat T cells over-expressing LILRB1; 3) promotes the activation of human primary T cells stimulated with allogenic DCs. Anti-LILRB1/LILRB2-mediatted LILRB2 blockad:1) enhances TNFα expression and inhibits IL10 expression in LPS-stimulated human PBMC; 2) reprograms both hMDMs and TAMs induced by tumor cells to polarize toward a more inflammatory phenotype, including enhanced T cell responses to stimulation by DCs and macrophages, increased expression of M1 markers and secreted proinflammatory cytokine TNFα, decreased expression of M2 markers and secreted anti-inflammatory cytokine IL10. Conclusions: We have generated dual antagonist anti-LILRB1/LILRB2 antibodies. The anti-LILRB1/LILRB2 can reprogram suppressive myeloid cells to a stimulatory state, enhance phagocytotic function of myeloid cells, and promote the activation of lymphoid cells. The data support further development of anti-LILRB1/LILRB2 as an immunotherapeutic for solid tumor malignancies. Citation Format: Guangan Hu, Quanju Zhao, Lai Shi, Nan Bing, Dong Zhang. Novel dual antagonist anti-LILRB1/LILRB2 antibodies reprogram myeloid cells and potentiate T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3433.

Abstract 128: A Chimeric Antigen Receptor Targeting MDA-LDL Activates Regulatory T Cells In The Presence Of Human Atherosclerotic Plaque

Introduction: Regulatory T cells (Tregs) suppress inflammation in atherosclerosis, and therefore have the therapeutic potential to reduce the risk of MI and stroke. However, there is currently no method to generate antigen specific Tregs that target atherosclerosis. We therefore engineered Tregs that express a Chimeric Antigen Receptor (CAR) targeting malonaldehyde-modified LDL (MDA-LDL), the most common form of oxidized LDL and a key molecular component of atherosclerosis. Methods: Novel single chain variable fragments (scFv) were synthesized using sequences from antibodies targeting human MDA-LDL. Oxidized-LDL specific CARs (ox-CARs) were subsequently engineered by fusing each scFv to an IgG4 hinge, CD28 transmembrane and CD28/CD3z cytoplasmic domains. CD4 + CD25 + CD127 low/- Tregs were purified from human blood via FACS and lentivirally transduced to express the novel ox-CARs (ox-CAR-Tregs). Human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy (CEA). Autologous ox-CAR-Tregs were analyzed for activation after ex-vivo co-culture with CEA samples. Results: A rationally designed panel of 42 ox-CARs were engineered using scFv derived from 12 antibodies targeting MDA-LDL. We first assessed CAR expression and activation in Jurkat T cells to identify promising ox-CAR variants for further evaluation in human Tregs. After culture in the presence of MDA-LDL, six ox-CAR-Treg variants consistently showed significant activation, compared to controls, based on CD71 expression, cytokine expression and proliferation in the absence of CD3/28 stimulation. Human atherosclerotic samples were identified to have substantial amounts of MDA-LDL epitopes using IHC. Autologous ox-CAR-Tregs showed a dose-dependent increase in CD71 expression after ex-vivo co-culture with atherosclerotic plaque. Conclusion: An optimized CAR targeting MDA-LDL activates Tregs when cultured with human atherosclerotic plaque ex-vivo.

Identification of highly selective type II kinase inhibitors with chiral peptidomimetic tails

Identification of highly selective type II kinase inhibitors is described. Two different chiral peptidomimetic scaffolds were introduced on the tail region of non-selective type II kinase inhibitor GNF-7 to enhance the selectivity. Kinome-wide selectivity profiling analysis showed that type II kinase inhibitor 7a potently inhibited Lck kinase with great selectivity (IC50 of 23.0 nM). It was found that 7a and its derivatives possessed high selectivity for Lck over even structurally conserved all Src family kinases. We also observed that 7a inhibited Lck activation in Jurkat T cells. Moreover, 7a was found to alleviate clinical symptoms in DSS-induced colitis mice. This study provides a novel insight into the design of selective type II kinase inhibitors by adopting chiral peptidomimetic moieties on the tail region.

