T-lymphocyte Cell Line Jurkat Research Articles

Delivery of a model lipophilic membrane cargo to bone marrow via cell-derived microparticles

Bone marrow (BM) is the central immunological organ and the origin of hematological diseases. Efficient and specific drug delivery to the BM is an unmet need. We tested delivery of fluorescent indocarbocyanine lipids (ICLs, DiR, DiD, DiI) as a model lipophilic cargo, via different carriers. Systemically injected T-lymphocyte cell line Jurkat delivered ICLs to the BM more efficiently than erythrocytes, and more selectively than PEGylated liposomes. Near infrared imaging showed that the delivery was restricted to the BM, lungs, liver and spleen, with no accumulation in the kidneys, brain, heart, intestines, fat tissue and pancreas. Following systemic injection of ICL-labeled cells in immunodeficient or immunocompetent mice, few cells arrived in the BM intact. However, between 5 and 10% of BM cells were ICL-positive. Confocal microscopy of intact BM confirmed that ICLs are delivered independently of the injected cells. Flow cytometry analysis showed that the lipid accumulated in both CD11b + and CD11b- cells, and in hematopoietic progenitors. In a xenograft model of acute myeloid leukemia, a single injection of 10 million Jurkat cells delivered DiD to ~15% of the tumor cells. ICL-labeled cells disappeared from blood almost immediately post-intravenous injection, but numerous cell-derived microparticles continued to circulate in blood. The microparticle particle formation was not due to the ICL labeling or complement attack and was observed after injection of both syngeneic and xenogeneic cells. Injection of microparticles produced in vitro from Jurkat cells resulted in a similar ICL delivery as the injection of intact Jurkat cells. Our results demonstrate a novel delivery paradigm wherein systemically injected cells release microparticles that accumulate in the BM. In addition, the results have important implications for studies involving systemically administered cell therapies.

Clickable Methyltetrazine-Indocarbocyanine Lipids: A Multicolor Tool Kit for Efficient Modifications of Cell Membranes.

Cell-based therapeutics are one of the most promising and exciting breakthroughs in modern medicine. Modification of the cell surface with ligands, biologics, drugs, and nanoparticles can further enhance the functionality. Previously, we described the synthesis of a dioctadecyl indocarbocyanine Cy3 analog (aminomethyl-DiI) for efficient and stable modification (painting) of mouse erythrocytes with small molecules, enzymes, and biologics. Here, we synthesized a near-infrared aminomethyl dioctadecyl derivative of Cy7 (aminomethyl-DOCy7) and systematically compared it to aminomethyl-DiI as an anchor for the modification of human erythrocytes, Jurkat cells, and primary T cells with immunoglobulin G. To enable copper-free click chemistry modification of cell membranes, we conjugated a methyltetrazine (MTz) group to the amino-indocyanine lipids via a polyethylene glycol (PEG) linker. DOCy7-PEG3400-MTz showed over 99% modification efficiency of human red blood cells (RBCs) at 25 μM. Reaction of trans-cyclooctene (TCO) modified immunoglobulin G (IgG) with DOCy7-PEG4-MTz-modified RBCs (2-step method) resulted in ∼80,000 IgG molecules per erythrocyte, whereas modification with a preconjugated DOCy7-PEG3400-IgG construct (1-step method) resulted in ∼20,000 IgG molecules per erythrocyte as detected by immuno dot-blot. The number of IgG/RBC was controlled by the concentration of IgG. The incubation of RBCs with DiI-PEG3400-MTz resulted in a similar number of IgG/RBC. Modification of the T-lymphocyte cell line Jurkat with IgG resulted in ∼1 × 106 IgG/cell with the 1-step and 2-step methods, and the efficiency was similar for DOCy7 and DiI constructs. Finally, we used DOCy7 and DiI constructs to demonstrate efficient modification of primary CD3+T cells from healthy donors. In conclusion, click indocarbocyanine conjugates represent a novel multicolor chemical biology tool kit for efficient surface modification of different cells types and can be used for potential imaging and drug delivery applications involving engineered cells.

Isolation and Stimulation of Peritoneal Macrophages withApoptotic Jurkat Cells to Produce IL-10.

Clearance of apoptotic cells by macrophages is critical to ensuring cellular homeostasis and suppression of autoimmunity. Macrophage recognition of apoptotic cells triggers an anti-inflammatory response, which is mediated by the release of IL-10, TGF-β etc. with concurrent inhibition of pro-inflammatory cytokines (such as TNFα, IL-12, IL-1β). To characterize cytokine profile produced by macrophages during phagocytosis of apoptotic cells, we developed an effective, more physiologic system using isolated murine peritoneal macrophages and T-lymphocyte cell line Jurkat as a source of apoptotic cells. Apoptosis of Jurkat cells is induced with staurosporine, a protein kinase C (PKC) inhibitor and detected by Annexin V/propidium iodide staining. This in vitro assay demonstrates that murine peritoneal macrophages produce large amounts of IL-10 following exposure to apoptotic Jurkat cells.

