Constitutive and mitogen-induced production of T cell growth factor by stable T cell hybridoma lines

Stable T cell growth factor- (TCGF; IL 2) producing cloned T cell hybridoma lines were constructed by fusing murine alloantigen-activated T cells with the 8-azaguanine-resistant lymphoma line, BW5147. Many, but not all, clones of one of these hybridomas, i.e., hybridoma 24, secreted TCGF constitutively, but production was markedly enhanced by stimulation with T cell mitogens. Large numbers of TCGF-secreting hybridoma cells in a stable functional state could be obtained from histocompatible mice inoculated with cloned T cell hybridomas. Moreover, such in vivo-derived hybridoma cells could be stimulated sequentially with mitogen at least twice to secrete their biologically-active product, resulting in larger TCGF yields from the same cells. The secreted product of these T cell hybridoma lines resembled TCGF isolated from other cellular sources in that it: a) supported the growth of a TCGF-dependent T cell line; b) provided help for the induction of alloantigen-reactive cytotoxic T lymphocytes from thymocyte precursors; c) facilitated concanavalin A-induced mitogenic responses of low thymocyte numbers; d) had an apparent m.w. of 30,000 to 40,000 by gel filtration chromatography; and e) was eluted from DEAE-Sephacel ion-exchange chromatography columns by salt concentrations of 30 to 150 mM NaCl. The ability of these T cell hybridomas to grow in vivo and retain their functional characteristics in a stable form should prove useful in terms of providing large numbers of TCGF-secreting cells and studying in vivo aspects of the production of TCGF as well as other immunoregulatory mediators.     

Production of cytotoxic factor(s) in human T cell lines transformed by a human retrovirus

Human T cell lines, MT-2, TCL-Ter, TCL-Haz, and TCL-Kan which were transformed by a human retrovirus, constitutively produced cytotoxic factor(s) (CF) in the culture supernatants. In these cell lines, MT-2 produced the largest amount of CF. The amount of CF produced by MT-2 was 9-10 or 3-4 times larger than that produced by a human B cell line, RPMI 1788, or normal peripheral blood leukocytes stimulated with mitogens and phorbol ester. The kinetics of the production by MT-2 was similar in media with and without serum. The activity was stable at 56 degrees C for 30 min but was lost at 80 degrees C for 30 min and at pH 2 for 20 hr. On gel filtration, the molecular weight of the factor produced by MT-2 was approximately 90,000. On isoelectric focusing, the activity was recovered in the fraction at pH 6.5-7.0.     

Regulation of T cell growth factor production: arrest of TCGF production after 18 hours in normal lectin-stimulated mouse spleen cell cultures

The detailed kinetics of TCGF accumulation in Con A-stimulated spleen cell cultures shows a maximum at 24 hr, with a subsequent decrease in activity. This decrease is not due to the appearance of inhibitory substances "masking" TCGF activity. Pulse experiments show that the rate of TCGF production falls sharply after 18 hr and is completely arrested after 24 hr of Con A stimulation. The arrest in TCGF production is the result neither of culture depletion in medium components nor of limiting accessory cell function or inactivation of the lectin, and it thus seem to be the result of inactivation of TCGF-producing T cells. This regulation is not the result of a TCGF-mediated feedback mechanism but rather of lectin-induced suppressive cells that appear in culture after 24 hr and turn off de novo production of TCGF in fresh cultures.     

Porphyra cell cultures: isolation,growth and polysaccharide production

A range of cell lines was isolated fromPorphyra umbilicalis L. (Rhodophyta) tissue using a variety of methods, the most successful involving exposure to a limpet acetone powder enzyme extract for 24 h, homogenisation and filtration through a series of polyester meshes. All established lines grew as 0.1–5 mm diameter aggregates in liquid culture; most were stable and have been grown in shake-flask or air-lift culture for periods in excess of 1 yr without reverting to the foliose growth form. An investigation of the medium used to grow these lines indicated that it was not nitrogen-deficient and that the sodium chloride concentration was optimal. The addition of an organic buffer increased the final cell yield. None of these cell lines grew heterotrophically in medium supplemented with a range of fixed carbon sources. The infrared spectra of polysaccharides isolated fromPorphyra aggregates and from tissue grown under identical conditions indicated that the structures of the two isolates were analogous.Presented at the XIIIth International Seaweed Symposium, University of British Columbia, Vancouver, Canada, August 1989.     

