Search PubMed⌕ Search

Biomedical subjects

M Nabholz

Publications and source records attributed to M Nabholz.

At least 55 records · Page 3Linked to original sources
PubMed1985

Antigen stimulation of cytolytic T lymphocyte precursors: minimal requirements for growth and acquisition of cytolytic activity.

Minimal requirements for growth and acquisition of cytolytic activity by alloantigen-stimulated cytolytic T lymphocyte precursors (CTL-P) have been investigated. CTL-P from C57BL/6 spleen were purified by staining with monoclonal anti-Lyt-2 antibodies followed by positive selection on a fluorescence-activated cell sorter. Microcultures containing 2000 purified Lyt-2+ cells were set up with irradiated P815 (DBA/2 mastocytoma) stimulating cells and pure interleukin 2 obtained by recombinant DNA technology (rIL2). Proliferation and generation of antigen-specific cytolytic activity were observed to be both antigen and IL2-dependent in such microcultures. Furthermore, P815 stimulating cells could be replaced by 5 other H-2d cell lines of differing tissue origin. In limiting dilution studies, 1-2% of Lyt-2+ CTL-P (contaminated by less than 0.2% macrophages) could be induced to grow and express cytolytic activity in the presence of irradiated P815 and rIL2. Taken together, these data indicate that neither accessory cells nor differentiation factors (other than IL2) are absolutely required for alloantigen-induced growth and differentiation of CTL-P.

Animals↗
PubMed1985

Appearance of cytolytic granules upon induction of cytolytic activity in CTL-hybrids.

Examining a CTL-line (B6.1) with specific cytolytic activity, we have confirmed the ability of CTL-lines to produce poly C9-like, tubular complexes of two sizes (polyperforin 1 and 2). Isolated cytoplasmic granules from this cell line can induce hemolysis and contain the lysosomal marker arylsulfatase. Electron microscopial studies show that granules contain precursor molecules which are assembled into polyperforin upon addition of Ca2+. Antibodies raised against granules predominantly react with a 27-kd protein. We compared the B6.1 cell with a hybrid, PC60, derived from a cross between B6.1 and a non-cytolytic rat-thymoma. This hybrid line exhibits inducible CTL-activity. In PC60 with induced cytolytic activity, polyperforins 1 and 2, cytolytic granules and granule-associated proteins are detected. Non-cytolytic PC60 cells lack cytolytic granules and polyperforin 1, but contain polyperforin 2. These results suggest that cytolytic granules appear in parallel with the expression of cytotoxicity during T-lymphocyte differentiation.

Animals↗
PubMed1985

Glucocorticoid resistance is a dominant trait in hybrids between cytolytic T-lymphocyte lines and AKR thymomas.

By screening several cytolytic T-lymphocyte lines, AKR thymomas, and CTL X AKR thymoma hybrids from two different crosses for their sensitivity to the glucocorticoid (GC) analog dexamethasone (dex), we have found that CTL lines and cytolytically active, IL-2-dependent (CTL-like) hybrids are resistant to the cytostatic or cytolytic effects of dex; AKR thymomas and thymoma-like hybrids (cytolytically inactive, IL-2-independent), however, are sensitive to these effects of the drug. The GC resistance behaves like a dominant trait in these crosses. Although they are resistant to GC, the CTL lines and the CTL-like hybrids do contain functional hormone receptors and macrophage-activating factor (MAF) release by the CTL lines and CTL-like hybrids is inhibited by dex.

Animals↗
PubMed1985

Antigenic stimulation regulates the expression of IL 2 receptors in a cytolytic T lymphocyte clone.

We have isolated and characterized an H-2Kb-specific CTL clone, B1.8, whose growth is strictly dependent on IL 2 and periodic exposure to stimulator cells. A single, brief encounter with antigen induces on resting B1.8 cells the expression of high-affinity IL 2 receptors, which is maintained for several days, thus allowing cells to undergo at least six rounds of division in the presence of affinity-purified IL 2. We show with the use of monoclonal anti-Lyt-2 or -H-2Kb antibodies, which block specific antigen recognition by B1.8 cells, that continuous antigenic stimulation is not required during this period of growth. After this time, B1.8 cells lose their ability to respond to IL 2 and revert to a resting state, a process that is reversed upon addition of fresh stimulator cells. The decline and subsequent arrest of cell growth correlates with a loss of IL 2 receptors; growth-arrested cells express a 10-fold lower number of receptors than actively growing cells.

