Jurkat T-cell Line Research Articles

Investigating role of Mycobacterium tuberculosis secretory antigens in altering activation of T cell signaling events in Jurkat T cells.

Mycobacterium tuberculosis is able to survive and persist as an intracellular pathogen by modulating its own metabolism and host immunity. The molecules and mechanisms utilized to accomplish this modulation are not fully understood. The present study elucidates the effects of M. tuberculosis secretory antigens on T-cell-receptor (TCR)/CD28-triggered signaling in Jurkat T-cells. In the present study, intracellular calcium mobilization was investigated in CD3-activated cells in response to M. tuberculosis antigens, Ag85A, early secretory antigenic target-6 (ESAT-6), and H37Rv. The activation of mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p-38 was also analyzed in CD3- and CD28-activated cells by western blotting. Our results showed CD3-triggered modulations in free intracellular calcium levels in Jurkat T-cells in response to M. tuberculosis antigens. In addition, we also noted M. tuberculosis antigens induced downregulation in phosphorylation of ERK1/2 and p-38. Overall, our results proposed that M. tuberculosis secretory antigens, particularly ESAT-6, impede TCR/CD28-induced signaling events which could be responsible for T-cell unresponsiveness, implicated in the progression of disease. The present study demonstrated M. tuberculosis secretory antigens induced alteration of T-cell signaling pathways in unsensitized Jurkat T-cell line which might be implied in T-cell dysfunctioning during the progression of the disease.

Bacterial pore-forming toxin pneumolysin: Cell membrane structure and microvesicle shedding capacity determines differential survival of cell types.

Bacterial infectious diseases can lead to death or to serious illnesses. These outcomes are partly the consequence of pore-forming toxins, which are secreted by the pathogenic bacteria (eg, pneumolysin of Streptococcus pneumoniae). Pneumolysin binds to cholesterol within the plasma membrane of host cells and assembles to form trans-membrane pores, which can lead to Ca2+ influx and cell death. Membrane repair mechanisms exist that limit the extent of damage. Immune cells which are essential to fight bacterial infections critically rely on survival mechanisms after detrimental pneumolysin attacks. This study investigated the susceptibility of different immune cell types to pneumolysin. As a model system, we used the lymphoid T-cell line Jurkat, and myeloid cell lines U937 and THP-1. We show that Jurkat T cells are highly susceptible to pneumolysin attack. In contrast, myeloid THP-1 and U937 cells are less susceptible to pneumolysin. In line with these findings, human primary T cells are shown to be more susceptible to pneumolysin attack than monocytes. Differences in susceptibility to pneumolysin are due to (I) preferential binding of pneumolysin to Jurkat T cells and (II) cell type specific plasma membrane repair capacity. Myeloid cell survival is mostly dependent on Ca2+ induced expelling of damaged plasma membrane areas as microvesicles. Thus, in myeloid cells, first-line defense cells in bacterial infections, a potent cellular repair machinery ensures cell survival after pneumolysin attack. In lymphoid cells, which are important at later stages of infections, less efficient repair mechanisms and enhanced toxin binding renders the cells more sensitive to pneumolysin.

Analysis of caecal mucosal inflammation and immune modulation during Anoplocephala perfoliata infection of horses.

Anoplocephala perfoliata is the commonest equine tapeworm, theadult parasites are attached in groups close to the ileocaecal valve causing marked inflammatory pathology. This work aimed to characterize the nature of the in vivo mucosal immune response to Aperfoliata, and to investigate the role of Aperfoliata excretory-secretory components in modulating in vitro immune responses. Real-time PCR detected elevation of IL13 and TGFβ transcription in early-stage Aperfoliata infection. In late-stage infection, IL-13, IL4 and Ifn transcripts were reduced while the regulatory cytokines, TGFβ, IL10 and the transcription factor FOXP3 were increased in tissue close to the site of Aperfoliata attachment; indicating downregulation of T-cell responses to Aperfoliata. In vitro, Aperfoliata excretory-secretory products induced apoptosis of the Jurkat T-cell line and premature cell death of ConA stimulated equine peripheral blood leucocytes. Analysis of cytokine transcription patterns in the leucocyte cultures showed a marked inhibition of IL-1 and IL-2 suggesting that a lack of T-cell growth factor transcription underlies the mechanism of the induced equine T-cell death. These preliminary findings suggest Aperfoliata may have the ability to down-regulate host T-cell responses.