Isolation of a myofibroblast growth factor from human breast carcinoma cell lines

Conditioned media of a series of well-established human breast carcinoma cell lines were screened for mitogenic activity on human myofibroblasts. Whereas all carcinoma lines derived from desmoplastic ductal breast carcinoma primaries exhibited moderate to high levels of mitogenic activity, the single line derived from a non-desmoplastic (medullary) carcinoma exhibited low activity. Levels of mitogenic activity were independent of estrogen receptor status and estrogen/antiestrogen treatment. Fractionation of the conditioned media revealed a cationic, hydrophobic mitogenic factor of M.W. 25,000. The factor did not stimulate the growth of endothelial or carcinoma cells nor the growth of NRK fibroblasts in soft agar.     

Correlation between T cell receptor gamma delta isotypic forms and cytotoxic activity: analysis with human T cell clones and lines

Three biochemically distinct isotypic forms of the human T cell receptor (TcR) gamma delta structure can be expressed at the cell membrane. This unique variation in structure of TcR, which is due to C gamma gene segments utilization, prompted us to look for isotype-association functional differences. In this regard, we have developed human T cell clones or lines from normal thymus or peripheral blood from several patients. In the present report, we have selected by phenotypic, biochemical, and TcR gene rearrangement analysis representative pairs of IL2-dependent clones or lines for each TcR gamma delta isotypic form. The results showed a lack of correlation between the TcR isotypes and the ability of the cells to proliferate in response to TcR stimulation mediated through the CD3 molecular complexes. By contrast, despite the fact that all of these representative cells exhibit an NK-like activity, as measured by their ability to kill K562, the strongest lytic activity was observed with the cells having the disulfide-bonded form of the receptor. Moreover only those latter cells were able to efficiently kill the LAK-sensitive Daudi cell line.     

T cell growth factor: parameters of production and a quantitative microassay for activity.

Several soluble factors have recently been associated with the proliferation and differentiation of thymus-derived lymphocytes. One of these factors present in medium conditioned by T cell mitogen-stimulated lymphocytes has the ability to promote the long-term culture of normal and antigen-specific cytotoxic T cells. We report a method to test for this proliferative stimulus in the form of a sensitive microassay based upon the tritiated-thymidine incorporation of continuous murine tumor-specific cytotoxic T cell lines (CTLL). The microassay requires microliter quantitites of sample fluid and is amenable to quantitative analysis. This highly reproducible, quantitative assay for T cell growth factor (TCGF) has allowed investigation as to the kinetics of TCGF generation and has revealed that T lymphocytes are required for its production. Further investigation has supported the notion that this nonspecies-specific factor is actively removed from tissue culture medium by the proliferation of either T cell mitogen-activated lymphocytes or CTLL.     

Tumor cytotoxic factor/hepatocyte growth factor from human fibroblasts: cloning of its cDNA, purification and characterization of recombinant protein.

Two different forms of cDNA for F-TCF were isolated from cDNA library prepared with mRNA from human embryonic lung fibroblast, IMR-90 cells. One of them was completely identical to the cDNA for placenta type hepatocyte growth factor (HGF) and the other one was a variant cDNA for the HGF with a deletion of 15 base pairs in the coding region. The cDNAs were expressed in CHO cells and recombinant proteins were purified and characterized. The deleted form of recombinant F-TCF (rF-TCF) was slightly lower in heparin affinity than the intact form. Both rF-TCFs showed almost same dose-response curves for cytotoxicity on Sarcoma 180 or Meth A sarcoma cells. Dose-response curves for the stimulation of DNA synthesis in rat hepatocytes were also almost same before reaching maximal activity at 12.5 ng/ml but significantly different at higher concentrations. The deleted form of rF-TCF maintained maximal activity in the dose range of 12.5 to 100 ng/ml, although the intact form decreased the activity dose-dependently at more than 25 ng/ml. This suggests that the deletion of five amino acids results in a conformational change which alters heparin binding and hepatocyte growth stimulating activities.     