A design and optimization of a high throughput valve based microfluidic device for single cell compartmentalization and analysis

The need for high throughput single cell screening platforms has been increasing with advancements in genomics and proteomics to identify heterogeneity, unique cell subsets or super mutants from thousands of cells within a population. For real-time monitoring of enzyme kinetics and protein expression profiling, valve-based microfluidics or pneumatic valving that can compartmentalize single cells is advantageous by providing on-demand fluid exchange capability for several steps in assay protocol and on-chip culturing. However, this technique is throughput limited by the number of compartments in the array. Thus, one big challenge lies in increasing the number of microvalves to several thousand that can be actuated in the microfluidic device to confine enzymes and substrates in picoliter volumes. This work explores the design and optimizations done on a microfluidic platform to achieve high-throughput single cell compartmentalization as applied to single-cell enzymatic assay for protein expression quantification. Design modeling through COMSOL Multiphysics was utilized to determine the circular microvalve’s optimized parameters, which can close thousands of microchambers in an array at lower sealing pressure. Multiphysical modeling results demonstrated the relationships of geometry, valve dimensions, and sealing pressure, which were applied in the fabrication of a microfluidic device comprising of up to 5000 hydrodynamic traps and corresponding microvalves. Comparing the effects of geometry, actuation media and fabrication technique, a sealing pressure as low as 0.04 MPa was achieved. Applying to single cell enzymatic assay, variations in granzyme B activity in Jurkat and human PBMC cells were observed. Improvement in the microfluidic chip’s throughput is significant in single cell analysis applications, especially in drug discovery and treatment personalization.

VAV1-GFP Fusion Protein Generated By Intron-Based Genome Editing through CRISPR/Cas9 Leads to Spontaneous Activation in TCR Signaling

Recurrent VAV1 mutations and gene fusions (VAV1-THAP4, VAV1-MYO1F, and VAV1-S100A7) have been identified in peripheral T-cell lymphoma (PTCL) including angioimmunoblastic T-cell lymphoma (AITL) patients. A common theme of these genetic aberrations is the loss of the auto-inhibitory C-terminal SH3 domain of VAV1 resulting in aberrant activation of VAV1 independent of normal activation events. Although mouse models support VAV1 mutation/fusion as having a driver oncogenic role in the pathogenesis of PTCL, investigations on VAV1 activity in human cells were performed mainly on the Jurkat cell line with exogenous expression of VAV1 fusion proteins. This approach has un-physiological expression of VAV1 and the functions of VAV1 fusion/mutation under normal endogenous regulation need to be explored. In this study, we introduced a fusion gene, similar to what has been observed in PTCL, into the endogenous VAV1 locus. The fusion gene was under normal regulatory controls instead of being over-expressed by a viral vector, thus providing a more accurate assessment of its function in vivo. To simulate VAV1 fusion, we knocked in a green fluorescence protein (GFP) sequence followed by a simian virus 40 (SV40) poly(A) signal into intron 25 of VAV1 locus by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. A homologous DNA repair (HDR) template with tandem homologous sequences of VAV1 gene, GFP gene, and a SV40 transcription poly(A) signal was electroporated into Jurkat cells together with the Cas9/sgRNA ribonucleoprotein (RNP) complex. This knock-in disrupted the transcription of exon 26 and exon 27, resulting in an in-frame fusion protein with GFP fused to the C-terminal of SH2 of VAV1 (VAV1SH2-GFP)(Figure A). Because our guide RNA targeted the intron 25 sequence by CRISPR/Cas9 system, any possible indels caused by non-homologous end joining will occur within the intron and will not change the protein sequence of the wild type VAV1. The GFP expressing cells were isolated from the edited cell population by FACS. The fusion of GFP with VAV1 in the sorted cells was confirmed by western blot (Figure B) and these cells displayed a heterozygous VAV1SH2-GFP fusion/wild type (WT) phenotype that mimicked the VAV1 translocations observed in PTCL patients. Jurkat cells with VAV1SH2-GFP showed spontaneous activation of the T-cell receptor (TCR) signaling pathway. Analysis of signaling events downstream of VAV1 demonstrated increased phosphorylation in ITK, LCK, and subsequent ERK in Jurkat cells with VAV1SH2-GFP compared with WT Jurkat cells by western blot (Figure B). We also observed consistently elevated pERK in Jurkat cells with VAV1SH2-GFP by flow cytometry (Figure C). Notably, this elevation in pERK was spontaneous and independent of TCR stimulation with anti-CD3 antibody. VAV1SH2-GFP fusion protein also led to marked activation of downstream NFAT and NF-κB pathways as shown by Luciferase reporter assays (Figure D). Similarly, the enhanced NFAT and NF-κB pathway activation in Jurkat with the fusion protein was independent of TCR stimulation. Interestingly, with antiCD3 stimulation, Jurkat cells with VAV1SH2-GFP showed significantly lower pERK, NFAT and NF-κB activity compared to WT Jurkat cells with anti-CD3 stimulation. In conclusion, in Jurkat cells genetically edited with VAV1SH2-GFP, spontaneous activation of TCR signaling and subsequently increased NFAT and NF-κB activity were observed. Our findings further support that VAV1 C-terminal SH3 domain plays an important regulatory role in blocking VAV1 activity in the absence of proper activation. Removing C-terminal SH3 domain or replacing it with GFP or other protein relieves this inhibition, allowing spontaneous activation independent of TCR stimulation. Our study also indicates the sensitivity of the TCR signaling pathway to the level of activation and hyperactivation is detrimental. To validate the function of VAV1SH2-GFP in normal T cells, we have also successfully edited primary CD4+ T cells with VAV1SH2-GFP and the TCR signaling pathway in edited primary CD4+ T cell is currently being evaluated with and without anti-CD3/CD28 stimulation. Figure Disclosures No relevant conflicts of interest to declare.