Evaluation of anticancer activity of α-defensins purified from neutrophils trapped in leukoreduction filters

AimsThe α-defensins or human neutrophil peptides (HNP 1–3) that exist in azurophilic granules are found to have anticancer activity. The pattern of disulfide bonds in α-defensins is crucial for the functional properties. Therefore, synthesis using the chemical and recombinant approaches is a challenging. A safe source for the production of α-defensins can be the use of leukoreduction filters in blood banks that contain large quantities of neutrophils and are discarded after use. The aim of this study was to purify α-defensins from neutrophils trapped in leukofilters and to investigate its anticancer activity. Materials and methodsImmunoprecipitation was performed to purify α-defensins and the presence of protein was confirmed by Western Blot. The Jurkat T-cell line was incubated with different concentrations (5, 10 and 15 μg/ml) of purified HNP1–3 for 16 h. Cell viability was measured using a WST-1 assay and apoptosis was analyzed for Annexin V/PI markers. Caspase-3/7 activity was determined using fluorescence assay. The effects of purified α-defensins were compared to commercial HNP 1–3. Key findingsPurified HNP 1–3 decreased the viability at 10 and 15 μg/ml and commercial HNP 1–3 at 15 μg/ml concentrations. Following to the purified HNP1–3 treatment, the percentage of Annexin V positive population and caspase-3 activity were significantly increased compared to control (p = 0.000 and p = 0.001, respectively) and commercial HNP1–3 (p = 0.034 and p = 0.018, respectively). SignificanceResults indicated the anticancer activity of HNP1–3 which can be used as future chemotherapeutic drugs. Furthermore, leukofilters can be considered as economic source for purifying these peptides.

Abstract A031: Engineering antigen density sensors for T-cell immunotherapy

Abstract T-cells can be redirected to kill tumor cells via synthetic T-cell receptors known as chimeric antigen receptors (CARs); this approach is becoming a highly promising therapeutic strategy for cancer treatment. Current CAR T-cells, while effective at killing cells expressing the target antigen, fail to discriminate between cells expressing high and low levels of common antigens. Potential target antigens are often also found on normal cells at lower expression levels, and CAR T-cells get activated even by low amounts of target antigen, resulting in potentially deadly toxic effects. We are approaching this problem of antigen density discrimination by using common biochemical mechanisms for cooperative recognition to design new synthetic T-cell receptors. CAR T-cells are highly sensitive to even low densities of the target antigen. Current strategies for generating CAR T-cells consist of selecting antibodies with high affinity since previous studies have shown that the CAR T-cell activity is inversely correlated with the antibody’s affinity. However, these CARs are unable to discriminate between cancer and normal cells based on antigen density. Recent studies suggest that lowering the receptor affinity on a CAR T-cell could increase its selectivity against targeT-cells with different antigen densities. Nevertheless, lowering the affinity is unlikely to provide a sharp density threshold of activation. Instead for a sharp threshold transition from off to on, one would require cooperativity and nonlinear recognition. We have designed a two-step recognition-activation circuit that involves two receptors. In this circuit an initial recognition event alters the potency of a subsequent response. We combined a weak receptor that turns on the activity of a high-affinity receptor that fully activates the cell. For this circuits we have made used of a new class of modular receptors called synthetic notch receptors (SynNotch). SynNotch receptors use antibody-based domains to recognize a target antigen, but when activated, they control transcription via a cleaved transcriptional domain. We have designed and expressed several synNotch/CAR circuits and have tested in vitro their ability to achieve antigen density sensing. These circuits use a synNotch receptor to control the expression of a CAR—all recognizing the same Her2 ligand. The constructs are introduced into T-cells via standard lentiviral infection, and sorted for receptor expression using flow cytometry (anti-myc stain). We have screened candidate receptors for expression and activation response in the human Jurkat T-cell line and human primary CD4+ and CD8+ T-cells from several donors, using standard flow-based assays (e.g. CD69 Activation, T-cell proliferation) and cytokine assays (e.g., IL-2, INF-γ). We are now taking the best circuit candidate and proceeding in mouse models. We have first characterized the growth curves of tumors made by engineered targeT-cells to measure the baseline tumor growth and then I have tested either conventional CAR T-cells or my new engineered T-cells in bilateral tumors into NSG immunocompromised mice, in which one side contains a low-density Her2 K562 tumor, and the other side will contain a higher-density Her2 K562 tumor. The engineered T-cells are injected into the tail vein, and allowed to traffic freely to either tumor. We are monitoring tumor size by caliper over the course of 4 weeks, and tracking the differential clearance of the high- and low-density tumors. We have confirmed that when using a fixed affinity standard CAR T-cell, then cells kill either high- or low-expression-level Her2 tumors with equal efficiency. I have begun parallel mouse experiments with the two receptor circuits that show improved antigen density discrimination in vitro. While still in progress, preliminary results look promising. Citation Format: Rogelio A. Hernandez-Lopez, Wendell A. Lim, Wei Yu. Engineering antigen density sensors for T-cell immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A031.

