Wild-type Jurkat Research Articles

Effect of peroxiredoxin 1 or peroxiredoxin 2 knockout on the thiol proteome of Jurkat cells.

Peroxiredoxins are important regulators of cellular peroxide metabolism. As antioxidants, they restrict oxidation of other cell proteins, but as signaling molecules they can act as sensors and promote thiol protein oxidation via a redox relay mechanism. The presence of peroxiredoxins could therefore influence other thiol proteins, even in cells experiencing endogenous redox activity. To investigate this for the two cytoplasmic peroxiredoxins, Prdx1 and Prdx2, we have compared the thiol proteome of wildtype Jurkat cells with cells in which either one was knocked out. Using mass spectrometry and isotope tagging, approximately 10,000 common CysSH-containing peptides were detected for each WT/KO comparison. Knockout of Prdx1 or Prdx2 resulted in a change in redox state of a small selection of Cys residues, with less than 100 giving more than a 2-fold difference. Strikingly, a large proportion of these, including those that showed the greatest change, were common to both KOs. Some Cys residues showed more oxidation in the knockouts, whereas others showed less. The candidate proteins have diverse functions and have not been known to be oxidant sensitive. No differences were seen in redox state of Cys residues of other Prdxs and oxidant sensitive proteins. A change in expression in Prdx2 knockout cells was indicated for seven cytoskeletal or regulatory thiol proteins, three of which were tested and validated by western blotting. Little firm evidence was found for thiol redox changes dependent on either Prdx that could be attributed to oxidation via a relay mechanism.

Dominant interfering CARD11 variants disrupt JNK signaling in T cells and impaired CARD11-JNK signaling increases expression of GATA3

CARD11 is a critical scaffold protein linking antigen receptor (AgR) engagement to transcriptional programs required for effector lymphocyte survival, proliferation, and differentiation. Prior work established that CARD11 mediates these effects by enabling AgR-induced signaling through NF-kB, mTORC1 and c-Jun N-terminal kinase 2 (JNK2). Although JNK influences helper CD4+ T cell differentiation, mechanisms governing JNK signal regulation and function in human lymphocytes remain incompletely understood. We therefore asked if/how dominant interfering (DI) CARD11 variants impact JNK signaling and JNK-specific transcriptomic changes in T cells. Wild-type (WT) and CARD11 knockout (KO) Jurkat T cells were transfected with plasmids expressing WT or DI CARD11 variants to assess effects on CARD11-dependent JNK signaling. Additionally, RNA sequencing was performed on mitogen-activated WT and CARD11 KO Jurkat T cells +/– JNK inhibition. Our results revealed that CARD11 is required for AgR-dependent activation of JNK1 and JNK2. Furthermore, heterozygous DI CARD11 mutations found in patients with CARD11-associated atopy with dominant interference of NF-kB signaling (CADINS) disease disrupted AgR-dependent JNK1/2 phosphorylation, c-Jun accumulation, and AP-1 transcriptional activity with variable potency, similar to the degree of impaired NF-kB activation. Intriguingly, JNK inhibition in Jurkat and primary CD4+ T cells enhanced AgR-induced expression of GATA3, the master transcriptional regulator of Th2 cell differentiation. These results were recapitulated in effector Tcells generated from CADINS patient PBMC, with greater GATA3 induction observed after mitogen activation relative to healthy controls. Intriguingly, simultaneous reactivation of JNK signaling through a CARD11-independent mechanism blunted AgR-induced GATA3 induction, suggesting JNK activity can directly modulate GATA3 expression. In summation, our novel findings imply that defective CARD11-dependent JNK signaling in CD4+ T cells contributes to enhanced Th2 differentiation and the atopic diathesis observed in CADINS patients, unveiling a new potential therapeutic target for ameliorating severe allergic disease manifestations.

Lack of Nck1 protein and Nck-CD3 interaction caused the increment of lipid content in Jurkat T cells

BackgroundThe non-catalytic region of tyrosine kinase (Nck) is an adaptor protein, which is ubiquitously expressed in many types of cells. In T cells, the Nck1 isoform promotes T cell receptor signalling as well as actin polymerisation. However, the role of Nck1 in the lipid metabolism in T cells is unknown. In the present study, we investigated the effect of the Nck1 protein and Nck–CD3 interaction on lipid metabolism and on the physical and biological properties of Jurkat T cells, using a newly developed holotomographic microscope.ResultsHolotomographic microscopy showed that Nck1-knocked-out cells had membrane blebs and were irregular in shape compared to the rounded control cells. The cell size and volume of Nck1-deficient cells were comparable to those of the control cells. Nck1-knocked-out Jurkat T cells had a greater lipid content, lipid mass/cell mass ratio, and lipid metabolite levels than the control cells. Interestingly, treatment with a small molecule, AX-024, which inhibited Nck–CD3 interaction, also caused an increase in the lipid content in wild-type Jurkat T cells, as found in Nck1-deficient cells.ConclusionsKnockout of Nck1 protein and hindrance of the Nck–CD3 interaction cause the elevation of lipid content in Jurkat T cells.