Correction to: TRPM7 channel activity in Jurkat T lymphocytes during magnesium depletion and loading: implications for divalent metal entry and cytotoxicity

The original article was published with an error. In Figure 9b there are 3 typographical errors: instead of the Greek mu letter it shows the unconverted data.

TRPM7 channel activity in Jurkat T lymphocytes during magnesium depletion and loading: implications for divalent metal entry and cytotoxicity.

TRPM7 is a cation channel-protein kinase highly expressed in T lymphocytes and other immune cells. It has been proposed to constitute a cellular entry pathway for Mg2+ and divalent metal cations such as Ca2+, Zn2+, Cd2+, Mn2+, and Ni2+. TRPM7 channels are inhibited by cytosolic Mg2+, rendering them largely inactive in intact cells. The dependence of channel activity on extracellular Mg2+ is less well studied. Here, we measured native TRPM7 channel activity in Jurkat T cells maintained in external Mg2+ concentrations varying between 400 nM and 1.4 mM for 1-3 days, obtaining an IC50 value of 54 μM. Maintaining the cells in 400 nM or 8 μM [Mg2+]o resulted in almost complete activation of TRPM7 in intact cells, due to cytosolic Mg2+ depletion. A total of 1.4 mM [Mg2+]o was sufficient to fully eliminate the basal current. Submillimolar concentrations of amiloride prevented cellular Mg2+ depletion but not loading. We investigated whether the cytotoxicity of TRPM7 permeant metal ions Ni2+, Zn2+, Cd2+, Co2+, Mn2+, Sr2+, and Ba2+ requires TRPM7 channel activity. Mg2+ loading modestly reduced cytotoxicity of Zn2+, Co2+, Ni2+, and Mn2+ but not of Cd2+. Channel blocker NS8593 reduced Co2+ and Mn2+ but not Cd2+ or Zn2+ cytotoxicity and interfered with Mg2+ loading as evaluated by TRPM7 channel basal activity. Ba2+ and Sr2+ were neither detectably toxic nor permeant through the plasma membrane. These results indicate that in Jurkat T cells, entry of toxic divalent metal cations primarily occurs through pathways distinct from TRPM7. By contrast, we found evidence that Mg2+ entry requires TRPM7 channels.

Downregulation of PD-L1 via amide analogues of brefelamide: Alternatives to antibody-based cancer immunotherapy.

