HT-2 Cells Research Articles

Abstract 2323: ZGGS18, a bispecific drug candidate of anti-VEGF and TGFbeta-trap, inhibiting tumor proliferation and improving tumor microenvironment with I/O therapy

Abstract ZGGS18 is a bifunctional fusion protein targeting both human vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β), which consist of a monoclonal antibody against VEGF and an engineered extracellular domain of TGF-β receptor II (TGF-βRII ECD). In vitro studies show ZGGS18 specifically and simultaneously binding towards various VEGFs and capturing different TGF-β isoforms by trap domains that play a synergistic role to block signaling of VEGF/TGF-β with their receptors. For instance, ZGGS18 inhibits human VEGF121 and 165 induced HUVEC proliferation; ZGGS18 also neutralizes TGF-β1 and -β3 related growth arrest of HT-2 cells. Further studies show that ZGGS18 does not induce ADCC- or CDC-dependent killing tumor cells expressing two soluble targets, suggesting its binding affinities towards Fc-gamma and complement C1q receptors without effector functions. These results support functional depletion of VEGF/TGF-β in tumor microenvironment (TME) by ZGGS18 essentially for its MOA to inhibit tumor growth. A dose-dependent in vivo anti-tumor efficacy of ZGGS18 (1, 3 and 15 mg/kg) has been found in PBMC humanized H460 lung cancer xenograft model. In addition, ZGGS18 has enhanced efficacies to kill tumor growth in MC38 xenograft models when it is combined with anti-PD-1 antibodies (keytruda), support the improvement of TME for I/O reagents by ZGGS18 in cooperation with its role in anti-proliferation and anti-angiogenesis. Toxicity and safety pharmacology studies with weekly repeat doses for four weeks in cynomolgus monkeys indicate that ZGGS18 is well tolerated over the dose range 13, 40 and 120 mg/kg without obviously toxic or adverse effects (NOAEL =120 mg/kg). PK/TK analyses indicate that ZGG18 has a prolonged half-life in plasma, attribute to its human neonatal Fc receptor (FcRn) binding and pH-dependent interaction for mAb recycling. The IND application of ZGGS18 has been approved by both FDA and Chinese NMPA for clinical trials in treatment of advanced tumors as monotherapy and/or in combined with PD-1/PD-L1 based I/O therapies. Citation Format: Ruifeng Liu, Alfonso Suarez, Tyler Liban, Bin Zhang, Tongcheng Dai, Margaret Karow, Jackie Sheng, Zelin Sheng, Binhua Lv, Bing Zhu. ZGGS18, a bispecific drug candidate of anti-VEGF and TGFbeta-trap, inhibiting tumor proliferation and improving tumor microenvironment with I/O therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2323.

Flow Cytometry Analysis of Anastasis using CellTrace Far Red (CTFR) cell tracker in Cultured Mammalian T Lymphocytes

Anastasis is a cellular recovery process that rescues apoptotic cells from the brink of death. Our lab has developed a model for studying anastasis using cultured mouse HT2 lymphocytes dependent on the cytokine interleukin 2 (IL2). Within 24 hours of IL2 deprivation, HT2 cells display characteristic changes underlying apoptosis, including cellular condensation, membrane blebbing and fragmentation into apoptotic bodies, phosphatidylserine externalization, caspase 3 activation as well as G1 cell cycle arrest. HT2 cells can subsequently be rescued by adding IL2 back into the culture medium. In this study, we have used quantitative flow cytometry in conjunction with CellTrace Far Red (CTFR) and SYTOX Green (live/dead indicator) to track the kinetics of anastasis and re‐entry of viable apoptotic cells into the cell cycle. HT2 lymphocytes continuously maintained in IL2 comprised a homogeneous, viable population of CTFR+ cells which decreased in fluorescence intensity with each subsequent cell cycle for up to 96 hours post‐CTFR labelling. To investigate apoptosis reversal, HT2 cells were labeled with CTFR, deprived of IL2 for 24 hours and then rescued with IL2 re‐addition for periods of 24, 48, and 72 hours. Our findings indicate that viable, CTFR+ HT2 cells gradually resumed cell cycle entry over the 72‐hour rescue period. As caspase activation represents a cellular marker of early‐mid stage apoptotic cells, we also investigated caspase 3/7 activity in viable CTFR+ cells using the Nucview‐488 fluorescent caspase 3 substrate. Three distinct populations of caspase‐high, medium and low, viable CTFR+ HT2 cells were observed at 24 hours following IL2 deprivation. IL2‐rescued cells subsequently segregated into caspase‐high and low populations, ultimately merging into a single, caspase‐low population by 72 hours post‐rescue. Collectively, our findings suggest that CTFR tracking can be used successfully as a tool to follow the kinetics of apoptosis reversal in IL2‐dependent mouse HT2 lymphocytes, and is a promising model for the study of the mechanistic basis of anastasis.Support or Funding InformationUnion College Faculty and Student Research Grants