Genetic Control of Splicing at SIRPG Modulates Risk of Type 1 Diabetes.

Signal regulatory protein SIRPγ (CD172G) is expressed on the surface of lymphocytes, where it acts by engaging its ligand, CD47. SIRPG, which encodes SIRPγ, contains a nonsynonymous coding variant, rs6043409, which is significantly associated with risk for type 1 diabetes. SIRPG produces multiple transcript isoforms via alternative splicing, all encoding potentially functional proteins. We show that rs6043409 alters a predicted exonic splicing enhancer, resulting in significant shifts in the distribution of SIRPG transcript isoforms. All of these transcript isoforms produced protein upon transient expression in vitro. However, CRISPR/Cas9 targeting of one of the alternatively spliced exons in SIRPG eliminated all SIRPγ expression in Jurkat T cells. These targeted cells formed fewer cell-cell conjugates with each other than with wild-type Jurkat cells, expressed reduced levels of genes associated with CD47 signaling, and had significantly increased levels of cell-surface CD47. In primary CD4+ and CD8+ T cells, cell-surface SIRPγ levels in response to anti-CD3 stimulation varied quantitatively by rs6043409 genotype. Our results suggest that SIRPG is the most likely causative gene for type 1 diabetes risk in the 20p13 region and highlight the role of alternative splicing in lymphocytes in mediating the genetic risk for autoimmunity.

CD63+ and MHC Class I+ Subsets of Extracellular Vesicles Produced by Wild-Type and CD47-Deficient Jurkat T Cells Have Divergent Functional Effects on Endothelial Cell Gene Expression.

T cells and endothelial cells engage in bidirectional communication that regulates angiogenesis and T cell transmigration. Extracellular vesicles (EVs) mediate intercellular communication by the transfer of bioactive molecules including RNAs. EVs produced by a given cell type are heterogeneous in their RNA content, but it is unclear how specific EV surface markers relate to their functional effects on target cells. Our previous work established that Jurkat T cell EVs bearing CD63, MHC-I, or CD47 surface markers contain distinct noncoding RNA populations. The present study reveals that CD63+ and MHC-I+ EVs from CD47-deficient Jurkat T cells are enriched in small non-coding RNAs relative to EVs from wild-type Jurkat T cells. CD47-deficient Jurkat T cells secrete more CD63+ and MHC-I+ EVs, but MHC-I+ EVs are selectively taken up more by human umbilical vein endothelial cells. Transcriptomics analysis of endothelial cells treated with CD63+ or MHC-I+ EVs showed surface marker- and CD47-dependent changes in gene expression in the target cells. Gene set enrichment analysis identified CD47-dependent, and surface marker-dependent effects of T cell EVs on VEGF and inflammatory signaling, cell cycle, and lipid and cholesterol metabolism. Thus, subsets of T cell EVs differentially regulate endothelial cell metabolism and inflammatory and angiogenic responses.

Impact of Humoral Immunogenicity (Anti-mCAR19 Antibodies) on CTL019 Cellular Kinetics, Efficacy, and Safety

