Specific versus nonspecific target cell destruction by T lymphocytes sensitized in vitro. I. Kinetics

An in vitro culture method giving a high cell recovery rate of 25–38% has been developed to generate a relatively pure population of T cell progeny reacting to xenogeneic cell antigen. The method is basically a modification of the Ginsburg method and utilizes mosaic monolayers that are comprised of syngeneic Lewis and xenogeneic BALB/c fibroblasts. Thoracic duct lymphocytes of Lewis rats were cultured for 6 days on these monolayers and the resulting lymphocyte population, rich in blast cells, was collected and separated free of contaminating fibroblasts by an additional 6 hr incubation without monolayers. Cytotoxic activity of these harvested lymphocytes was assessed by a modification of the Hellström method in which embryonic target fibroblasts, Lewis or BALB/c, were plated at a critical density of 2200 cells/cm² of culture area. This, coupled with the use of a newly developed culture medium, allowed the demonstration of specific as well as nonspecific activity of up to 100% suppression at 48 hr of test incubation. The specific activity was clarified and determined critically by comparing the activity of these lymphocytes to that of lymphocytes grown on syngeneic monolayers, though rich in blast cells, reacting only to the horse serum in the medium. The nonspecific activity was not due to the deterioration of test cultures and needed the continued presence of fully active blast cells for complete suppression of the target cell growth in the later phase of test cultures. By changing the ratio of lymphocytes to target cells and following the kinetics of target cell destruction, it was shown that specific activity effects an active killing of target cells, whereas the nonspecific activity was primarily the suppression of target cell growth. The specific activity was effective with far less lymphocytes per target cell; was nearly complete by 12 hr of test cultures while the nonspecific activity was complete at 48 hr. Yet, the increase in the specific activity was nearly proportional to that of nonspecific activity and even the nonspecific activity seemed to kill sensitive Lewis target cells at its full intensity. These observations are in accordance with an interpretation of the mechanism of the specific target cell destruction as involving contact and recognition followed by the release of lymphotoxin into or onto target cells.     

Nonspecific cytotoxic effects of antigen-transformed lymphocytes. Kinetics, cell-requirements and the role of recruitment

PPD-stimulated human peripheral blood lymphocytes have been shown to exert a nonspecific cytotoxic effect on allogeneic and xenogeneic target cells labeled with ⁵¹Cr. Chromium release induced by washed transformed lymphocytes was linear with time. At lymphocyte to target cell ratios of 120:1, significant killing could be demonstrated as early as $\text{1}\text{2}$ hr. At 8 hr, killing occurred with ratios as low as 3:1. The cytotoxic effect was related to the ability of the lymphocytes to transform to PPD, but reached an earlier peak than either DNA or RNA synthesis. Supernatants from lymphocyte cultures were not cytotoxic: instead, viable transformed cells were required. Partial removal of macrophages from the original cultures increased the cytotoxic effect.Lymphocytes could also be nonspecifically recruited to exert a cytotoxic effect by a mitogenic-like factor produced in transforming cultures. Preliminary evidence suggested that this factor acted independently of PPD and of histocompatibility antigens. These results are discussed with reference to possible amplification mechanisms for late cytotoxic effects, and to delayed hypersensitivity reactions in vivo.     

Activation of nonspecific killer cells by interleukin 2-containing supernatants

In the absence of specific antigen stimulation, nonspecific killer cells were induced by culturing C57BL/6 lymph node or spleen cells with interleukin 2-containing supernatants. These supernatants were obtained from stimulation of either rat spleen cells with concanavalin A or a variant of the T cell lymphoma, EL4 (H-2b) with phorbol myristic acetate. The ability of the EL4 supernatant to induce nonspecific killer cells was abrogated by absorption with an interleukin 2-dependent T cell line or by concanavalin A-stimulated spleen cell blasts, but not by lipopolysaccharide-stimulated spleen cell blasts or by a non-interleukin 2-producing EL4 line. Partially purified interleukin 2 from EL4 supernatants could also support nonspecific killer cell induction. The induction of cytolytic cells by interleukin 2 is sensitive to gamma-irradiation and has a D omicron of 120 rad. The nonspecific killer cells induced are likely cytotoxic T lymphocytes; the majority of the precursor and effector cells bear the Thy-1 alloantigen marker. These nonspecific killer cells killed a broad spectrum of target cells, including concanavalin A- and lipopolysaccharide-induced splenic blasts of syngeneic or allogeneic mice, a syngeneic tumor, and a cloned allogeneic cytotoxic T cell line. The frequency of precursors for nonspecific killer cells in C57BL/6 lymph node and spleen cells are 1/7000 and 1/12,000, respectively. Clonal analyses revealed that these nonspecific killers exhibit heterogeneity with respect to their target cell specificities. The induction of nonspecific killers by interleukin 2-containing supernatants is partially dependent on nylon wool-adherent cells; in antigen-stimulated cultures the most specific killer cells were obtained from cultures in which nylon wool-nonadherent lymph node responder cells were stimulated with nylon wool-nonadherent allogeneic splenic stimulator cells that were treated with anti-Thy-1 antibody and complement. The relevance of these findings with respect to the frequencies and fine specificities of cytotoxic T lymphocytes generated in interleukin 2-supplemented cultures is discussed.     