Suitability of Polyelectrolyte Shells Modified with Fullerene Derivate for Immunoisolation of Cells. Experimental Study

The polymeric permiselective membranes application for immunoisolation of cells separating the transplanted cells from the host immunological system may eliminate immunosuppressive therapy during transplantation. The suitability of polyelectrolyte modified nanocoatings for immunoisolation of cells was assessed. The polymeric shells modified with incorporated fullerene derivate were applied for encapsulation of human T-lymphocyte cell line Jurkat or rat pancreatic islets of Langerhans using layer-by-layer technique. Hydroxylated fullerene was incorporated to the polyelectrolyte shell for hydrophility increase as well as for layer stability improvement. Evaluation with AFM, FTIR, fluorescence microscopy confirmed the nanocoating presence on the encapsulated cells. It was observed that polylysine-polyethyleneimine membrane with incorporated fullerenol allowed for encapsulated cells functioning in vitro. Membrane conformation applied for encapsulation of pancreatic rat islets allowed for glucose level decline during xenotransplantation into mice. The elaborated nanocoating may be recommended as the possible alternative to the space consuming microencapsulation for biomedical purposes.

Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5μM DON for 3, 6 and 24h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4μM DON for 6 and 24h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs.

Aggregation behavior and in vitro biocompatibility study of octopus-shaped macromolecules based on tert-butylcalix[4]arenes

A series of products based on tert-butylcalix[4]arene have been synthesized by anionic polymerization of ethylene oxide. The resulting products are amphiphilic octopus-shaped macromolecules, consisting of a hydrophobic calix[4]arene core and four arms of hydrophilic poly(ethylene oxide) chains. In aqueous solutions the polyoxyethylated tert-butylcalix[4]arenes were found to self-associate above certain CMC determined by dye solubilization technique. The light scattering study reveals that the polyoxyethylated tert-butylcalix[4]arenes form aggregates of narrow size distribution and hydrodynamic diameters ranging from about 155 to 245 nm and aggregation numbers from tens to hundreds macromolecules per particle depending on the degree of polymerization of the PEO chains. An in vitro biocompatibility study showed that the tested compounds are practically devoid of intrinsic cytotoxic and hemolytic effects and moreover they failed to modulate the mitogen-induced interleukin-2 release from the human T-lymphocyte cell line Jurkat E6-1. Taken together the excellent in vitro biocompatibility profile and the favorable physicochemical characteristics of the tested polyoxyethylated calix[4]arenes give us reason to consider them as promising for further evaluation as drug delivery platforms.

Conformal Nano-Thin Modified Polyelectrolyte Coatings for Encapsulation of Cells

Encapsulation of cells in polymeric shells allows for separation of biological material from produced factors, which may find biotechnological and biomedical applications. Human T-lymphocyte cell line Jurkat as well as rat pancreatic islets were encapsulated using LbL technique within shells of polyelectrolyte modified by incorporation of biotin complexed with avidin to improve cell coating and to create the potential ability to elicit specific biochemical responses. The coating with nano-thin modified shells allowed for maintenance of the evaluated cells' integrity and viability during the 8-day culture. The different PE impact may be observed on different biological materials. The islets exhibited lower mitochondrial activity than the Jurkat cells. Nevertheless, coating of cells with polyelectrolyte modified membrane allowed for functioning of both model cell types: 10 μm leukemia cells or 150 μm islets during the culture. Applied membranes maintained the molecular structure during the culture period. The conclusion is that applied modified membrane conformation may be recommended for coating shells for biomedical purposes.

Ca2+ release and Ca2+ entry induced by rapid cytosolic alkalinization in Jurkat T-lymphocytes.

4-Aminopyridine (4-AP), a compound usually known as a K(+)-channel inhibitor, induced rapid cytosolic alkalinization from pH 7.15 to pH 7.4, and subsequently Ca2+ mobilization in the T-lymphocyte cell line Jurkat. Other weak bases, such as NH4Cl or triethanolamine, induced a smaller and/or slower increase in cytosolic pH, resulting in a lower or no detectable Ca2+ signal. In the presence of extracellular Ca2+, 4-AP mediated a rapid and sustained increase in the free cytosolic Ca2+ concentration similar to that obtained by T-cell receptor-mediated stimulation. In the absence of extracellular Ca2+, 4-AP transiently released Ca2+ from an intracellular store that is most likely identical with the agonist- and Ins(1,4,5)P3-sensitive Ca2+ pool of Jurkat T-cells. As possible mechanisms for Ca2+ release from this particular pool as induced by 4-AP we examined (i) formation of Ins(1,4,5)P3 and (ii) sensitization of the Ins(1,4,5)P3-receptor/Ca(2+)release system by increasing intracellular pH. Although 4-AP did not induce formation of inositol polyphosphates, as demonstrated by h.p.l.c. analysis, in permeabilized cells the dose-response curve for Ins(1,4,5)P3 was shifted to the left by changing the intracellular pH from 7.2 to 7.4. This indicated that sensitization of the Ins(1,4,5)P3-receptor/Ca(2+)-release system was responsible for the effects of 4-AP seen in intact cells. In conclusion, 4-AP appears a novel tool for depletion of the agonist-sensitive Ca2+ pool of T-cells without simultaneous formation of Ins(1,4,5)P3, thereby inducing capacitative Ca2+ entry in these cells.