Background: The humoral immunogenicity of CAR19, a chimeric antigen receptor with a murine scFV domain developed for treatment with CTL019 (tisagenlecleucel) in relapsed/refractory (R/R) pediatric and young adult acute lymphoblastic leukemia (ALL), was evaluated in 2 studies (ELIANA [NCT02435849]; ENSIGN [NCT02228096]). To date, little is known about the presence and impact of preexisting or treatment-induced anti-murine (m)CAR19 antibodies in patients treated with CAR T-cell therapy.Methods: Anti-mCAR19 antibodies were measured in patients from ELIANA and ENSIGN trials before and after CTL019 infusion to evaluate the impact of anti-mCAR19 antibodies on cellular kinetics, efficacy, and safety. Data were pooled from ELIANA (n = 68) and ENSIGN (n = 29). Anti-mCAR19 antibodies were captured by mCAR19-transfected Jurkat cells and wild-type Jurkat cells. Following incubation and washing, an R-phycoerythrin (PE)-labeled anti-IgG/M F(ab')2 fragment was added for detection of anti-mCAR19 antibodies using flow cytometry and reported as median fluorescence intensity (MFI). Exploratory assay development included assessment of whether the presence of immunoglobulins after IVIG therapy in clinical samples would interfere with the anti-mCAR19 antibody assay. Impact of preexisting immunogenicity, as detected by MFI levels, was evaluated using cellular kinetic, efficacy, and safety endpoints. The impact of treatment-induced immunogenicity (defined as having an increase in anti-mCAR19 antibody levels over baseline) was assessed by calculating the fold change between preexisting (ie, baseline) levels and postinfusion levels.Results: Approximately 90% of patients displayed preexisting anti-mCAR19 antibodies; a similar incidence was detected in healthy volunteer samples, as observed during assay development. Approximately 35% of patients developed treatment-induced anti-mCAR19 antibodies. No apparent relationship between CTL019 expansion (AUC0-28d) and preexisting or treatment-induced anti-mCAR19 antibodies was identified (r2 < 0.001 and r2 = 0.006, respectively); similar results were seen for Cmax (Figure 1a). Presence of anti-mCAR19 after infusion did not appear to influence the persistence of transgene or day 28 response (Figure 1b). Kaplan-Meier curves showed that the presence of preexisting and treatment-induced anti-mCAR19 antibodies did not appear to impact the duration of response or event-free survival (Figure 1c). Strip plots showed consistent levels of preexisting and treatment-induced anti-mCAR19 antibodies across patients with safety events (grade 0/1/2/3/4), including CRS, neutropenia, thrombocytopenia, and neurological events. There was no apparent relationship between treatment- induced anti-mCAR19 antibodies with B-cell recovery categories (recovery ≤ 3 months, > 3 months and ≤ 6 months, > 6 months, and ongoing sustained B-cell aplasia). There was also no association in patients at the time of B-cell recovery with the presence of treatment-induced anti-mCAR19 antibodies following CTL019 infusion. B-cell aplasia requiring immunoglobulin replacement therapy (IVIG) occurred following CTL019 in the majority of patients. The CTL019 concentration time profiles for patients with treatment-induced anti-mCAR19 antibodies were categorized by the time following IVIG administration (within 1 week, 2 weeks, or > 3 weeks of IVIG administration). The results revealed no impact of the time at which IVIG was administered on the in vivo expansion and persistence of the transgene.Conclusions: We report the first comprehensive assessment of the impact of anti-mCAR antibodies on clinically relevant endpoints for CAR therapy. Determination of immunogenicity is an important endpoint required by health authorities for biologic drugs, and these analyses demonstrate the importance of characterizing the impact on clinical endpoints for CAR therapies. These analyses in patients with pediatric R/R ALL revealed a surprisingly high frequency of baseline anti-mCAR19 antibodies and showed that the presence of preexisting and treatment-induced anti-mCAR19 antibodies did not impact the cellular kinetics, safety, and efficacy of CTL019. Cell-mediated immunity studies are ongoing. [Display omitted] DisclosuresMueller:Novartis Pharmaceuticals Corporation: Employment. Waldron:Novartis Pharmaceuticals Corporation: Employment. Sickert:Novartis Institutes for BioMedical Research: Employment. Grupp:University of Pennsylvania: Patents & Royalties; Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; Adaptimmune: Consultancy; Jazz Pharmaceuticals: Consultancy. Maude:Novartis Pharmaceuticals: Consultancy, Other: Medical Advisory Boards. Levine:Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees. Laetsch:Novartis Pharmaceuticals Corporation: Consultancy. Pulsipher:Novartis Pharmaceuticals Corporation: Consultancy, Other: Phase II steering committee; Chimerix: Other: Advisory board Dec 2015; Jazz Pharmaceuticals: Other: advisory board, education; CSL Behring: Other: advisory board Feb 2017; Adaptive: Other: Advisory board 6/17. Boyer:Novartis Pharmaceuticals Corporation: Honoraria. August:Novartis Pharmaceuticals Corporation: Consultancy. Awasthi:Novartis Pharmaceuticals Corporation: Employment. Levine:GE Healthcare: Consultancy; Novartis Pharmaceuticals Corporation: Patents & Royalties, Research Funding; Brammer Bio: Consultancy; Tmunity Therapeutics: Equity Ownership, Research Funding. June:WIRB/Copernicus Group: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celldex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Taran:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Wood:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Myers:Novartis Pharmaceuticals Corporation: Consultancy.

Targeted knockout of &amp;lt;italic&amp;gt;Kcna3&amp;lt;/italic&amp;gt; and its function in Jurkat T cells using the CRISPR/Cas9 system

To explore the use of the CRISPR/Cas9 system to target Kv1.3 channel-encoding genes Kcna3 and elucidate the pathophysiological function mediated by Kv1.3 channels in Jurkat T cells. The first exon sgRNA of human Kcna3 in pX458-sgRNA recombinant vector was transferred into human Jurkat T cells via electroporation. T7EN1 digestion, Genomic PCR products and TA cloning sequencing, Western blot, and functional assays, such as electrophysiology, live-cell calcium imaging and ELISA were used to validate Kcna3 knockout effect in Jurkat T cells. The results of the sequencing analysis showed that the CRISPR/Cas9 recombinant plasmids pX458-sgRNA targeting Kcna3 were successfully constructed. The designed sgRNA2 can efficiently cleave genomic DNA. Kv1.3 protein expression could not be detected using Western blot analysis. Genomic PCR products and TA cloning sequencing showed the presence of Indel mutation. Whole-cell patch clamp experiments showed that Kv1.3 channel currents could not be recorded in Kcna3 knockout Jurkat T cells. Live-cell calcium imaging showed that the increase in free Ca2+ concentration in Kcna3 knockout Jurkat T cells was significantly lower than that in wild type Jurkat T cells. ELISA results showed that the IL-2 level of Kcna3 knockout Jurkat T cells was significantly decreased compared with that in wild-type Jurkat T cells ( P Kcna3 in Jurkat T cells using the CRISPR/Cas9 system strongly proved that Kv1.3 mediated the immune inflammatory response of Jurkat T cells, which provides a novel cell model for further study of the pathophysiological function of the Kv1.3 channel.