Identity of a tumor cytotoxic factor from human fibroblasts and hepatocyte growth factor

Human embryonic lung diploid fibroblast, IMR-90 cells secreted a tumor cytotoxic factor. The fibroblast-derived tumor cytotoxic factor (F-TCF) has a cytotoxic activity to Sarcoma 180 and a cytostatic and degenerative activities to KB cells. F-TCF has been purified about 540,000-fold with 23.3% recovery from 75 liters of the conditioned medium containing 5% newborn calf serum. The purified F-TCF is a basic glycoprotein with isoelectric point values of 7.4 to 8.6. It was stable in the pH range from 6.0 to 9.0 and was stable at the heating temperature of 60 degrees C for 10 min, but completely inactivated by reducing it with 2-mercaptoethanol. F-TCF has molecular weight of 76 to 80 kD on SDS-PAGE under non-reducing conditions and is a heterodimer consisting of a large alpha subunit with 52 to 56 kD and a small beta subunit with 30 to 34 kD. F-TCF was identified as one of human hepatocyte growth factors by the physicochemical properties including N terminal and a few internal amino acid sequences. We have confirmed that F-TCF has an ability to dramatically stimulate DNA synthesis in adult rat hepatocytes in the low dose range of 1 to 10 ng/ml.     

A functional analysis of hematopoietic growth factor production from class I and class II human alloreactive T cell clones

Class I and Class II human alloreactive T cell clones or their conditioned media were mixed with progenitor cell-enriched null cells to assess their ability to stimulate human hematopoietic progenitor cell (HPC) growth. Optimal release of erythroid, myeloid or megakaryocyte colony-stimulating factors occurred after 72 hours and required contact of cloned T cells with irradiated stimulator cells expressing the appropriate major histocompatibility complex (MHC) determinants recognized by the T cells. Individual clones were quite heterogeneous in their capacity to release hematopoietic growth factors. Clones that produced optimal levels of factors that stimulated granulocyte-macrophage colony growth did not always produce equivalent amounts of factors that stimulated erythroid colony growth and vice versa when tested against identical target cells. Class II clones released nearly twice as much interleukin 3 activity as Class I clones. Class II clones that lacked cell-mediated lympholytic (CML) activity against B or T lymphoblastoid targets were consistent stimulators of HPC growth. In contrast, Class I or Class II clones that contained CML activity either poorly stimulated or inhibited HPC growth. These CML-positive clones produced greater amounts of gamma interferon. Our findings may have important implications for HPC growth following allogeneic mismatched bone marrow transplantation.     

Resistance of primary CD8+ cytotoxic T lymphocytes to lysis by cytotoxic granules from cloned T cell lines

Recent evidence has shown that cloned, murine CTL cell lines are resistant to the cytotoxic components of the toxic granules they release upon specific interaction with their target cells. Inasmuch as the resistance might be due to selection in culture over many months by repeated exposure to these cytolytic components (which are released repeatedly as a result of the cultured CTL being periodically stimulated by target cells), we asked whether primary CTL are also resistant. The primary CTL were elicited in vivo by i.p. injection of allogeneic tumor cells or in vitro by 5- to 6-day MLC or by 48-h exposure to the lectin Con A. The responding cells were separated into purified CD8+ (i.e., CD4-, CD8+) and purified CD4+ (i.e., CD4+, CD8-) T cell populations that were analyzed for cytolytic activity and for resistance to lysis by toxic secretory granules derived from cloned CTL cell lines. The CD8+ T cells were highly cytolytic and relatively resistant; they retained their cytolytic activity and were lysed to a minimal extent (0 to 10%) by quantities of isolated granules that lysed 80 to 90% of the P815 tumor cell line (tested as a representative standard cell line). The CD4+ T cells, in contrast, had only minimal cytolytic activity and were far more susceptible to granule-mediated lysis. Although the resistance of primary CD8+ T cells is impressive, it is not as pronounced as the resistance of the cloned CTL cell lines, indicating that during long-term culture there is some selection for increased resistance to granule-mediated lysis. In contrast to T cells (especially CD8+ T cells), Ia+ macrophages, isolated from primary immune peritoneal exudates, were highly susceptible to granule-mediated lysis.     