Activation of latent transforming growth factor-β1, a conserved function for pregnancy-specific beta 1-glycoproteins.

Do all 10 human pregnancy-specific beta 1-glycoproteins (PSGs) and murine PSG23 activate latent transforming growth factor-β1 (TGF-β1)? All human PSGs and murine PSG23 activated latent TGF-β1. Two of the 10 members of the PSG1 family, PSG1 and PSG9, were previously shown to activate the soluble small latent complex of TGF-β1, a cytokine with potent immune suppressive functions. Recombinant PSGs were generated and tested for their ability to activate the small latent complex of TGF-β1 in a cell-free ELISA-based assay and in a bioassay. In addition, we tested the ability of PSG1 and PSG4 to activate latent TGF-β bound to the extracellular matrix (ECM) or on the membranes of the Jurkat human T-cell line. Recombinant PSGs were generated by transient transfection and purified with a His-Trap column followed by gel filtration chromatography. The purified PSGs were compared to vehicle (PBS) used as control for their ability to activate the small latent complex of TGF-β1. The concentration of active TGF-β was measured in an ELISA using the TGF-β receptor II as capture and a bioassay using transformed mink epithelial cells that express luciferase in response to active TGF-β. The specificity of the signal was confirmed using a TGF-β receptor inhibitor. We also measured the binding kinetics of some human PSGs for the latent-associated peptide (LAP) of TGF-β using surface plasmon resonance and determined whether PSG1 and PSG4 could activate the large latent complex of TGF-β1 bound to the ECM and latent TGF-β1 bound to the cell membrane. All experiments were performed in triplicate wells and repeated three times. All human PSGs activated the small latent complex of TGF-β1 (P < 0.05 vs. control) and showed similar affinities (KD) for LAP. Despite the lack of sequence conservation with its human counterparts, the ability to activate latent TGF-β1 was shared by a member of the murine PSG family. We found that PSG1 and PSG4 activated the latent TGF-β stored in the ECM (P < 0.01) but did not activate latent TGF-β1 bound to glycoprotein A repetitions predominant (GARP) on the surface of Jurkat T cells. The affinity of the interaction of LAP and PSGs was calculated using recombinant proteins, which may differ from the native proteins in their post-translational modifications. We also utilized a truncated form of murine PSG23 rather than the full-length protein. For the studies testing the ability of PSGs to activate membrane-bound TGF-β1, we utilized the T-cell line Jurkat and Jurkat cells expressing GARP rather than primary T regulatory cells. All the studies were performed in vitro. Here, we show that all human PSGs activate TGF-β1 and that this function is conserved in at least one member of the rodent PSG family. In vivo PSGs could potentially increase the availability of active TGF-β1 from the soluble and matrix-bound latent forms of the cytokine contributing to the establishment of a tolerogenic environment during pregnancy. None. The research was supported by a grant from the Collaborative Health Initiative Research Program (CHIRP). No conflicts of interests are declared by the authors.