Description of the leukocyte function-associated antigen 1 (LFA-1 or CD11a) promoter.

The CD11a/CD18 (leukocyte function-associated antigen 1 or LFA-1) leukocyte integrin is expressed at high levels on the cell surface of T lymphocytes and macrophages, where it mediates homotypic and heterotypic adherence between leukocytes and other cell types by binding to intracellular adhesion molecules 1 and 2 on the conjugate cell. To initiate studies of the molecular regulation of expression of the CD11a molecule, we isolated genomic clones corresponding to the 5'-flanking region of CD11a, identified the transcriptional start sites for CD11a, and characterized the CD11a promoter sequence in transient expression assays. The CD11a promoter (1.7 kb) directed functional activity of a heterologous reporter gene in the T-lymphocyte cell line Jurkat and the myeloid cell line HL-60 but did not direct functional activity in three different nonleukocyte cell lines. Deletional analysis of the CD11a promoter sequence indicated the presence of distinct, cell-type-specific regulatory sequences with the region from -40 to -17 relative to the transcription start sites responsible for most of the in vitro activity of the CD11a promoter in the Jurkat T-cell line, and the promoter sequence located within the first 17 bp relative to the transcription start sites responsible for CD11a promoter activity in the HL-60 cell line. Identification of the CD11a promoter provides the opportunity to identify unique cis-acting elements and trans-acting factors responsible for the cell-type-specific expression of CD11a in human leukocytes. Further, the CD11a promoter may be useful in transgenic constructs and in retroviral vectors to direct expression of heterologous genes selectively in leukocytes.

Functional and immunological responses of Jurkat lymphocytes transfected with the substance P receptor.

1. We have transfected the rat substance P receptor (SPR) cDNA into the leukemic T-lymphocyte cell line Jurkat (J-wt) in order to study the effects of substance P (SP) on lymphocyte signaling mechanisms and the resultant neuropeptide-induced immunological changes. 2. The SPR cDNA was transfected into J-wt by the method of electroporation. Clones expressing SPRs were selected using a functional assay that measured SP-induced mobilization of intracellular Ca2+ ([Ca2+]i) in a fluorescence activated cell sorter (FACS) and by their expression of specific 125I-SP binding. 3. One clone, J-SPR, was identified and shown by Northern blot and 125I-SP saturation binding techniques to express the 2.2-kb SPR message and approximately 50,000 SPRs/cell with a Kd of 0.3 nM, respectively. Stimulation of J-SPR by SP resulted in the rapid mobilization of [Ca2+]i. This response was dose dependent in the range 10(-11)-10(-6) M SP and was maximal at 10(-7) M SP, with an EC50 of 0.3-0.5 nM SP. We further demonstrated that the SPR is rapidly desensitized following SP stimulation and by activation of the cell's T-cell receptor (TCR). Whole-cell patch-clamp experiments on J-SPR show that SP stimulation induces a Cl- current by a Ca2+ mediated process dependent on Ca2+/calmodulin-dependent protein kinase (CaMK). 4. Stimulation of J-SPR by SP results in changes in the cell surface expression of a number of molecules that play important roles in cell adhesion and activation: the expression of LFA-1 is decreased, and CD2 and IL-2 receptors are increased by 30 min, 6 hr, and 24 hr, respectively, following stimulation, as assessed by antibody staining in a FACS. 5. The expression of functional SPRs in Jurkat lymphocytes will not permit a detailed examination of how the activation of SPRs result in altered immune responses and further elucidate the role this neuropeptide receptor plays in inflammation.