Flow Cytometry Analysis of Anastasis in Cultured Mammalian Lymphocytes

Apoptosis is a physiological form of programmed cell death characterized by phosphatidylserine externalization, caspase activation, and genomic DNA fragmentation. Recent studies have shown that apoptosis may be reversed even in its late stages, calling the previous notion of a ‘point of no return’ into question. This recovery process has been termed anastasis (‘rising to life’), but its underlying mechanisms are not well understood. Our lab has developed a model of studying anastasis using cultured mouse HT‐2 lymphocytes dependent on the cytokine interleukin 2 (IL2). Our previous findings have shown that HT‐2 cells accumulate in the G1 stage of the cell cycle following 24 hours of IL2 deprivation and can be rescued by adding IL2 back into the culture medium. In this study, we have used quantitative flow cytometry to investigate which apoptotic events are congruent with anastasis. HT2 cells were deprived of IL2 for 24 hours and then rescued for periods of 0, 24, 48, and 72 hours. CD25 surface expression (high‐affinity IL2 receptor) and phosphatidylserine externalization (annexin V staining) were subsequently monitored prior to and following recovery, as markers of T cell activation and early stage apoptosis, respectively. Control HT2 lymphocytes continuously maintained in IL2 comprised a single viable population of CD25HIGHAnnexin−cells. Cells deprived of IL2 for 24 hours were observed to fall into two populations: CD25LOWAnnexin+early apoptotic and CD25LOWAnnexinnon‐apoptotic cells. In both populations, CD25 expression was reduced approximately 10‐fold when compared to IL2‐maintained controls. During anastasis, three distinct viable cell populations emerged: 1) CD25LOWAnnexin+cells that did not progress into the later stages of apoptosis, 2) CD25LOWAnnexin−non‐apoptotic cells, and 3) CD25HIGHAnnexin−non‐apoptotic cells. With advancing recovery time, viable CD25LOWpopulations decreased as CD25HIGHcells increased. By 72 hours post‐recovery, the majority of cells were actively cycling, annexin negative cells. These findings suggest that IL2‐dependent mouse HT‐2 lymphocytes may be a promising model to study the mechanisms of anastasis.Support or Funding InformationUnion College Faculty and Student Research GrantsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Flow Cytometry Cell Cycle Analysis of Apoptotic Mouse HT‐2 T Lymphocytes