Non-Functionalized Ultrasmall Silica Nanoparticles Directly and Size-Selectively Activate T Cells.

Sub-micron-sized silica nanoparticles, even as small as 10-20 nm in diameter, are well-known for their activation of mononuclear phagocytes. In contrast, the cellular impact of those <10 nm [ i.e., ultrasmall silica nanoparticles (USSN)] is not well-established for any cell type despite anticipated human exposure. Here, we synthesized discrete populations of USSN with volume median diameters between 1.8 to 16 nm and investigated their impact on the mixed cell population of human primary peripheral mononuclear cells. USSN 1.8-7.6 nm in diameter, optimally 3.6-5.1 nm in diameter, induced dose-dependent CD4 and CD8 T-cell activation in terms of cell surface CD25 and CD69 up-regulation at concentrations above 150 μM Sitotal (∼500 nM particles). Induced activation with only ∼2.4 μM particles was (a) equivalent to that observed with typical positive control levels of Staphylococcal enterotoxin B (SEB) and (b) evident in antigen presenting cell-deplete cultures as well as in a pure T-cell line (Jurkat) culture. In the primary mixed-cell population, USSN induced IFN-γ secretion but failed to induce T-cell proliferation or the secretion of IL-2, IL-10, or IL-4. Collectively, these data indicate that USSN initiate activation, with Th1 polarization, of T cells via direct particle-cell interaction. Finally, similarly sized iron hydroxide particles did not induce the expression of T-cell activation markers, indicating some selectivity of the ultrasmall particle type. Given that humans may be exposed to ultrasmall particles and that these materials have emerging bioclinical applications, their off-target immunomodulatory effects via direct T-cell activation should be carefully considered.

Abstract A74: T cell activation standardization for therapeutic assay development

Abstract Introduction: Several cancer immunotherapy strategies rely on T-cell activation (1). While T-cell activation mechanisms are well established, there is a dearth of quantified standardization of these activation pathways. Standardization with quantified end-points [induced by known activators and pathways] allows the development of useful assays to evaluate emerging immunotherapies. Our initial work has focused on T-cell activation. Materials and Methods: Activation of the Jurkat T-cell line was induced by the combined action of ionomycin and PMA (phorbol 12-myristate 13-acetate) (2). Jurkat cells were cultured in 24 well plates in RPMI 1640 media (with 10% FBS and 1% Penicillin/Streptomycin) at a density of 106 cells/ml. We looked at a wide concentration range for both activators (500 - 1500 ng/ml for ionomycin and 10 - 100 ng/ml for PMA), in order to identify their most synergistic combination for T-cell activation as quantified by biomarker expression (i.e. IL-2 ELISA). The negative controls were 1% DMSO (used for solubilizing the activators), and PBS (phosphate buffered saline). For a specific combination of 1000 ng/ml ionomycin and 100 ng/ml PMA, the designed time points were 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. The cells and supernatant were preserved at the end of these time-points. The viability of the cells was tested using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay and the IL-2 production was detected using ELISA (Enzyme-Linked ImmunoSorbent Assay). Results: In addition to identifying the optimal activating concentrations of ionomycin and PMA, the obtained results characterized the time-course of maximal Jurkat cell activation over a 24 h period. During this 24 h period, the IL-2 levels and the cell proliferation rates progressively increased before starting to plateau. The production of IL-2 started increasing at 4 h from 157.6 pg/ml and continued to go up until 24 h to 687.6 pg/ml. Cell proliferation decreased a little during the first 2 h and increased from 57% to 114% (of the PBS control) between 8 to 24 h. Conclusions and Discussion: Our work establishes a standardized basis to determine the extent and the time-course of maximal T-cell activation through a given mechanism - in this case through the mechanism of calcium ionophore induced (ionomycin) and phorbol ester induced (PMA) T-cell activation (3). Such quantified standardization and maximization of T-cell activation in turn presents a cell-culture model (or assay) to evaluate candidate therapeutics designed to work through the same activation pathway. In addition to T-cells, we are working on the quantified standardization of the activation of NK-cells (4) and dendritic cells (5), with activation measured by the end-points of biomarker expression profiling (ELISA/Luminex), proliferation (MTT assay), microscopy and flow cytometry (CD receptor expression profiling).