Animals↗
PubMed1985

Interleukin 2 is both necessary and sufficient for the growth and differentiation of lectin-stimulated cytolytic T lymphocyte precursors.

Minimal requirements for the growth and differentiation of lectin-stimulated cytolytic T lymphocyte precursors (CTL-P) have been investigated. CTL-P from murine spleen were purified by staining with monoclonal anti-Lyt-2 antibodies, followed by positive selection on a fluorescence-activated cell sorter. Microcultures containing 2000 purified Lyt-2+ cells were set up with optimal concentrations of lectin (leukoagglutinin or concanavalin A) and pure interleukin 2 (IL 2) obtained either by immunoaffinity chromatography or recombinant DNA technology. Proliferation and generation of CTL activity in lectin-stimulated Lyt-2+ cells were IL 2 dose-dependent. Furthermore, no increased response was observed when crude supernatants containing other putative CTL differentiation factors were added, even when IL 2 concentrations were suboptimal. When highly purified Lyt-2+ cells contaminated by 1% Lyt-2- cells and less than 0.1% macrophages were cultured at limiting dilution, as few as 20 responding cells proliferated and differentiated into CTL in the presence of leukoagglutinin and pure IL 2; in fact, CTL activity and proliferation were strongly correlated in individual microcultures. In addition, 10 to 30% of individual CTL-P were growth-inducible under these experimental conditions. Together, these data show that the proliferation and differentiation of lectin-stimulated CTL-P can occur in pure IL 2 without any demonstrable requirement for other differentiation factors or accessory cells.

Animals↗
PubMed1985

High and low affinity IL 2 receptors: analysis by IL 2 dissociation rate and reactivity with monoclonal anti-receptor antibody PC61.

In this report we characterize two classes of interleukin 2 (IL 2) binding sites on the basis of their differential IL 2 dissociation rate and of their reactivity with PC61, a monoclonal anti-IL 2 receptor (IL 2-R) antibody. PC61 inhibited the binding of IL 2 to both classes of receptor, but IL 2 did not inhibit the binding of PC61. This indicates that PC61 recognizes a determinant that is distal to the actual IL 2 binding site of the receptor. Dissociation experiments showed that the addition of excess unlabeled IL 2 resulted in a biphasic release of radiolabeled IL 2; 80 to 90% was dissociated rapidly (dissociation half-time, t1/2 of 60 sec) and the remainder more slowly (t1/2 60 to 90 min). The proportion of high and low affinity IL 2-R, as well as the relative difference in dissociation rates fit very well with the estimates derived previously from Scatchard plot analysis of equilibrium IL 2 binding. The addition of PC61 caused an accelerated dissociation of IL 2 from both high and low affinity IL 2-R (t1/2 of 16 and 120 sec respectively).

Animals↗
PubMed1984

Characterization of soluble factors that induce the cytolytic activity and the expression of T cell growth factor receptors of a T cell hybrid.

A rat X mouse T cell hybrid (PC60) proliferates in the absence of T cell growth factor (TCGF) and its cytolytic activity can be induced by culture in mixed leukocyte culture supernatants or concanavalin A-activated rat spleen cell supernatant (CS) to lyse 51Cr-labeled tumor target cells. To characterize the factor(s) responsible for this reversible induction, serum-free CS was fractionated by reverse phase high performance liquid chromatography and by phenyl-Sepharose chromatography. A cytotoxicity-inducing activity (CIA) was separated from TCGF and macrophage-activating factor/interferon-gamma. CIA was found to be a macromolecule with an apparent molecular weight of 12,000-18,000 and a pI of 5.0 and 6.2. Its activity on PC60 cells depended on the addition of TCGF. Thus TCGF may have other effects on T cells than the induction of entry into cell cycle. The number of TCGF surface receptors on PC60 cells was measured using purified 3H-TCGF. TCGF receptors were undetectable on noninduced cells but appeared during induction. The expression of TCGF receptors was not induced either by TCGF or by CIA-containing supernatants or fractions alone, only by a combination of both. These results show that TCGF plays a role in the regulation of the expression of its own receptors.