The therapeutic blockade of immune checkpoint has emerged as an effective treatment option for a broad range of tumors. However, the objective tumor response is still limited to a small number of cases and tumor types. The full utility of monoclonal antibody (mAb)-based treatment is hindered by several inherent limitations. Thus, there is an urgent requirement to explore alternative modalities targeting the same pathways. In the present study, two amide analogues of brefelamide, TPFS-201 and TPFS-202, were identified as small molecular immune checkpoint inhibitors, as they downregulated PD-L1 expression in tumor cells. PD-L1 was suppressed in cancer cells treated with TPFD compounds at both mRNA and protein levels, as detected by reverse transcription quantitative PCR and flow cytometric analysis, respectively. Reporter assays using a PD-L1 promoter luciferase construct confirmed the transcriptional inhibition of PD-L1 by TPFS compunds. TPFS compound-mediated PD-L1 downregulation in cancer cells consequently restored T cell activity, as identified by the reduction of apoptosis and an increase in interleukin-2 promoter activity in Jurkat T cells, which were co-cultured with TPFS compound-treated A549 cells. TPFS compound-mediated PD-L1 inhibition was partially abolished by the disruption of the putative transcriptional co-activator with PDZ (TAZ)/TEA domain (TEAD)-binding motif in the PD-L1 promoter. The inhibitory effect of TPFS compounds on PD-L1 was markedly inhibited in mouse cell lines, which is consistent with previous research demonstrating that PD-L1 regulation by TAZ is not conserved in mice due to distinct promoter sequences flanking the TAZ/TEAD-binding motif. Together, the data of the current study indicated the potential utility of the brefelamide amide analogues as small molecule immune checkpoint inhibitors, thereby providing therapeutic alternatives, which could be used as monotherapy or in combination with mAbs-based treatment.

SQSTM-1/p62 potentiates HTLV-1 Tax-mediated NF-\u03baB activation through its ubiquitin binding function

The NF-κB pathway is constitutively activated in adult T cell leukemia, an aggressive malignancy caused by Human T Leukemia Virus type 1 (HTLV-1). The viral oncoprotein Tax triggers this constitutive activation by interacting with the ubiquitin-rich IKK complex. We previously demonstrated that Optineurin and TAX1BP1, two members of the ubiquitin-binding, Sequestosome-1 (SQSTM-1/p62)-like selective autophagy receptor family, are involved in Tax-mediated NF-κB signaling. Here, using a proximity-dependent biotinylation approach (BioID), we identify p62 as a new candidate partner of Tax and confirm the interaction in infected T cells. We then demonstrate that p62 knock-out in MEF cells as well as p62 knock-down in HEK293T cells significantly reduces Tax-mediated NF-κB activity. We further show that although p62 knock-down does not alter NF-κB activation in Jurkat T cells nor in infected T cells, p62 does potentiate Tax-mediated NF-κB activity upon over-expression in Jurkat T cells. We next show that p62 associates with the Tax/IKK signalosome in cells, and identify the 170–206 domain of p62 as sufficient for the direct, ubiquitin-independent interaction with Tax. However, we observe that this domain is dispensable for modulating Tax activity in cells, and functional analysis of p62 mutants indicates that p62 could potentiate Tax activity in cells by facilitating the association of ubiquitin chains with the Tax/IKK signalosome. Altogether, our results identify p62 as a new ubiquitin-dependent modulator of Tax activity on NF-κB, further highlighting the importance of ubiquitin in the signaling activity of the viral Tax oncoprotein.

Effect of temperature on raft-dependent endocytic cluster formation during activation of Jurkat T cells by concanavalin A

Temperature plays an important role in the immune response. Acclimatization occurs when there are changes in ambient temperature over a long period. In this study, we used the human leukemic Jurkat T cell line to study the effect of temperature on the immune system using concanavalin A (ConA), a plant-derived immunostimulant, as a trigger for T-cell activation. Previously, we have reported endocytic intracellular cluster formation during T-cell activation by ConA with the aid of rafts and polymerization of the cytoskeleton (actin and microtubules). Here, we investigated the effect of temperature on cluster formation (with the aid of three-dimensional images of the cells) and on the stability of rafts, actin, and microtubules. When the temperature was changed between 23°C and 37°C (physiological temperature), clusters could be observed throughout this temperature range. Raft structure was stabilized at lower temperatures but destabilized at higher temperatures. Actin was stable when the temperature was higher than 27°C. When actin was depolymerized, clustering was not observed at 37°C but could be observed at 23°C. There were no changes in microtubules within this temperature range. Thus, raft clustering may be associated with raft stability at lower temperatures (<27°C) and with actin at higher temperatures (≥27°C). Hence, we provided insight into the associations between temperature, rafts, actin, and microtubules in the immune response.