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA &lt;em&gt;In Situ&lt;/em&gt; Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and METΔ14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors α and β in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay.

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and METΔ14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors α and β in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Construction of an M1GS Ribozyme for Targeted and Rapid mRNA Cleavage; Application on the Ets-2 Oncogene.

RNase P-mediated cleavage of target RNAs has been proposed as a promising tool for gene silencing. Ets-2 proto-oncogene controls the expression of a wide variety of genes involved in cancer and immunity. Construction of a functional RNase P-based ribozyme (M1GS303) that targets Ets-2 mRNA. The accessible sites for targeting of Ets-2 mRNA were identified by footprinting analysis. M1GS303 ribozyme was constructed by cloning. The activity of the ribozyme in the presence or absence of spiramysin in E. coli cells and human cell lines was quantified by RT-PCR. The efficiency of the ribozyme in silencing the endogenous expression of Ets-2 in human cell lines was examined by RT-PCR, western blot and immunofluorescence analysis. In E. coli cells co-transformed with plasmids bearing M1GS303 and the ets-2 target gene, Ets-2 mRNA was decreased by 93% 12h after IPTG induction in the absence, and after 4h in the presence of spiramycin. Ets-2 was rapidly downregulated in the human embryonic kidney cell line HEK293 and the T-cell line Jurkat transfected with an M1GS303 plasmid; the silencing effect of M1GS303 was considerably faster when the cells were cultured with spiramycin. In Jurkat cells, Ets-2-downregulation resulted in upregulation of the expression of IL-2, IL-4 and IFN-α cytokine genes that have Ets-2 binding sites on their promoters, whereas it had no effect on the expression of the IL-10 gene that lacks Ets-2 binding sites on its promoter. M1GS303 ribozyme cleaves effectively Ets-2 mRNA in bacteria and mammalian cells, and its activity is enhanced by spiramycin. Downregulation of ets-2 gene in the T-cell line Jurkat upregulates IL-2, IL-4 and IFN-α cytokine genes. M1GS technology may be a better alternative to conventional gene-interference therapies and the delineation of the effects of gene silencing in various pathologies.

The Toxoplasma gondii ME-49 strain upregulates levels of A20 that inhibit NF-\u03baB activation and promotes apoptosis in human leukaemia T-cell lines

BackgroundAcute T-lymphocyte leukaemia is a form of haematological malignancy with abnormal activation of NF-κB pathway, which results in high expression of A20 and ABIN1, which constitute a negative feedback mechanism for the regulation of NF-κB activation. Clinical studies have found that acute T-lymphocyte leukaemia patients are susceptible to Toxoplasma gondii infection; however, the effect of T. gondii on the proliferation and apoptosis of human leukaemia T-cells remains unclear. Here, we used the T. gondii ME-49 strain to infect human leukaemia T-cell lines Jurkat and Molt-4, to explore the effect of T. gondii on proliferation and apoptosis, which is mediated by NF-κB in human leukaemia T-cells.MethodsThe Tunel assay was used to detect cell apoptosis. Cell Counting Kit-8 was used to detect cell proliferation viability. The apoptosis level and the expression level of NF-κB related proteins in human leukaemia T-cells were detected by flow cytometry and Western blotting.ResultsWestern blotting analyses revealed that the T. gondii ME-49 strain increased the expression of A20 and decreased both ABIN1 expression and NF-κB p65 phosphorylation. By constructing a lentiviral-mediated shRNA to knockdown the A20 gene in Jurkat T-cells and Molt-4 T-cells, the apoptosis levels of the two cell lines decreased after T. gondii ME-49 infection, and levels of NF-κB p65 phosphorylation and ABIN1 were higher than in the non-konckdown group. After knockingdown ABIN1 gene expression by constructing the lentiviral-mediated shRNA and transfecting the recombinant expression plasmid containing the ABIN1 gene into two cell lines, apoptosis levels and cleaved caspase-8 expression increased or decreased in response to T. gondii ME-49 infection, respectively.ConclusionsOur data suggest that ABIN1 protects human leukaemia T-cells by allowing them to resist the apoptosis induced by T. gondii ME-49 and that the T. gondii ME-49 strain induces the apoptosis of human leukaemia T-cells via A20-mediated downregulation of ABIN1 expression.