Apoptosis is an essential form of programmed cell death (PCD) in multicellular animals, in which cells are dismantled and disposed of in a controlled fashion. In the vertebrate immune system, apoptosis prevents the emergence of autoreactive lymphocytes, and is essential for self‐tolerance. In mouse HT‐2 T lymphocytes, apoptosis and internucleosomal DNA fragmentation can be initiated by cytokine deprivation (interleukin‐2 or IL‐2), addition of the protein kinase inhibitor staurosporine, or addition of the topoisomerase inhibitor camptothecin. We used flow cytometry to determine if apoptosis in HT‐2 cells involved accumulation in a specific phase of the cell cycle, and whether the mode of induction affected cell cycle distribution. Our findings indicate that IL‐2 deprivation induces cell cycle arrest in the G1 phase, prior to the accumulation of cells with sub‐G1 DNA content. In contrast, HT‐2 cells treated with staurosporine and camptothecin accumulated in G2/M and S phase, respectively. We used western blotting and quantitative chemiluminescence to demonstrate that Kip‐1 (p‐27), a CDK‐inhibitor that regulates the progression of cells from G1 to S phase, is up‐regulated in a time‐dependent fashion following IL‐2 deprivation and staurosporine. When compared to HT‐2 control cultures (+IL‐2) and Beta‐tubulin protein levels, Kip‐1 levels were significantly increased 24 hours following IL‐2 deprivation, and 6 hours post‐staurosporine addition. These findings suggest that Kip‐1 up‐regulation may be involved in multiple apoptotic pathways of T lymphocytes. Studies are currently underway to quantitatively determine protein levels of S (PCNA) and M (phospho‐histone H3) phase‐specific markers in HT‐2 cells in response to apoptotic stimuli.

Overproduction of recombinant human transforming growth factor beta 3 in Chinese hamster ovary cells

Transforming growth factor beta 3 (TGFβ3) is an important cytokine, functioning in cell proliferation and differentiation, and has been considered to have therapeutic potential for treating various diseases and for scar reduction in adult wound healing. In the current study, a Chinese hamster ovary (CHO) cell line overexpressing recombinant human TGFβ3 (rhTGFβ3) was established. Through a 15-day fed-batch culture process in a 7.5-l bioreactor (5-l working volume) using chemically defined medium, the established cells could produce over 133mg/l of rhTGFβ3 protein. The rhTGFβ3 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 65%, with protein purity greater than 97%. The N-terminal amino acid sequences of the purified rhTGFβ3 were confirmed by N-terminal sequencing analysis. The purified rhTGFβ3 was further demonstrated to be functionally active by measuring the inhibition of growth of HT-2 cells, revealing a half-maximal effective concentration of 42.11pg/ml and specific activity of 1.84×107U/mg.

BCL-2 Is a Downstream Target of ATF5 That Mediates the Prosurvival Function of ATF5 in a Cell Type-dependent Manner

ATF5 loss of function has been shown previously to cause apoptotic cell death in glioblastoma and breast cancer cells but not in non-transformed astrocytes and human breast epithelial cells. The mechanism for the cell type-dependent survival function of ATF5 is unknown. We report here that the anti-apoptotic factor BCL-2 is a downstream target of ATF5 that mediates the prosurvival function of ATF5 in C6 glioma cells and MCF-7 breast cancer cells. ATF5 binds to an ATF5-specific regulatory element that is downstream of and adjacent to the negative regulatory element in the BCL-2 P2 promoter, stimulating BCL-2 expression. Highlighting the critical role of BCL-2 in ATF5-dependent cancer cell survival, expression of BCL-2 blocks death of C6 and MCF-7 cells induced by dominant-negative ATF5, and depletion of BCL-2 impairs ATF5-promoted cell survival. Moreover, we found that BCL-2 expression is not regulated by ATF5 in non-transformed rat astrocytes, mouse embryonic fibroblasts, and human breast epithelial cells, where expression of BCL-2 but not ATF5 is required for cell survival. These findings identify BCL-2 as an essential mediator for the cancer-specific cell survival function of ATF5 in glioblastoma and breast cancer cells and provide direct evidence that the cell type-specific function of ATF5 derives from differential regulation of downstream targets by ATF5 in different types of cells.

Synthesis of Dideoxymycobactin Antigens Presented by CD1a Reveals T Cell Fine Specificity for Natural Lipopeptide Structures

Mycobacterium tuberculosis survival in cells requires mycobactin siderophores. Recently, the search for lipid antigens presented by the CD1a antigen-presenting protein led to the discovery of a mycobactin-like compound, dideoxymycobactin (DDM). Here we synthesize DDMs using solution phase and solid phase peptide synthesis chemistry. Comparison of synthetic standards to natural mycobacterial mycobactins by nuclear magnetic resonance and mass spectrometry allowed identification of an unexpected alpha-methyl serine unit in natural DDM. This finding further distinguishes these pre-siderophores as foreign compounds distinct from conventional peptides, and we provide evidence that this chemical variation influences the T cell response. One synthetic DDM recapitulated natural structures and potently stimulated T cells, making it suitable for patient studies of CD1a in infectious disease. DDM analogs differing in the stereochemistry of their butyrate or oxazoline moieties were not recognized by human T cells. Therefore, we conclude that T cells show precise specificity for both arms of the peptide, which are predicted to lie at the CD1a-T cell receptor interface.