Animals↗
PubMed1984

Regulation of the rate of cell cycle progression in quiescent cytolytic T cells by T cell growth factor: analysis by flow microfluorometry.

We have previously shown that greater than 90% of B6.1 cells, a murine cytolytic T lymphocyte (CTL) cloned line which is solely dependent on T cell growth factor (TCGF) for continuous growth in vitro, accumulates in the G1 phase of the cell cycle after transfer into culture medium containing no TCGF. Moreover, when such quiescent cells are exposed again to TCGF, greater than 85% reenter the S phase and subsequently divide in a relatively synchronous fashion. In this study, the regulation of the rate of cell cycle progression of quiescent B6.1 cells after exposure to TCGF was analyzed using two complementary DNA staining techniques, namely, the propodium iodide method (to enumerate cells entering the S phase) and the Hoechst 33342-bromodeoxyuridine substitution technique (to enumerate cells which have gone through mitosis). After TCGF addition, quiescent B6.1 cells resumed DNA synthesis and divided after a lag phase of 10 and 20 h, respectively. The duration of the lag phase was found to be dependent on the length of time during which quiescent B6.1 cells had been deprived of TCGF, but was independent of the concentration of TCGF used for restimulation. In contrast, the proportion of cells responding to TCGF as well as the rate of their first passage through mitosis was dependent on TCGF concentration. The presence of TCGF for at least 6 h was required for a maximal response. Moreover, direct evidence was obtained that TCGF by itself was able to stimulate proliferation of quiescent B6.1 cells in the absence of other growth factors and serum constituents other than bovine serum albumin, transferrin, and lipids.

Animals↗
PubMed1984

Karyotypic analysis of murine cytolytic T-cell lines.

G-banding techniques have been used to analyze the karyotypes of four independently derived murine cytolytic T lymphocyte (CTL) lines established in culture 2-4 yr earlier. For two of them, several subclones were included in the analysis. The modal arm number of three of the lines was close to diploid, whereas in the fourth line, both near-diploid and near-tetraploid subclones were found. The most frequent type of rearrangement was the formation of metacentrics by centromeric fusion. A comparison of the abnormalities (markers) occurring in the different CTL lines showed the formation of similar chromosomal rearrangements in independent lines, thus suggesting nonrandom chromosomal changes in these cells. Several of the metacentrics are derived from the fusion of normal murine chromosomes that are apparently prone to form such "markers," as the occurrence of similar abnormalities has been reported in other established cell lines and tumors. On the other hand, some of the biarmed and telocentric marker chromosomes seem to characteristically occur in CTL but not in other types of cell lines.

Animals↗
PubMed1983

Selective killing of target cells by antibody-ricin A chain or antibody-gelonin hybrid molecules: comparison of cytotoxic potency and use in immunoselection procedures.

Ricin A chain and gelonin, two plant proteins that can powerfully reduce the protein synthetic capacity of ribosome preparations, were covalently coupled to a monoclonal anti-Thy-1.2 antibody. Two conjugates were prepared by using N-succinimidyl-3-2(-pyridyldithio)propionate, which generates a disulfide linkage between the component molecules. Both conjugates specifically killed Thy-1.2 antigen-bearing EL-4 cells, but had no effect on Thy-1.2- BW5147 cells. The cytotoxic potency of both reagents was determined by comparing the cloning efficiency of E1-4 target cells after treatment with the conjugate. The frequency of cells surviving treatment with 45 micrograms/10(6) cells of the gelonin conjugate was 1/72, whereas this frequency was 1/836 after exposure to 7 micrograms of the Ricin A chain conjugate. Both reagents could be successfully used to select Thy-1.2- cells from cell mixtures consisting of Thy-1.2+ and Thy-1.2- cells.

Animals↗
PubMed1982

Correlated expression of T cell growth factor dependence, sensitivity to Vicia villosa lectin, and cytolytic activity in hybrids between cytolytic T cells and T lymphomas.