P-A1 CBF-1 promotes the establishment and maintenance of HIV latency by recruiting Polycomb repressive complexes, PRC1 and PRC2, at HIV LTR

The type of chromatin structure around the LTR promoter of integrated HIV provirus provides critical signals that regulate transcription during both productive and latent HIV infections. The C-promoter binding factor-1 (CBF-1) is a potent and specific inhibitor of the HIV-1 transcription, which perform its function after binding to specific sites at HIV LTR. Here we demonstrate that the knockdown of endogenous CBF-1 in latently infected primary CD4+ T cells, using specific small hairpin RNAs (shRNA), resulted in the reactivation of latent HIV proviruses. By performing Chromatin immunoprecipitation (ChIP) assays, using latently infected primary T cells and Jurkat T-cell lines, we demonstrated that CBF-1 induces the establishment and maintenance of HIV latency by recruiting Polycomb Group (PcG/PRC) corepressor complexes or Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Knockdown of CBF-1 resulted in the dissociation of PRCs corepressor complexes and enhanced the recruitment of RNA polymerase II (RNAPII) at HIV LTR. Knockdown of certain components of PRC1 and PRC2 leads to the reactivation of latent proviruses. Similarly, treatment of latently infected primary CD4+ T cells with the EZH2 inhibitor, 3-deazaneplanocin A (DZNep), led to their reactivation. Importance: Instead of inhibiting individual enzymes, targeting factors such as CBF-1, which mediate the establishment of multiple repressive epigenetic changes, could be more beneficial in designing strategies to reactivate and subsequently eliminate latent HIV reservoirs.

Role of a non-canonical splice variant of the Helios gene in the differentiation of acute lymphoblastic leukemic T cells.

T-cell acute lymphoblastic leukemia is a hematopoietic malignant disease, which arises from a genetic defect in the T-cell maturation signaling pathway. As a result, it is necessary to identify the molecules that impact T-cell development and control lymphoid-lineage malignancy. The present study utilized Jurkat T lymphoblastic cells as a well-established approach for the investigation into the function of the non-canonical alternative splice variant of Helios for the in vitro study of T-cell differentiation and leukemogenesis. In the present study, the Jurkat T-cell lines with stable overexpression of the wild-type (Helios-1) or the non-canonical short isoform (Helios-Δ326-1431), were established. RNA microarray, reverse transcription-quantitative polymerase chain reaction and flow cytometry were used to assess changes in the gene expression profiles and to monitor the cell surface markers during T-cell differentiation. Multiple genes associated with T-cell differentiation and leukemogenesis were identified as being either activated or suppressed. In addition, the results indicated that the stable overexpression of the Helios isoforms stimulated the differentiation pathway of the T-lineage lymphoblastic cells. Therefore, these results suggest that full-length Helios-1 has a tumor suppressor-like and immunomodulatory role, in contrast to the oncogenic function of the non-canonical short isoform Helios-Δ326-1431.

Mechanogenetics for the remote and noninvasive control of cancer immunotherapy.