Transforming growth factor beta in human milk and some infant formula are biologically active in HT‐2 cells

Transforming growth factor beta (TGFβ) of human milk (HM) is thought to play an important role in maintaining appropriate immune function in infants. In the present study we assessed and compared total and TGFβ2 specific activity in HM and infant formulas (IF) in HT‐2 cells. This bioassay is used to study regulation of T‐cell growth by TGFβ inhibiting the S‐phase progression of HT‐2 cells stimulated with IL‐4. Mature HM were sampled from mothers in the US and Mexico, while IF samples (Enfamil® LIPIL®/A+®) were obtained from US, China, Mexico, Thailand, and the Philippines from the manufacturer. HM inhibited cell growth in a dose‐dependent manner. HM at a concentration of TGFβ equivalent to that found of Enfamil (4900 ± 1400 pg/ml) exhibited a 2‐folds higher total bioactivity, which may be explained in part by additional functional factors in HM that also inhibit cell proliferation, differences in physiochemical properties of the milks, or potential matrix effects. TGFβ related activity in HM did not differ between US and Mexican mothers; similarly TGFβ related activity in IF did not vary by the manufacturer location. TGFβ2 specific neutralizing antibody decreased activity in IF and HM by 2‐3 folds and 2‐7 folds respectively, suggesting that some, but not all, of the activity is due to TGFβ2. These results demonstrate that both infant formula and HM exerted similar TGFβ related activities in HT‐2 cells. Supported by MJN.

A role for STAT5 in the pathogenesis of IL-2-induced glucocorticoid resistance.

Glucocorticoids (GC) are highly effective in the control of diseases associated with T cell activation. However, a subset of individuals is GC insensitive. Previous studies have demonstrated that IL-2 can induce steroid resistance in mouse T cells. However, the mechanism for this phenomenon is unknown. In the current study we found that the murine cell line (HT-2) is steroid resistant when incubated with IL-2, but steroid sensitive when grown in IL-4. Furthermore, when HT-2 cells are treated with IL-2, the glucocorticoid receptor (GCR) does not translocate to the cell nucleus after dexamethasone treatment. In contrast, the GCR in IL-4-stimulated HT-2 cells does translocate into the cell nucleus after dexamethasone treatment. IL-2-induced steroid insensitivity in HT-2 cells appears to be a signaling event as the effects of IL-2 on nuclear translocation of the GCR occurred within 30 min even in the presence of cycloheximide. Indeed, preincubation of HT-2 cells with a Janus-associated kinase 3 inhibitor restored nuclear translocation of the GCR even in the presence of IL-2. Immunoprecipitation experiments revealed that phosphorylated STAT5 and GCR formed immune complexes. This association may lead to retardation of GCR nuclear translocation because IL-2 was not able to induce steroid insensitivity in splenocytes from STAT5 knockout mice. This study demonstrates a novel role for STAT5 in IL-2-induced steroid insensitivity.

Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors

To test the effect of co-expression of immunomodulatory molecules, together with target antigen, two recombinant Streptococcus gordonii strains were constructed which secreted either murine interleukin-2 (IL-2) or interferon-gamma (IFN-γ) in addition to a surface anchored test antigen (the conserved C-repeat region (CRR) of the M6 protein of Streptococcus pyogenes). The secretion of functional cytokines by S. gordonii was achieved by in-frame fusion of sequences encoding mature IL-2 or IFN-γ to the sequences encoding the leader signal of the M6 protein. Expression of the M protein CRR region from a separate chromosomal site produced double recombinants expressing a secreted cytokine and the M protein CRR region anchored to the surface. Protein expression was verified by streak blot, immunoblot, and ELISA on both the single and double recombinants. A cytokine bioassay using HT-2 cells verified biological activity of recombinant IL-2 secreted from S. gordonii. When mice were immunized subcutaneously with the different S. gordonii expression strains, cytokine co-expression apparently modulated the systemic immune response. These results show that streptococci can deliver biologically active molecules such as cytokines along with antigens to the immune system. These results demonstrate that a cytokine-secreting, noninvasive, bacterial vaccine vector can be used to modulate immune responses to a co-expressed antigen.

Genetic Evidence for an Additional Factor Required for Erythropoietin-Induced Signal Transduction

Erythropoietin (EPO) and its receptor (EPOR) are required for the development of mature erythrocytes. After binding of ligand, the EPOR activates a variety of signaling pathways that ultimately control cellular proliferation, survival, and specific gene expression. Although erythroid progenitors appear to be the principal EPO-responsive cell type in vivo due to the restricted expression of the EPOR, many growth factor-dependent cell lines expressing the EPOR can respond to EPO by activating many or all of these pathways. In the present study, we have identified a cellular context (the interleukin-2 [IL-2]-dependent HT-2 line) in which the EPO stimulation of the EPOR fails to support cellular proliferation, STAT-5 induction, or MAPK activation, despite efficient phosphorylation of the EPOR and JAK2 and inhibition of apoptosis after withdrawal of IL-2. Interestingly, when we fused HT-2 cells expressing the EPOR with Ba/F3 cells in a complementation assay, the resulting hybridomas proliferated and potently activated STAT-5 and MAPK in response to EPO. These data indicate that an unidentified cellular factor is needed to mediate signaling by the EPOR. Moreover, Ba/F3 cells apparently express this factor(s) and somatic fusions can, therefore, confer EPO-responsiveness to HT-2 cells that lack this factor.

Increased Zap-70 Association with CD3ζ in CD4 T Cells from Old Mice

Aging diminishes the amount of phosphotyrosine in the CD3ζ chains of resting and activated mouse CD4 T cells by about threefold and might therefore be expected to a corresponding decline in Zap-70 association with CD3ζ and in Zap-70 kinase function in CD3ζ complexes. We show here that aging leads, unexpectedly, to an approximately twofold increase in the amount of Zap-70 associated with CD3ζ in resting CD4 T cells. There is, however, no effect of age on total intracellular Zap-70 content. Cross-linking CD3 to CD4 leads to an increase of only 50% in the functional activity of Zap-70 in CD3ζ complexes from freshly isolated CD4 T cells of young donors. Compared to Jurkat and HT-2 cells, fresh T cells show both higher baseline levels and lower induced levels of Zap-70 function in CD3ζ complexes. CD4 T cells from old mice have baseline levels of Zap-70 activity similar to those seen in activated T cells from young mice, and these levels do not increase after CD3/CD4 cross-linking. Tyrosine-specific phosphorylation of Zap-70 is also higher at rest in old T cells than in young T cells and inducible only in cells from young donors. These data suggest that age-related defects in T cell activation are not likely to be attributable simply to a decline in Zap-70 association with CD3ζ or to diminished Zap-70 phosphorylation. The increase with age in CD3ζ-Zap association, despite the loss with age in CD3ζ tyrosine phosphorylation, suggests that the pattern of tyrosine phosphate groups among CD3ζ ITAM groups may be different in T cells from young and old donors or that access to ITAM regions within CD3ζ may be blocked by inter- or intramolecular steric hindrance in young CD4 T cells.