Determination of inositol polyphosphates from human T-lymphocyte cell lines by anion-exchange high-performance liquid chromatography and post-column derivatization

The intracellular amounts of several inositol tris-, tetrakis- and pentakisphosphates and inositol hexakisphosphate were determined in resting and stimulated cells from human T-lymphocyte lines. The inositol polyphosphates were separated by anion-exchange high-performance liquid chromatography and were detected on-line by a recently developed post-column dye system. In the human T-lymphocyte cell line Jurkat, basal intracellular concentrations ranged between 25±10 pmol per 10 9 cells for inositol 1,4,5-trisphosphate to 6380±355 pmol per 10° cells for inositol hexakisphosphate. Similar basal concentrations were observed in the human T-lymphocyte cell line HPB.ALL, with the exception that inositol hexakisphosphate was approximately 665±10 pmol per 10 9 cells. Stimulation of the human T-lymphocyte cell line Jurkat via the T-cell receptor by a monoclonal antibody directed against the T-cell receptor-CD3 complex induced time-dependent changes in the intracellular concentrations of multiple inositol polyphosphate isomers, including inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4,6-tetrakisphosphate, an as yet unidentified inositol tetrakisphosphate isomer, inositol 1,3,4,5,6-pentakisphosphate, inositol 1,2,3,4,6-pentakisphosphate and dl-inositol 1,2,4,5,6-pentakisphosphate. Inositol 1,4,5-trisphosphate increased only transiently after 5 min, whereas dl-inositol 1,4,5,6-tetrakisphosphate (determined as the enantiomeric mixture) increased after 20 min.

T-cell receptor-mediated metabolism of inositol polyphosphates in Jurkat T-lymphocytes. Identification of a D-myo-inositol 1,2,3,4,6-pentakisphosphate-2-phosphomonoesterase activity, a D-myo-inositol 1,3,4,5,6-pentakisphosphate-1/3-phosphatase activity and a D/L-myo-inositol 1,2,4,5,6-pentakisphosphate-1/3-kinase activity.

Stimulation of the human T-lymphocyte cell line Jurkat via the T-cell receptor/CD3 complex by an anti-CD3 antibody (OKT3) induced time-dependent changes in the intracellular concentrations of multiple inositol polyphosphate (InsPn) isomers. Quantitative mass analysis by anion-exchange HPLC and a recently developed postcolumn dye system (Mayr, G. W. (1988) Biochem. J. 254, 585-591) revealed basal intracellular concentrations between less than 5 pmol/10(9) cells for Ins(1,3,4,5)P4 and 6380 +/- 355 pmol/10(9) cells for InsP6. Time course analysis of samples from stimulated Jurkat T-cells showed an increase of Ins(1,3,4,5)P4 to 1125 +/- 125 pmol/10(9) cells within 10 min and remained elevated over more than 30 min. Moreover, increases of the intracellular concentrations of Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, and/or Ins(3,4,5,6)P4 (determined as the enantiomeric mixture), Ins(1,3,4,5,6)P5, Ins(1,2,3,4,6)P5 and InsP6 were observed. In contrast, the concentration of Ins(1,2,4,5,6)P5 and/or Ins(2,3,4,5,6)P5 (determined as the enantiomeric mixture) decreased after stimulation. Using cytosolic extracts from Jurkat T-lymphocytes incubated with purified Ins(1,3,4,5,6)P5, Ins(1,2,3,4,6)P5, or Ins(1,2,4,5,6)P5/Ins(2,3,4,5,6)P5 three enzyme activities were observed. Ins(1,3,4,5,6)P5 was dephosphorylated by a phosphatase removing a phosphate group at the 1 and/or 3 position resulting in the formation of Ins(1,4,5,6)P4 and/or Ins(3,4,5,6)P4 (determined as the enantiomeric mixture). Ins(1,2,3,4,6)P5 was metabolized by a specific phosphatase that cleaved the phosphate group at the 2 position, thereby generating the product Ins(1,3,4,6)P4. On the other hand, Ins(1,2,4,5,6)P5/Ins(2,3,4,5,6)P5 was phosphorylated by a 1/3-kinase activity to InsP6. Together novel receptor-mediated metabolic pathways of inositol polyphosphates were demonstrated in human T-lymphocytes, and corresponding enzyme activities for the inositol pentakisphosphate metabolism were found in cell lysates.

The cytosolic-binding protein for the immunosuppressant FK-506 is both a ubiquitous and highly conserved peptidyl-prolyl cis-trans isomerase.

We have recently isolated an abundant cytosolic protein from human T-cells which specifically binds the immunosuppressive agent, FK-506. The FK-506-binding protein (FKBP) is a member of a novel class of proteins possessing peptidyl-prolyl cis-trans isomerase activity. These proteins are believed to play an important role in accelerating the rate at which proteins fold into their native conformations. In the present study, we demonstrate that FKBP is not a lymphoid-specific protein, but is widely distributed and phylogenically conserved. FKBP, purified from three sources (a human T-lymphocyte cell line JURKAT, bovine calf thymus, and Saccharomyces cerevisiae) exhibit identical molecular weights, immunological cross-reactivities, and a high degree of NH2-terminal amino acid sequence homology. In addition, FKBP from all sources possesses peptidyl-prolyl cis-trans isomerase activity which can be specifically inhibited by FK-506. We conclude that FKBP may serve an important biological function in all eukaryotic cells.