Production of macrophage activating factor by human leukemic T cell lines

Human leukemic T cell lines were tested for their ability to produce a macrophage activating factor. When mouse peritoneal macrophages were cultured for 48 hr in the presence of culture supernatants from cell lines HPB-ALL, CCRF-CEM, or MOLT-4, glucose oxidation via the hexose monophosphate pathway was enhanced by five to seven fold. Culture supernatants from cell line HPB-MLT stimulated the oxidation to a lesser extent. However, cell line CCRF-HSB-2 was essentially inactive as a producer. The active supernatants also stimulated the release of hydrogen peroxide from macrophages, whereas the inactive one did not. Since treatment of the cell lines with 12-o-tetradecanoyl phorbol acetate or phytohemagglutinin had little effect on the production of the factor except HPB-ALL, the cell lines seemed to secrete the factor constitutively. The stimulatory effect was dose-dependent and evident at a concentration as low as a 1/80 dilution. The factor was resistant to heat treatment at 100 C for 20 min, nondialysable and sensitive to protease digestion. The activating factor could be partially purified by anion exchange and gel filtration chromatographies.     

The suppressor T cell induced by syngeneic splenic cell antigen down-regulates hapten-specific cytotoxic T cells by elaborating a factor inhibitory for IL2-dependent cell replication

An in vitro study has been made of the mechanism by which a suppressor T cell, that is induced in lymph nodes by a syngeneic splenic cell antigen, prevents generation of cytotoxic T cells specific for hapten-altered self antigens. When popliteal lymph node cells exposed in vivo to syngeneic splenic cells were immunized in vitro with heat-treated syngeneic TNP-coupled thymocytes and excess helper factors, the Ts remained inactive. In this condition the exposed popliteal lymph node cells routinely demonstrated approximately twice the CTL response developed by lymph node cells from normal mice. Nevertheless, when triggered in vitro by splenic antigen on either X-irradiated B or T cells, the exposed but not the normal lymph node cells exhibited reduced hapten-altered self-specific CTL responses. Furthermore, T cells within spleen cell-exposed popliteal lymph node cell populations when reexposed to splenic T cells made a factor that was found to be suppressive of CTL generation by normal lymph node cells in vitro. The nondialyzable T-cell suppressor factor (TsF) did not appear to act on lymph node precursor CTLs, nor on helper T cells but instead acted at the level of utilization of helper factors in the development of CTLs. In an examination of the effect of TsF on cellular replication, TsF was found to be nontoxic for CTLL-20, an IL-2-dependent T cell, and it did not hinder the uptake of IL-2 by receptor blockade of this cell. Nevertheless, the replication of CTLL-20 that is IL-2 driven was diminished in the presence of TsF. Similarly, TsF was found to be inhibitory for T-cell proliferation stimulated by mitogen but had no effect on a B myeloma cell proliferative response. Thus, TsF appears to act as an inhibitor of a T cell's capability to replicate despite the presence of the stimulus for replication, namely, IL-2.     