PGE2INHIBITS IL-2 AND IL-4-DEPENDENT PROLIFERATION OF CTLL-2 AND HT2 CELLS

The cytotoxic T cell line CTLL-2 and the T helper cell line HT2 proliferative in response to interleukin 2 (IL-2) or IL-4 without requiring stimulation by antigen through the T cell receptor and therefore lend themselves for studies of IL-2 and IL-4-dependent proliferation and signalling through their cognate receptors. Here we have used CTLL-2 and HT2 cells to investigate the effect of the inflammatory mediator prostaglandin E2(PGE2) on IL-2- and IL-4-dependent proliferation. PGE2inhibited IL-2- as well as IL-4-dependent proliferation of both CTLL-2 and HT2 cells, with IL-4-dependent proliferation being more sensitive than IL-2-dependent proliferation and CTLL-2 cells being more sensitive than HT2 cells. A quantitative dose–effect analysis revealed a two-step increase of inhibition (around 10−10M and 10−5M PGE2) for all combinations of cells and cytokines approaching 100% at high concentrations of PGE2. The data suggest that even in cases where synthesis of IL-2 and IL-4 is differently affected by PGE2, IL-2- and IL-4-dependent T cells may still be similarly sensitive to PGE2by way of their cytokine responsiveness. Furthermore, the effects of PGE2may be mediated by more than one functional binding site or receptor subtype. PGE2levels are an important consideration when CTLL-2 and HT2 cells are used for the measurement of IL-4 and IL-2.

Molecular role of TGF-beta, secreted from a new type of CD4+ suppressor T cell, NY4.2, in the prevention of autoimmune IDDM in NOD mice.

A new type of CD4+ T cell clone (NY4.2) isolated from pancreatic islet-infiltrated lymphocytes of acutely diabetic non-obese diabetic (NOD) mice prevents the development of insulin-dependent diabetes mellitus (IDDM) in NOD mice, as well as the recurrence of autoimmune diabetes in syngeneic islet-transplanted NOD mice. It has been demonstrated that the cytokine TGF-beta, secreted from the cells of this clone, is the substance which prevents autoimmune IDDM. This investigation was initiated to determine the molecular role TGF-beta plays in the prevention of autoimmune IDDM by determining its effect on IL-2-induced signal transduction in Con A-activated NOD mouse splenocytes and HT-2 cells. First, we determined whether TGF-beta, secreted from NY4.2 T cells, inhibits IL-2-dependent T cell proliferation in HT-2 cells (IL-2-dependent T cell line) and NOD splenocytes. We found that TGF-beta suppresses IL-2-dependent T cell proliferation. Second, we determined whether TGF-beta inhibits the activation of Janus kinases (JAKs), as well as signal transducers and activators of transcription (STAT) proteins, involved in an IL-2-induced signalling pathway that normally leads to the proliferation of T cells. We found that TGF-beta inhibited tyrosine phosphorylation of JAK1, JAK3, STAT3 and STAT5 in Con A blasts from NOD splenocytes and HT-2 cells. Third, we examined whether TGF-beta inhibits the cooperation between STAT proteins and mitogen-activated protein kinase (MAPK), especially extracellular signal-regulated kinase 2 (ERK2). We found that TGF-beta inhibited the association of STAT3 and STAT5 with ERK2 in Con A blasts from NOD splenocytes and HT-2 cells. On the basis of these observations, we conclude that TGF-beta may interfere with signal transduction via inhibition of the IL-2-induced JAK/STAT pathway and inhibition of the association of STAT proteins with ERK2 in T cells from NOD splenocytes, resulting in the inhibition of IL-2-dependent T cell proliferation. TGF-beta-mediated suppression of T cell activation may be responsible for the prevention of effector T cell-mediated autoimmune IDDM in NOD mice by TGF-beta-producing CD4+ suppressor T cells.

Dynamic equilibrium between calcineurin and kinase activities regulates the phosphorylation state and localization of the nuclear factor of activated T-cells.