Upregulation of TNF-α by Triglycerides is Mediated by MEK1 Activation in Jurkat T Cells

Upregulation of TNF-α by Triglycerides is Mediated by MEK1 Activation in Jurkat T Cells

Purification of nasulysin-1: A new toxin from Porthidium nasutum snake venom that specifically induces apoptosis in leukemia cell model through caspase-3 and apoptosis-inducing factor activation

Nasulysin-1, a new zinc-metalloproteinase from the snake venom of the hognose pit viper Porthidium nasutum, was purified to homogeneity using molecular exclusion chromatography and high performance liquid chromatography on a reverse phase column. The molecular mass of the purified enzyme was 25,900 kDa and pI 4.1, as determined by 1D and 2D polyacrylamide gel electrophoresis. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of the N-terminal amino acid sequence (1FSPRYIELVVVADHGMFKKYNSNLNTIR28; 1TASLANLEVWSK12; 1DLLPR6) of the purified nasulysin-1, shows close structural homology with other snake venom metalloproteinases isolated from different snake venoms. The purified nasulysin-1 showed specific apoptosis-inducing activity in Jurkat and K562 cells, a T-cell acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (AML) cell model, respectively, without affecting the viability of human lymphocyte cells. After 48 h treatment, nasulysin-1 (20 μg/mL) induced loss of the mitochondrial membrane potential (ΔΨm), activated the apoptosis-inducing factor (AIF), activated the protease caspase-3, and induced chromatin condensation and DNA fragmentation, all hallmarks of apoptosis. These results strongly suggest that nasulysin-1 selectively induces apoptosis to eliminate leukemia cells. Thus, these data warrant further investigation into the use of the metalloproteinase protein, nasulysin-1 as a potential therapeutic agent for treating leukemia.

Functional analysis of differences in transcriptional activity conferred by genetic variants in the 5' flanking region of the IL12RB2 gene.

Interleukin 12 receptor β chain (IL12RB2) is a crucial regulatory factor involved in cell-mediated immune responses, and genetic variants of the gene encoding IL12RB2 are associated with susceptibility to various immune-related diseases. We previously demonstrated that haplotypes with single nucleotide polymorphisms (SNPs) in the 5' flanking region of IL12RB2, including -1035A>G (rs3762315) and -1023A>G (rs3762316), affect the expression of IL12RB2, thereby altering susceptibility to leprosy and periodontal diseases. In the present study, we identified transcription factors associated with the haplotype-specific transcriptional activity of IL12RB2 in T cells and NK cells. The -1023G polymorphism was found to create a consensus binding site for the transcription factor activating protein (AP)-1, and enzyme-linked immunosorbent assay (ELISA)-based binding assays showed that these SNPs enhanced AP-1 binding to this region. In reporter assays, suppression of JunB expression using siRNA eliminated differences in the -1035G/-1023G and -1035A/-1023A regions containing IL12RB2 promoter activity in Jurkat T cells and NK3.3 cells. These results suggested that the -1035/-1023 polymorphisms created differential binding affinities for JunB that could lead to differential IL12RB2 expression. Moreover, the -1035G and -1035A alleles formed binding sites for GATA-3 and myocyte enhancer factor-2 (MEF-2), respectively. Our data indicated that in addition to JunB, the SNP at -1035/-1023 influenced GATA-3 and MEF-2 binding affinity, potentially altering IL12RB2 transcriptional activity. These findings confirm the effects of rs3762315 and rs3762316 on IL12RB2 transcription. These genetic variants may alter cellular activation of T cells and NK cells and modify cell-mediated immune responses.