Cis-trimethoxy resveratrol induces intrinsic apoptosis via prometaphase arrest and prolonged CDK1 activation pathway in human Jurkat T cells.

Cis-trimethoxy resveratrol (cis-3M-RES) induced dose-dependent cytotoxicity and apoptotic DNA fragmentation in Jurkat T cell clones (JT/Neo); however, it induced only cytostasis in BCL-2-overexpressing cells (JT/BCL-2). Treatment with 0.25 μM cis-3M-RES induced G2/M arrest, BAK activation, Δψm loss, caspase-9 and caspase-3 activation, and poly (ADP-ribose) polymerase (PARP) cleavage in JT/Neo cells time-dependently but did not induce these events, except G2/M arrest, in JT/BCL-2 cells. Moreover, cis-3M-RES induced CDK1 activation, BCL-2 phosphorylation at Ser-70, MCL-1 phosphorylation at Ser-159/Thr-163, and BIM (BIMEL and BIML) phosphorylation irrespective of BCL-2 overexpression. Enforced G1/S arrest by using a G1/S blocker aphidicolin completely inhibited cis-3M-RES-induced apoptotic events. Cis-3M-RES-induced phosphorylation of BCL-2 family proteins and mitochondrial apoptotic events were suppressed by a validated CDK1 inhibitor RO3306. Immunofluorescence microscopy showed that cis-3M-RES induced mitotic spindle defects and prometaphase arrest. The rate of intracellular polymeric tubulin to monomeric tubulin decreased markedly by cis-3M-RES (0.1-1.0 μM). Wild-type Jurkat clone A3, FADD-deficient Jurkat clone I2.1, and caspase-8-deficient Jurkat clone I9.2 exhibited similar susceptibilities to the cytotoxicity of cis-3M-RES, excluding contribution of the extrinsic death receptor-dependent pathway to the apoptosis. IC50 values of cis-3M-RES against Jurkat E6.1, U937, HL-60, and HeLa cells were 0.07-0.17 μM, whereas those against unstimulated human peripheral T cells and phytohaemagglutinin A-stimulated peripheral T cells were >10.0 and 0.23 μM, respectively. These results indicate that the antitumor activity of cis-3M-RES is mediated by microtubule damage, and subsequent prometaphase arrest and prolonged CDK1 activation that cause BAK-mediated mitochondrial apoptosis, and suggest that cis-3M-RES is a promising agent to treat leukemia.

The Nrf2 activators tBHQ and CDDO-Im have both Nrf2-dependent and – independent effects on human Jurkat T cell activation, as determined by CRISPR-Cas9 gene editing

Abstract Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress activated transcription factor, activated by such stimuli as reactive oxygen and electrophilic stress. Upon activation Nrf2 accumulates in the nucleus and activates a battery of target genes, many of which are cytoprotective in function. We have previously shown that a common synthetic food additive, tBHQ, is a potent Nrf2 activator and suppresses certain events of human T cell activation, namely, IL-2 secretion, CD25 expression, and transactivation ability of NFkB. The purpose of our current studies was to develop a Nrf2-null human model, in which it can be determined whether the effects of tBHQ upon the early events of T cell activation are Nrf2-dependent. Treatment of both Wild-type and Nrf2-null Jurkat T cells revealed that the suppression of IL-2 and NFkB by tBHQ is largely Nrf2-independent, whereas the suppression of CD25 is partially dependent upon Nrf2. Although interesting, these studies left the question as to the role of Nrf2 in T cell activation largely unanswered. Therefore, we utilized a more potent and selective Nrf2 activator, CDDO-Im. CDDO-Im treatment resulted in a Nrf2-dependent suppression of IL-2 secretion and an inhibition of NFkB activity that was partially Nrf2-dependent. Taken together, our model is the first to show that activation of Nrf2 suppresses early events of human T cell activation (This work is funded by NIH grants ES018885, ES024966 and GM092715).

Nrf2-Dependent and -Independent Effects of tert-Butylhydroquinone, CDDO-Im, and H2O2 in Human Jurkat T Cells as Determined by CRISPR/Cas9 Gene Editing.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress-activated transcription factor activated by stimuli such as electrophilic compounds and other reactive xenobiotics. Previously, we have shown that the commonly used food additive and Nrf2 activator tert-butylhydroquinone (tBHQ) suppresses interleukin-2 (IL-2) production, CD25 expression, and NFκB activity in human Jurkat T cells. The purpose of the current studies was to determine whether these effects were dependent upon Nrf2 by developing a human Nrf2-null T cell model using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. The current studies show that suppression of CD25 expression by tBHQ is partially dependent on Nrf2, whereas inhibition of IL-2 secretion is largely Nrf2-independent. Interestingly, tBHQ inhibited NFκB activation in an Nrf2-independent manner. This was an unexpected finding since Nrf2 inhibits NFκB activation in other models. These results led us to investigate another more potent Nrf2 activator, the synthetic triterpenoid 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im). Treatment of wild-type and Nrf2-null Jurkat T cells with CDDO-Im resulted in an Nrf2-dependent suppression of IL-2. Furthermore, susceptibility to reactive oxygen species was significantly enhanced in the Nrf2-null clones as determined by decreased mitochondrial membrane potential and cell viability. Importantly, this study is the first to describe the generation of a human Nrf2-null model, which is likely to have multiple applications in immunology and cancer biology. Collectively, this study demonstrates a role for Nrf2 in the effects of CDDO-Im on CD25 and IL-2 expression, whereas the effect of tBHQ on these parameters is complex and likely involves modulation of multiple stress-activated transcription factors, including NFκB and Nrf2.