While cell-based immunotherapy, especially chimeric antigen receptor (CAR)-expressing T cells, is becoming a paradigm-shifting therapeutic approach for cancer treatment, there is a lack of general methods to remotely and noninvasively regulate genetics in live mammalian cells and animals for cancer immunotherapy within confined local tissue space. To address this limitation, we have identified a mechanically sensitive Piezo1 ion channel (mechanosensor) that is activatable by ultrasound stimulation and integrated it with engineered genetic circuits (genetic transducer) in live HEK293T cells to convert the ultrasound-activated Piezo1 into transcriptional activities. We have further engineered the Jurkat T-cell line and primary T cells (peripheral blood mononuclear cells) to remotely sense the ultrasound wave and transduce it into transcriptional activation for the CAR expression to recognize and eradicate target tumor cells. This approach is modular and can be extended for remote-controlled activation of different cell types with high spatiotemporal precision for therapeutic applications.

Interleukin 2 Secretion by T Cells for Detection of Biologically Active Staphylococcal Enterotoxin Type E

Staphylococcus aureus is a significant worldwide source of clinical infections and foodborne illnesses; it acts through the synthesis of a group of enterotoxins (SEs) that cause gastroenteritis and also function as superantigens that activate T cells, resulting in massive cytokine production, yielding life-threatening toxicity. It is important that methods for detection and quantification of these toxins respond to their activity and not just the presence of the toxin molecule, which may be deactivated. Traditionally, live animals have been used to test for emesis following administration of the toxin-containing sample. Here, we present results studying cell-based alternatives for the assay of active staphylococcal enterotoxin type E (SEE), a toxin subtype identified in foodborne outbreaks in the United States, the United Kingdom, and France. We found that interleukin 2 production by T cells can be used as a specific biological marker for the quantitative detection of SEE as compared with subtypes SEA and SEB. Our assay shows a dose-response relationship between IL-2 secretion by Jurkat T-cell line and SEE concentration as low as 1 pg/mL.

DNA methylation of Th2 lineage determination genes at birth is associated with allergic outcomes in childhood.

There is now increasing evidence that asthma and atopy originate in part in utero, with disease risk being associated with the altered epigenetic regulation of genes. To determine the relationship between variations in DNA methylation at birth and the development of allergic disease, we examined the methylation status of CpG loci within the promoter regions of Th1/2 lineage commitment genes (GATA3, IL-4, IL-4R, STAT4 and TBET) in umbilical cord DNA at birth in a cohort of infants from the Southampton Women's Survey (n=696) who were later assessed for asthma, atopic eczema and atopy. We found that higher methylation of GATA3 CpGs -2211/-2209 at birth was associated with a reduced risk of asthma at ages 3 (median ratio [median methylation in asthma group/median methylation in non-asthma group]=0.74, P=.006) and 6-7 (median ratio 0.90, P=.048) years. Furthermore, we demonstrated that the GATA3 CpG loci associated with later risk of asthma lie within a NF-κB binding site and that methylation here blocks transcription factor binding to the GATA3 promoter in the human Jurkat T-cell line. Associations between umbilical cord methylation of CpG loci within IL-4R with atopic eczema at 12months (median ratio 1.02, P=.028), and TBET with atopy (median ratio 0.98, P=.017) at 6-7years of age were also observed. Our findings provide further evidence of a developmental contribution to the risk of later allergic disorders and suggest that involvement of epigenetic mechanisms in childhood asthma is already demonstrable at birth.

Evaluation of the Antiproliferative Activity of Maerua edulis (Capparaceae)on Jurkat-T cells

Plant species serve as a rich source of many novel biologically active compounds, with cancer treatment being developed from natural products as early as the 1960s. This study was carried out to investigate the anti-proliferative activity of Maerua edulis against human leukemic Jurkat-T cell line. Anticancer activity of the aqueous, acetone, hexane and methanol extracts of M. edulis root were evaluated against Jurkat-T cells, using 1, 3-bis (2-chloroethyl)-1-nitrosourea (BCNU) as a reference drug. The effects of incubating BCNU and reduced glutathione (GSH) on the activity of the extracts as well as the reversibility of the effects extract were determined. Only the methanol extract significantly inhibited the growth of Jurkat-T cells with a GI50 of 18.54 μg.ml-1. There was no interaction with BCNU and methanol extract, though there was a decline in cell viability with increasing M. edulis concentration. When incubated with the antioxidant GSH, the sensitivity of cells to methanol extract did not change. It was noted that the effects of M. edulis on Jurkat-T cells were irreversible. M. edulis may be a potential source of lead compounds with anti-cancer activity and further studies are required in order to isolate these active compounds.