Somatic cell fusion between cytolytically active, T cell growth factor- (TCGF) dependent murine T cell lines (CTL lines) and noncytolytic, TCGF-independent murine T lymphoma lines has yielded two types of somatic cell hybrids (5): cytolytic hybrids, growth of which is dependent on TCGF, and hybrids with very weak or undetectable cytolytic activity which grow at the same rate with or without TCGF. Here we report that the former can produce stable variants that resemble the latter type. Some of these TCGF-independent variants still have TCGF receptors. High susceptibility to the cytotoxic effects of Vicia villosa lectin, a marker distinguishing the parental CTL lines from T lymphomas, is expressed by the TCGF-dependent hybrids but not by the TCGF-independent variants. The two types of hybrids also differ in the expression of surface glycoproteins. We propose that there exists a genetic element in the CTL line that represses the TCGF-independent replication mechanism of the T lymphoma parent in the TCGF-dependent hybrids and that this genetic element is lost or switched off in the TCGF-independent variants.

Animals↗
PubMed1982

An efficient method for purification of human T-cell growth factor.

A 1000-fold purification of human T-cell growth factor (TCGF) was achieved starting from supernatants of human spleen cells stimulated with phytohaemagglutinin (PHA) in culture medium containing 0.5% serum. The purification scheme involved precipitation with ammonium sulphate, gel filtration and blue-Sepharose chromatography. The use of polyethylene glycol 6000 (PEG 6000) was critical during the chromatographic steps in order to obtain high final recoveries or activity (40-50%). Purified preparations of TCGF labelled with 125I by the chloramine T method revealed that the activity co-migrated with 2 molecular species of 14,000-17,000 daltons in SDS-PAGE under non-reducing conditions.

Animals↗
PubMed1982

Cell cycle regulation of cloned cytolytic T cells by T cell growth factor: analysis by flow microfluorometry.

In an attempt to better define the mode of action of TCGF we have investigated the effect of complete removal of TCGF on the cell cycle kinetics of a TCGF-dependent murine CTL cloned line (B6.1). Cells that had been cultured in the presence or absence of TCGF were stained with propidium iodide, a DNA binding dye, and the distribution of DNA content was analyzed by flow cytometry. Results indicated that the proportion of cells entering the S phase of the cell cycle started to decrease 6 hr after removal of TCGF from the culture medium. This fraction continued to decrease as a function of time, and after 30 hr without TCGF 93% of the cells were in the G1 phase of the cell cycle in comparison to 38% in control cultures containing TCGF. Direct evidence that B6.1 cells completed one cell cycle after removal of TCGF before they accumulated in the G1 phase was obtained with a technique combining the use of BUdR, a thymidine analog, and the DNA binding dye Hoechst 33342. When TCGF was added again to cultures of B6.1 cells that were arrested in the G1 phase, the majority of cells entered the S phase in a synchronous fashion after a lag phase of 10 to 12 hr. The duration of this lag phase was independent of the concentration of TCGF used to restimulate quiescent B6.1 cells. However, the number of cells entering S phase 10 hr after re-addition of TCGF was concentration dependent. Additional experiments indicated that the cytolytic activity was not dependent on TCGF, since no decrease in the activity of B6.1 cells was noted after these cells had been deprived of TCGF for up to 30 hr.

Animals↗
PubMed1980

Cytolytically active murine T-cell hybrids.

Following the demonstration that hybrids between normal B-lymphocytes and myeloma cell lines continue to secrete antibodies with the same specificity as those produced by the parental B-cells, many groups have tried to use this approach to obtain cell lines expressing T-lymphocyte functions by crossing thymoma lines not expressing any measurable activity with various types of T-cell populations. Although there have been reports that hybrids could be isolated which secrete T-cell products with immunological activity, efforts to produce functionally active hybrids from cytolytic T-cells have all been unsuccessful (refs 6, 7, and M. N. and H. D. Engers, unpublished). We have fused an established, T-cell growth factor (TCGF)-dependent murine cytolytic T-lymphocyte (CTL) line with a mouse thymoma line and have obtained hybrids with cytolytic activity when we selected the hybrids in TCGF-containing medium, while hybrids isolated in the absence of growth factor showed no detectable cytolytic potential.

Animals↗