The Nrf2 activator, tBHQ, inhibits IL-2 production by human T cells in a largely Nrf2-independent manner

Abstract Nrf2 is a transcription factor that is activated by cell stress, such as oxidative insult and exposure to xenobiotics. Upon activation, Nrf2 translocates to the nucleus, where it upregulates its target genes, many of which arecytoprotective in function. Nrf2 has been shown to modulate immune function; Nrf2-null mice are more sensitive to many models of inflammatory disease, including experimental autoimmune encephalitis and sepsis induced by cecal ligation and puncture. We have previously shown that activation of Nrf2 in primary mouse CD4 T cells promotes Th2 differentiation, which may have implications on the development of allergy and asthma. However, the role of Nrf2 in human CD4 T cell function remains uncharacterized. Thus, the purpose of the present studies was to determine the role of Nrf2 in the early events following CD4 T cell activation. Primary human CD4 T cells from peripheral blood were treated with varying concentrations of tBHQ (a known Nrf2 activator) prior to activation with anti-CD3/anti-CD28. Although treatment of human T cells with tBHQ suppressed the production of early cytokines, such as IL-2, the model fell short in determining whether these effects were Nrf2-dependent. Toward that end, Nrf2-null T cells were generated by implementation of CRISPR-CAS9 gene editing, in human Jurkat T cells. Treatment of Nrf2-null and Wild-type Jurkat T cells demonstrated that the effects of tBHQ upon cytokine production are largely independent of Nrf2. In contrast, suppression of CD25 expression by tBHQ is partially Nrf2-dependent. The current studies demonstrate that many of the effects of tBHQ on early events following CD4 T cell activation are independent of Nrf2. (This work was funded by NIH grants ES018885 and GM092715)

140. Point Mutation Correction for ADA SCID

Adenosine deaminase (ADA) deficiency is a rare genetic disease characterised by the absence of the ADA enzyme (EC 3.5.4.4) involved in purine salvage and degradation. Patients lacking ADA build up adenosine and deoxyadenosine metabolites in the organism, something that in turn leads to an accumulation of other deoxynucleotides and gives rise to functional defects in all lymphocyte lineages and severe combined immunodeficiency (ADA SCID). This SCID phenotype can be corrected by hematopoietic stem cell transplant, enzyme replacement therapy, and integrative gene therapy protocols employing autologous CD34+ cells. ADA deficiency confers a positive selective advantage to corrected lymphocytes over deficient cells, thus offering a great system for the study of novel molecular therapies such as gene editing. The advent of the CRISPR/Cas9 system has allowed for the swift tailoring of novel gene editing strategies in laboratories without access to advanced cloning platforms. We believe this democratisation of gene editing has also made more practicable the therapeutic reversal of genetic mutations into wild type sequences, the ultimate form of genetic correction. We have designed a CRISPR/Cas9 strategy that can target the nonsense Q3X (ADA c7C>T) point mutation endemic to ADA SCID patients of Somali origin residing in the UK, as well as its equivalent site in wild type sequences. In the present work we show evidence of the Q3X site non-homologous end joining (NHEJ) gene ablation in Jurkat cells by means of DNA sequencing and ADA protein readouts (approximately 70% efficiency). Moreover, we have designed a donor for the Q3X repair step that contains an adjacent traceable silent mutation in order to track any homology directed repair (HDR) when working with wild type cells. Here we show preliminary evidence of Q3X site HDR in wild type Jurkat cells in DNA sequencing results by detection of the aforementioned traceable mutation, albeit with low efficiency (less than 3% efficiency). We are currently attempting the translation of this approach into human CD34+ cells, the ultimate target cell population for the correction of the SCID phenotype in ADA deficient patients.

Study of antitumor effect of selected vanadium and molybdenum organometallic complexes in human leukemic T-cells

This work describes cytotoxic effect of non-platinum metal-based compounds on the human T-leukemic cells with different p53 status (p53 wild-type MOLT-4 and p53-deficient Jurkat cells). The cytotoxic and apoptosis-inducing effect of the vanadium complex [(η5-C5H5)2V(5-NH2-phen)]OTf (V1) and molybdenum complex [(η3-C3H5)Mo(CO)2(phen)Cl] (Mo1) were studied using flow cytometry, spectrophotometry and Western blotting. We found that the cytotoxic effect of both tested complexes after 24 h is higher against the both examined cell lines than that of cis-platin (cis-DDP). At later investigated time intervals of 48 and 72 h, the cytotoxic effect of the cis-DDP increased but the values of the cytotoxicity of the tested V1 and Mo1 complexes remained unchanged, with the cytotoxicity of V1 comparable to that of cis-DDP. Furthermore we observed that the apoptotic process was induced by the activation of the caspases 9 (intrinsic pathway) and 8 (extrinsic pathway) in cells exposed to evaluated complexes. In case of the p53 wild-type MOLT-4 cells, the expression of the tumor-suppressor protein p53 and its form phosphorylated at the serine 15 increased after both V1 and Mo1 treatment, similar to the effect of cis-DDP.