The nuclear factor of activated T-cells (NFATp) is a phosphorylated transcription factor that resides in the cytoplasm of unactivated T-cells. T-cell activation results in the activation of the phosphatase calcineurin (CaN), which leads to the dephosphorylation and subsequent nuclear localization of NFATp. We have investigated the role of kinases in the phosphorylation state and subcellular localization of NFATp. The phosphorylation state and nuclear/cytoplasmic location of NFATp were determined in unstimulated murine HT-2 cells treated with a panel of kinase inhibitors. Two of the seven kinase inhibitors, staurosporine (St) and bisindolylmaleimide I (BI), resulted in the dephosphorylation and nuclear localization of NFATp. These St-induced effects were inhibited by pretreatment with FK506, indicating that CaN activity was required for the observed effects on NFATp. Treatment of cells with ionomycin resulted in NFATp dephosphorylation and nuclear localization. Removal of ionomycin from the cells resulted in the reappearance of phosphorylated NFATp in the cytosol. St and BI also inhibited the re-accumulation of NFATp in the cytoplasm and its re-phosphorylation after ionomycin removal. The re-accumulation of NFATp in the cytosol after ionomycin withdrawal was shown to be energy- and temperature-dependent. Taken together, these results suggest that in unstimulated cells NFATp is actively maintained in the cytoplasm by kinases acting in opposition to basal CaN activity.

An Acid Phosphatase Assay for Quantifying the Growth of Adherent and Nonadherent Cells

We describe an acid phosphatase assay for determination of cell growth based on quantification of cytosolic acid phosphatase activity. The assay is based on the hydrolysis of thep-nitrophenyl phosphate by intracellular acid phosphatases in viable cells to producep-nitrophenol. For all cell types examined, absorbance ofp-nitrophenol at 405 nm is directly proportional to the cell number in the range of 103–105cells. The assay can quantify as few as 1000 cells per well in 96-well microtiter plates. The acid phosphatase assay was used to count various adherent and nonadherent cells, including human tumors, L6, and HT-2 cells. We also demonstrate the utility of this assay for analysis of growth factor and cytokine bioactivity on mammalian cells in culture. In comparison to [3H]thymidine incorporation, the acid phosphatase assay has similar sensitivity but a wider linear response range. The method also shows higher sensitivity and reproducibility in comparison to cell proliferation assays based on the reduction of tetrazolium salts. Because of the ease of use, sensitivity, and low cost, the acid phosphatase method is especially suited to applications where a large number of samples are assayed.

Application of murine T-T hybridoma cells to in vitro potency assay of human synthetic peptide vaccines

Recently, it has been shown that the immunization of mice with an 18 amino acid synthetic peptide corresponding to the third hypervariable region of MHC class II β chain can induce a specific antibody response against MHC class II molecules, and can be utilized in the prevention and treatment of experimental allergic encephalomyelitis (EAE) [ Proc. Natl. Acad. Sci. 1994, 91, 8005–8009] 1. Based on this finding, a chemically-modified synthetic peptide with the amino acid sequence corresponding to residues of β 57–76 from human HLA-DR4Dw4 ( DR4 1 peptide) is being clinically investigated for the treatment of rheumatoid arthritis in human. The present study describes the development of a novel in vitro potency assay for human HLA-DR4 1 peptide using cloned murine T-T hybridoma cells. Several mouse strains were immunized with the DR4 1 peptide and their lymph node T cell proliferation was measured in the presence of syngeneic APCs and the DR4 1 peptide. T cells isolated from the peptide primed-B10.PL mouse strain, which showed the highest recall response in this assay, were fused with BW5147 lymphoma cells to generate DR4 1 peptide-specific T-T hybridoma clones. Cloned hybridoma cells were characterized for peptide specificity and MHC class II restriction, and used to monitor the biological activity of various DR4 1 peptide preparations. The potency of peptide batches were assessed by measuring the IL-2 secretion of cloned T-T hybridoma cells upon TCR engagement in an antigen-specific manner. The quantitative detection of IL-2 was performed by measuring [ 3H]thymidine incorporation of HT-2 cells or directly by ELISA. These results demonstrate that peptide-specific murine T-T hybridoma clones can be successfully utilized to monitor biological activity of synthetic peptides by measuring T cell-mediated immunological responses. Development of such in vitro potency assay for synthetic peptides may have broad applications for vaccines related to immunological disorders.