Argonaute 2 immunoprecipitation revealed large tumor suppressor kinase 1 as a novel proapoptotic target of miR-21 in T cells.

MicroRNA (miR)-21 is an important suppressor of T-cell apoptosis that is also overexpressed in many types of cancers. The exact mechanisms underlying the antiapoptotic effects of miR-21 are not well understood. In this study, we used the Jurkat T-cell line as a model to identify apoptosis-associated miR-21 target genes. We showed that expression of miR-21 rapidly increases upon αCD3/αCD28 activation of Jurkat cells. Inhibition of miR-21 reduced cell growth which could be explained by an increase in apoptosis. MicroRNA target gene identification by AGO2 RNA-immunoprecipitation followed by gene expression microarray (RIP-Chip) resulted in the identification of 72 predicted miR-21 target genes that were at least twofold enriched in the AGO2-IP fraction of miR-21 overexpressing cells. Of these, 71 were at least twofold more enriched in the AGO2-IP fraction of miR-21 overexpressing cells as compared to AGO2-IP fraction of control cells. The target gene for which the AGO2-IP enrichment was most prominently increased upon miR-21 overexpression was the proapoptotic protein LATS1. Luciferase reporter assays and western blot analysis confirmed targeting of LATS1 by miR-21. qRT-PCR analysis in primary T cells showed an inverse expression pattern between LATS1 transcript levels and miR-21 upon T-cell stimulation. Finally, LATS1 knockdown partially rescued the miR-21 inhibition-induced impaired cell growth. Collectively, these data identify LATS1 as a miR-21 target important for the antiapoptotic function of miR-21 in T cells and likely also in many types of cancer.

Tenofovir alafenamide (TAF) does not deplete mitochondrial DNA in human T-cell lines at intracellular concentrations exceeding clinically relevant drug exposures

HIV-infected patients treated with certain nucleoside reverse transcriptase inhibitors (NRTIs) have experienced adverse effects due to drug-related mitochondrial toxicity. Tenofovir alafenamide (TAF) is a novel prodrug of the NRTI tenofovir (TFV) with an improved safety profile compared to tenofovir disoproxil fumarate (TDF). Prior in vitro studies have demonstrated that the parent nucleotide TFV has no significant effects on mtDNA synthesis. This study investigated whether clinically relevant TAF and TDF exposures affect mtDNA content in human lymphocytes. First, activated or resting peripheral blood mononuclear cells (PBMCs), as well as MT-2 and Jurkat T-cell lines, were continuously treated with ddC for 10 days to establish their susceptibility to mtDNA depletion. PBMCs had low sensitivity to NRTI-mediated mtDNA depletion in vitro. In contrast, ddC treatment of rapidly dividing MT-2 and Jurkat cells resulted in a dose-dependent decrease in mtDNA. Therefore, these two T-cell lines were selected for evaluating TAF and TDF treatment effects. MT-2 and Jurkat cells were pulse-treated with TAF or TDF every 24 h for 10 days to mimic pharmacologically relevant drug exposures. Pulse treatment of cells with 3.3 μM TAF or 1.1 μM TDF for 10 days resulted in 2- to 7-fold greater steady-state intracellular TFV-diphosphate (TFV-DP) levels than those observed clinically in TAF- or TDF-treated patients. At these concentrations, no significant TAF- (106.7% and 84.1% of control; p = 0.77 and 0.12 for MT-2 and Jurkat, respectively) or TDF- (100.6% and 91.0% of control; p = 0.91 and 0.37, respectively) associated reduction in mtDNA content was observed compared with untreated control cells. This study demonstrates that, despite delivering higher intracellular levels of TFV-DP than TDF, TAF does not inhibit mtDNA synthesis in vitro at concentrations exceeding the clinically relevant intracellular drug exposures. Thus, TAF has a low potential for mitochondrial toxicity in T-cells of HIV-infected patients.