Abstract 2442: XPO1 is selinexor prime target: validation by mutating cysteine 528 on both XPO1 alleles using CRISPR/Cas9 genome editing

Abstract Human Exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports a variety of different cargo proteins out of the cell's nucleus. Inhibition of XPO1-mediated protein export results in nuclear retention of cargo proteins including tumour suppressor proteins and is a novel therapeutic strategy for cancer. Selinexor, an orally bioavailable XPO1 inhibitor, effectively demonstrates potent activity against multiple types of cancer. Here we directly demonstrate the high selectivity of selinexor for XPO1 inside living cells. Selinexor binds to the cargo-binding pocket of XPO1 and inhibits the formation of XPO1-cargo complex. The binding of selinexor to XPO1 involves a slowly reversible Michael addition type interaction of its acrylate moiety with cysteine 528 residue of XPO1. Using CRISPR/Cas9 genome editing we have now created a homozygous Jurkat T-ALL cell line carrying a Cys528Ser mutation in XPO1. This cell line was used to study the specificity of selinexor for XPO1 inside the cancer cell. Selinexor was highly toxic to wild-type Jurkat cells in the low nanomolar range while the mutant cells were resistant up to micromolar concentrations. These results were confirmed by resistance to induction of caspase dependent apoptosis as well as cell cycle arrest in mutant cells. The single Cys528Ser mutation thus confers resistance to selinexor. In addition, selinexor failed to accumulate cargo proteins in the nucleus of mutant cells. Moreover, the Cys528Ser substitution conferred resistance to the direct interaction of this drug to XPO1 in cells. In conclusion, these results validate XPO1 as the selinexor's prime target and pinpoint its unique selectivity to the Cys528 residue of XPO1. Our findings also demonstrate the general utility of CRISPR/Cas9 genome editing for introducing point mutations in the genome of cancer cells for drug-target specificity validation. Citation Format: Jasper E. Neggers, Thomas Vercruysse, Maarten Jacquemyn, Els Vanstreels, Erkan Baloglu, Sharon Shacham, Marsha Crochiere, William Senapedis, Yosef Landesman, Dirk Daelemans. XPO1 is selinexor prime target: validation by mutating cysteine 528 on both XPO1 alleles using CRISPR/Cas9 genome editing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2442. doi:10.1158/1538-7445.AM2015-2442

Glycosylphosphatidylinositol deficiency attenuates the production of infectious HIV-1 and renders the virions sensitive to complement attack (VIR1P.1128)

Abstract Human immunodeficiency virus type 1 (HIV-1) escapes complement-mediated destruction by incorporating regulators of complement activation (RCA) from the host cells. Abrogation of RCA function leads to complement-mediated virolysis. However, little is known about the mechanism by which HIV-1 incorporates RCA from the host cell. Here we used glycosylphosphatidylinositol (GPI)-deficient Jurkat (Jurkat-7) cells that only express intracellular CD59 (a key member of RCA), but not on the surface, to study how HIV-1 incorporates CD59 from an infected cell. Compared to wild type Jurkat cells, Jurkat-7 cells are less susceptible to HIV-1 infection, and produces lower amount of virions due to the impaired GPI biosynthesis. Interestingly, virions produced from Jurkat-7 cells did not contain CD59, while virions from wild-type Jurkat cells did contain CD59 with a certain amount of viral protein expression. This indicates that HIV-1 incorporates CD59 from the surface of host cell rather than from the intracellular compartments. Virions produced from Jurkat-7 cells are less infectious, but more susceptible to complement-mediated virolysis due to the failed CD59 incorporation into the virus surface. Furthermore, transient restoration of GPI anchor in Jurkat-7 cells resulted in recovered surface expression of CD59. These results reveal a critical role of GPI anchor and GPI-anchored proteins in HIV-1 life cycle and incorporation of CD59, probably other host cell proteins into the HIV-1 virions.

Caspase Activation and Aberrant Cell Growth in a p53+/+ Cell Line from a Li-Fraumeni Syndrome Family

Wild-type p53 is well known to induce cell cycle arrest and apoptosis to block aberrant cell growth. However, p53's unique role in apoptosis and cell proliferation in Li-Fraumeni Syndrome (LFS) has not been well elucidated. The aim of this study is to characterize the activity of wild-type p53 protein in LFS family dominated by a germline negative mutant p53. As expected, etoposide-treated wild-type p53-containing cell lines, LFS 2852 and control Jurkat, showed a greater rate of caspase- and annexin V-induced apoptotic cell death compared to the p53-mutant LFS 2673 cell line although mitochondrial and nuclear assays could not detect apoptosis in these organelles. The most intriguing part of the observation was the abnormal proliferation rate of the wild-type p53-containing cell line, which grew twice as fast as 2673 and Jurkat cells. This is important because apoptosis inducers acting through the mitochondrial death pathway are emerging as promising drugs against tumors where the role of p53 is not only to target gene regulation but also to block cell proliferation. This study casts a long shadow on the possible dysregulation of p53 mediators that enable cell proliferation. The deregulation of proliferation pathways represents an important anticancer therapeutic strategy for patients with the LFS phenotype.