Direct Demonstration of NFATp Dephosphorylation and Nuclear Localization in Activated HT-2 Cells Using a Specific NFATp Polyclonal Antibody

Nuclear factor of activated T cells (NFAT) regulates transcription of a number of cytokine genes, and NFAT DNA binding activity is stimulated following T cell activation. Several lines of evidence have suggested that NFAT is a substrate for calcineurin, a serine/threonine phosphatase. Using a polyclonal antibody to murine NFATp, Western blot analysis of various mouse tissues demonstrated that the 110-130-kDa NFATp protein was highly expressed in thymus and spleen. Treatment of immunoprecipitated NFATp from untreated HT-2 cells with calcineurin resulted in the dephosphorylation of NFATp, demonstrating that NFATp is an in vitro substrate for calcineurin. NFATp immunoprecipitated from 32P-labeled HT-2 cells migrated as an approximately 120-kDa protein that was localized to the cytosol of the cells. Treatment of the cells with ionomycin resulted in a decrease in the molecular weight of NFATp and a loss of 32P, consistent with NFATp dephosphorylation. The dephosphorylation of NFATp was accompanied by localization of the protein to the nuclear fraction. Both of these events were blocked by preincubation of the cells with FK506, a calcineurin inhibitor, consistent with the hypothesis that NFATp is a calcineurin substrate in cells.

Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity.

To explore the possible involvement of STAT factors ("signal transducers and activators of transcription") in the interleukin 2 receptor (IL-2R) signaling cascade, murine HT-2 cells expressing chimeric receptors composed of the extracellular domain of the erythropoietin receptor fused to the cytoplasmic domains of the IL-2R beta or -gamma c chains were prepared. Erythropoietin or IL-2 activation of these cells resulted in rapid nuclear expression of a DNA-binding activity that reacted with select STAT response elements. Based on reactivity with specific anti-STAT antibodies, this DNA-binding activity was identified as a murine homologue of STAT-5. Induction of nuclear expression of this STAT-5-like factor was blocked by the addition of herbimycin A, a tyrosine kinase inhibitor, but not by rapamycin, an immunophilin-binding antagonist of IL-2-induced proliferation. The IL-2R beta chain appeared critical for IL-2-induced activation of STAT-5, since a mutant beta chain lacking all cytoplasmic tyrosine residues was incapable of inducing this DNA binding. In contrast, a gamma c mutant lacking all of its cytoplasmic tyrosine residues proved fully competent for the induction of STAT-5. Physical binding of STAT-5 to functionally important tyrosine residues within IL-2R beta was supported by the finding that phosphorylated, but not nonphosphorylated, peptides corresponding to sequences spanning Y392 and Y510 of the IL-2R beta tail specifically inhibited STAT-5 DNA binding.

Mutation of Asp20 of human interleukin-2 reveals a dual role of the p55 alpha chain of the interleukin-2 receptor.

Mutation of Asp20 in human interleukin-2 (IL-2) to Lys is known to result in an IL-2 molecule with unchanged binding to the p55 subunit of the IL-2 receptor, but with greatly decreased affinity for the p75 subunit (Collins, L., Tsien, W.-H., Seals, C. et al. Proc. Natl. Acad. Sci USA 1988. 85: 7709). Here we demonstrate that Lys20 IL-2 competed with a reduced (10-fold) affinity for high-affinity IL-2 receptors on two murine cell lines HT2 and CTLL. In parallel with this difference in receptor interaction, Lys20 IL-2 stimulated half-maximal HT2 cell proliferation at a 10-fold higher concentration than wild-type IL-2. However, half-maximal stimulation of CTLL cells required a 100-fold higher concentration of Lys20 IL-2. A similar 100-fold reduction in bioactivity of Lys20 IL-2 was observed for primary, activated, human or murine lymphocytes. Anti-p55 antibodies increased the concentration of Lys20 IL-2 required to stimulate HT2 cells to that required for CTLL cells. These data suggest that CTLL cells, while able to bind Lys20 IL-2 with high affinity, are lacking a p55-dependent function necessary for optimal stimulation. Therefore, p55 has a dual role, being important both for high-affinity IL-2 binding and for optimal cell triggering.