Rollinia mucosa cell suspension cultures: Establishment and growth conditions

Different types and concentrations of plant growth regulators were tested in order to obtain the best callus and cell suspension culture growth conditions of Rollinia mucosa (Jacq.) Baill. (Annonaceae). Picloram was shown to be the most efficient for induction and production of friable calluses, independent of the concentration used. Cellular morphology and viability, fresh and dry weights, pH and medium sugar concentration were determined for cell suspension cultures. Dissimilation curves were used for the characterization of the growth of cell suspension cultures. Picloram provided the most rapid growth and produced the highest biomass, with little variation in morphology (differentiated cells). It also provided the highest dissimilation, when compared with cell suspension cultures maintained in media with 2,4-D or NAA + BA + GA₃. Stable cell suspension cultures can be established in MS medium supplemented with 20.8 μM picloram. This revised version was published online in August 2006 with corrections to the Cover Date.     

Immunologic typing of splenic lymphocytes from human fetuses--donors of pancreatic islet cell cultures

To determine the HLA-phenotype of a potential donor of pancreatic islet cells, use was made of lymphocytes from 18-25-week-old human fetuses. The HLA-phenotype was clearly established in 39 out of 52 cases. In 13 cases, the authors failed to reveal histocompatibility antigens because of low viability of lymphocytes. The relationship was ascertained between the detectability of HLA-antigens in fetal donors and cytophysiological characteristics of islet cell cultures prepared from the pancreas.     

A model for light-limited continuous cultures: growth, shading, and maintenance

Light-limited growth in continuous cultures of phototrophic organisms is modeled. It is assumed that light energy up-take rate depends hyperbolically on light intensity and that the maintenance costs are proportional to biomass. Modeling the light distribution caused by shading within the vessel is necessary to explain the existence of steady state in light-limited chemostats. The model fits well to experimental data from literature on light-limited chemostats and turbidostats. Attention is given to the implications of the model for the estimation of the specific maintenance rate constant in light-limited continuous cultures.     

Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7

Development of generic differentiation protocols that function in a range of independently-derived human embryonic stem cell (hESC) lines remains challenging due to considerable diversity in culture methods practiced between lines. Maintenance of BG01 and HUES-7 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual- and trypsin-passaging, respectively. We adapted both lines to trypsin-passaging on feeders or on Matrigel in feeder-free conditions and assessed proliferation and cardiac differentiation. On feeders, undifferentiated proliferation of BG01 and HUES-7 was supported by all three media tested (BG-SK, HUES-C and HUES-nL), although incidence of karyotypic instability increased in both lines in BG-SK. On Matrigel, KSR-containing conditioned medium (CM) promoted undifferentiated cell proliferation, while differentiation occurred in CM containing Plasmanate or ES-screened Fetal Bovine Serum (FBS) and in unconditioned medium containing 100 ng/ml bFGF. Matrigel cultures were advantageous for transfection but detrimental to embryoid body (EB) formation. However, transfer of hESCs from Matrigel back to feeders and culturing to confluence was found to rescue EB formation. EBs formed efficiently when hESCs on feeders were treated with collagenase, harvested by scraping and then cultured in suspension in CM. Subsequent culture in FBS-containing medium produced spontaneously contracting EBs, for which the mean beat rate was 37.2 +/- 2.3 and 41.1 +/- 3.1 beats/min for BG01-EBs and HUES-7-EBs, respectively. Derived cardiomyocytes expressed cardiac genes and responded to pharmacological stimulation. Therefore the same culture and differentiation conditions functioned in two independently-derived hESC lines. Similar studies in other lines may facilitate development of universal protocols.     

Thymoma production of T cell growth factor (Interleukin 2)

Phorbol-12-myristate-13-acetate stimulates a subline of mouse EL-4 thymoma cells to produce, in vitro, in very high titer, T cell growth factor (Interleukin 2, IL 2). The EL-4-derived IL 2 has the same m.w. (30,000) and isoelectric point heterogeneity (pI 3.8-4.4) as the IL 2 produced by Con A-stimulated spleen cells. In addition, the thymoma-derived IL 2 exhibits the same spectrum of biologic activities as has been reported for spleen cell-derived IL 2.