Glycopeptide-Specific Chimeric Antigen Receptor Targeting of T Cell Leukemia

BackgroundAdoptive transfer of T cells redirected with chimeric antigen receptors (CARs) has shown tremendous efficacy in targeting CD19-expressing leukemic cells; however, these CAR T cells also deplete normal CD19-expressing B cells. Although B cell lymphopenia can be overcome by gamma-globulin supplementation, other forms of on-target, off-site toxicity emphasizes the need for cancer cell-specific epitopes in CAR T cell therapy. To date, T cell leukemia has not been targeted due to possible fratricide because of shared antigens expressed on leukemic T cells and CAR T cells. Cellular transformation often produces altered glycosylation patterns, making cancer-specific glycoantigens attractive targets for CAR development. Here we demonstrate the successful eradication of the Jurkat T cell leukemia with Tn-specific CAR T cells.MethodsThe Jurkat E6.1 T cell leukemia line contains a frameshift mutation in the T synthase chaperone protein Cosmc that prevents core 1 structure formation on O-linked protein glycosylation, resulting in the presentation of Tn antigen on many glycoproteins. Here we describe the development of a CAR using a scFv sequence derived from the 5E5 mAb, which was developed against the Tn antigen of Mucin-1 (Muc1). A CAR was constructed by linking the 5E5 scFv to CD8alpha hinge and transmembrane region to the signaling endodomain comprised of 4-1BB and CD3zeta. The resultant Tn specific CAR was expressed in a lentiviral vector.ResultsIn preliminary experiments Tn-CAR T cells had cytotoxicity to a variety of primary ovarian, breast and pancreatic tumor cells. Specific killing of Jurkat T leukemic cells was demonstrated by lysis of wild type Jurkat while the CAR T cells failed to kill Jurakat after overexpression of Cosmc. In specific, expression of full-length Cosmc expression in the Jurkat E6.1 cell line abolished staining by the 5E5 mAb, restoredT synthase activity, and protected the cells from lysis by 5E5-CAR T cells. Human T cells expressing the 5E5-CAR demonstrate rapid clearance of established Jurkat tumors in NSG mouse xenograft models, as assessed by serial bioluminescence imaging and prolonged survival compared to mice expressing a control CD19-specific CAR T cells (P<0.005).ConclusionsThese results demonstrate that the 5E5-CAR is specific for the Tn antigen and provides a potent cancer-specific immunotherapy for the targeting of non-dispensable tissues such as T cell leukemias. DisclosuresJune:Novartis: IP licensed by University of Pennsylvania to Novartis. Author entitled to royalties from the University. Patents & Royalties, Research Funding.

Abstract 646: Bi-specific T-cell engaging antibody activates T-cells to target the tumor associated antigen PR1

Abstract Acute (AML) and chronic (CML) myeloid leukemia together account for approximately 50% of newly diagnosed leukemias. Both AML and advanced CML have dismal survival outcomes but are susceptible to immunotherapy. We discovered PR1 (VLQELNVTV) as an effective human leukocyte antigen (HLA)-A2 restricted peptide and have targeted it in clinical trials using PR1 peptide vaccine. PR1 is derived from the serine proteases proteinase-3 and neutrophil elastase which are highly expressed in myeloid leukemia blasts. PR1 was shown to be immunogenic in myeloid malignancies, and we are currently developing several PR1 targeting therapies. We have completed the development and humanization of a T-cell receptor-like monoclonal antibody (h8F4) that targets PR1/HLA-A2 and eliminates primary human AML xenografts in NSG mice. To improve the efficacy of h8F4, we have developed a bi-specific T-cell engaging (BiTE) antibody that targets leukemia PR1/HLA-A2 and the T-cell receptor CD3. This would enhance the anti-leukemia immune response by bringing the T-cell in close proximity to the leukemia cell, and by further activating the T-cells to eliminate the target leukemia. Here we demonstrate successful construction and expression of the h8F4 BiTE in CHO cells. The antibody was purified from cell culture supernatant via Ni-NTA column chromatography, and identity was confirmed using western blot analysis. Next, flow cytometry was used to test the binding specificities of each targeting region of the BiTE antibody. The h8F4 BiTE antibody showed increased binding to T2 cells pulsed with PR1 peptide compared with T2 pulsed with irrelevant CMV peptide pp65 (MFI=627 and 72.4, respectively). In addition, the h8F4 BiTE demonstrated 6.8 fold higher binding to wild type Jurkat cells, which express CD3, in comparison with the CD3-lacking mutant Jurkat cell line J.RT3-T3.5. These results indicate that both regions of the h8F4 BiTE can efficiently recognize their intended targets. Next, we investigated whether h8F4 BiTE engagement with both PR1/HLA-A2 on T2 cells and CD3 on healthy donor PBMC could activate T-cells. We show with flow cytometry that upon 24h incubation in the presence of h8F4 BiTE and T2 cells pulsed with PR1 peptide, both CD4 and CD8 T-cells increase their surface expression of CD69 (21 fold in CD4; 10.3 fold in CD8), CD25 (35 fold in CD4; 3.5 fold in CD8) and CD71 (26.6 fold in CD4; 640 fold in CD8) compared to co-incubation in the absence of BiTE protein. In conclusion, this study shows that we have successfully developed a h8F4 BiTE which recognizes both the CD3 on T cells and PR1/HLA-A2 complexes on myeloid leukemia. Furthermore, the h8F4 BiTE can activate multiple T-cell subsets from healthy donor PBMC as early as 24h following co-incubation with PR1-pulsed T2 cells. This novel BiTE antibody could provide a safer and more potent treatment option for patients with aggressive myeloid leukemias that will improve upon currently available highly toxic standard therapies. Citation Format: Amanda Herrmann, Jin Im, Sijie Lu, Jeffrey Molldrem. Bi-specific T-cell engaging antibody activates T-cells to target the tumor associated antigen PR1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 646. doi:10.1158/1538-7445.AM2014-646

5-Lipoxygenase contributes to PPARγ activation in macrophages in response to apoptotic cells

Background: One hallmark contributing to immune suppression during the late phase of sepsis is macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells (AC). Taking the important role of the nuclear receptor PPARγ for this phenotype switch into consideration, it remains elusive how AC activate PPARγ in macrophages. Therefore, we were interested to characterize the underlying principle. Methods: Apoptosis was induced by treatment of Jurkat T cells for 3 hours with 0.5 μg/ml staurosporine. Necrotic cells (NC) were prepared by heating cells for 20 minutes to 65°C. PPARγ activation was followed by stably transducing RAW264.7 macrophages with a vector encoding the red fluorescent protein mRuby after PPARγ binding to 4 × PPRE sites downstream of the reporter gene sequence. This readout was established by treatment with the PPARγ agonist rosiglitazone (1 μM) and AC (5:1). Twenty-four hours after stimulation, mRuby expression was analysed by fluorescence microscopy. Lipid rafts of AC, NC, as well as living cells (LC) were enriched by sucrose gradient centrifugation. Fractions were analysed for lipid raft-associated marker proteins. Lipid rafts were incubated with transduced RAW264.7 macrophages as described above. 5-Lipoxygenase (5-LO) involvement was verified by pharmacological inhibition (MK-866, 1 μM) and overexpression. Results: Assuming that the molecule responsible for PPARγ activation in macrophages is localized in the cell membrane of AC, most probably associated to lipid rafts, we isolated lipid rafts from AC, NC and LC. Mass spectrometric analysis of lipid rafts of AC showed the expression of 5-LO, whereas lipid rafts of LC did not. Moreover, incubating macrophages with lipid rafts of AC induced mRuby expression. In contrast, lipid rafts of NC and LC did not. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated for 30 minutes with the 5-LO inhibitor MK-866 (1 μM) before apoptosis induction. In line with our hypothesis, these AC did not induce mRuby expression. Finally, although living Jurkat T cells overexpressing 5-LO did not activate PPARγ in macrophages, mRuby expression was significantly increased when AC were generated from 5-LO overexpressing compared with wild-type Jurkat cells. Conclusion: Our results suggest that induction of apoptosis activates 5-LO, localizing to lipid rafts, necessary for PPARγ activation in macrophages. Therefore, it will be challenging to determine whether 5-LO activity in AC, generated from other cell types, correlates with PPARγ activation, contributing to an immune-suppressed phenotype in macrophages.

Nanosecond pulsed electric fields (nsPEFs) activate intrinsic caspase-dependent and caspase-independent cell death in Jurkat cells

NsPEF ablation induces apoptosis markers, but specific cell death pathways have not been fully defined. To identify nsPEF-activated cell death pathways, wildtype human Jurkat cells and clones with deficiencies in extrinsic and intrinsic apoptosis pathways were investigated. NsPEFs activated caspase isozymes and induced identical electric field-dependent cell death in clones deficient in FADD or caspase-8, indicating that extrinsic apoptosis pathways were not activated. This was confirmed when cytochrome c release was shown to be unaffected by the pan caspase inhibitor, z-VAD-fmk. NsPEF-treated APAF-1-silenced cells did not exhibit caspase-3/7 and -9 activities and corresponding electric field-dependent cell death in this clone was attenuated compared to its vector control at low, but not at high electric fields. These data demonstrate that nsPEFs induce intrinsic apoptosis activate by cytochrome c release from mitochondria through an APAF-1- and caspase-dependent pathway as well as through caspase-independent mechanisms that remain to be defined. Furthermore, the results establish that nsPEFs can overcome natural and oncogenic mechanisms that promote cell survival through inhibition of apoptosis and other cell death mechanisms.