Attachment of porcine blastocysts to fibroblast monolayers in vitro

The objective of this study was to compare the ability of porcine blastocysts to attach to various cellular and non-cellular substrates $\text{in vitro}$. One hundred twenty-two hatched blastocysts were collected from 17 handmated gilts and sows at slaughter. Blastocysts were randomly assigned to one of four treatments: Minimal Essential Medium (MEM) supplemented with 10% (v/v) heat treated fetal calf serum (HTFCS), monolayers of bovine uterine fibroblasts in MEM + 10% HTFCS (Buf), monolayers of bovine testicular fibroblasts in MEM + 10% HTFCS (Btes), and MEM + 10% HTFCS exposed to uterine fibroblasts for 24 hr to condition the medium (cMEM). Embryos were cultured individually in 24 well Linbro culture plates at 37 C in a humidified gas atmosphere of 5% CO₂ in air. Embryos were observed at 24 hr intervals by phase contrast microscopy (100X) and measured with an ocular micrometer. Blastocyst attachment was greater (P < .01) in Buf ($\text{21}\text{35}$) compared to MEM ($\text{7}\text{32}$), cMEM ($\text{9}\text{28}$), and Btes ($\text{1}\text{27}$). Embryo diameter was greater (P < .05) 24 hr prior to attachment in Buf compared to the other treatments. In addition, trophoblast monolayers continued to proliferate for 20 days when cocultured with uterine fibroblasts. These observations suggest that uterine fibroblasts provide a superior substratum for blastocyst attachment and the maintenance of swine trophoblast cells $\text{in vitro}$.     

Regulation of polyamine biosynthesis in normal and transformed hamster cells in culture

Several aspects of polyamine biosynthesis were compared in low-passage hamster embryo fibroblasts and transformed hamster fibroblasts. Earlier studies had demonstrated a larger and longer-lasting induction of ornithine decarboxylase activity in transformed cells than in hamster embryo fibroblasts. The increases in intracellular polyamine concentrations after serum stimulation were much greater in chemically transformed HE68BP cells than in normal hamster fibroblasts. Treatment of confluent cultures with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, greatly potentiated ornithine decarboxylase induction by fresh medium in HE68BP cells, but not in hamster fibroblasts. A similar synergistic effect was observed when transformed cells, but not normal cells, were treated with the combination of insulin and promoter. HE68BP cells were capable of growth in medium containing serum concentrations as low as 0.5%, whereas only concentrations of 5% or more supported the growth of hamster embryo fibroblasts. Low serum concentrations induced ornithine decarboxylase in HE68BP cells but not in normal cells, and a given serum concentration always produced a greater induction of ornithine decarboxylase in transformed than in normal cells.Another enzyme involved in polyamine synthesis, $\text{S-}\text{adenosyl-}\text{L}\text{-methionine}$ decarboxylase was induced in normal and transformed cells by serum-containing medium or tetradecanoylphorbol acetate, but in contrast to ornithine decarboxylase, no synergistic effect was seen in transformed cells exposed to the combination of fresh medium and the tumor promoter. A macromolecular inhibitor of ornithine decarboxylase was readily detected in hamster fibroblast cultures treated with high concentrations of putrescine, but little or none of this inhibitor was found in HE68BP cultures. In both cell types, however, serum induction of ornithine decarboxylase was inhibited under conditions of excess putrescine.The results demonstrate several differences between normal and transformed hamster cells in the regulation of polyamine synthesis.     

Comparative studies between microbiological culture and uptake of uridine/uracil to detect mycoplasmal infection of cell cultures.

Controlled, prospective studies were performed to compare detection of cell culture mycoplasmas by ratio of uptake of tritiated uridine (UdR) to tritiated uracil (U) and by microbiological culture. Culture was by standard agar and broth inoculation with aerobic and anaerobic incubation; immunofluorescent staining of indicator cell cultures was used to detect M. hyorhinis. The ratio of uptake of UdR to U ($\text{UdR}\text{U}$) and interpretation of test results were by standard published methods and performed in triplicate. 115 cell cultures were simultaneously assayed by the two techniques. 84 cultures (73.1%) yielded agreement between the 2 methods; 2 cultures (1.7%) yielded conflicting results, and 29 cultures (25.2%) yielded $\text{UdR}\text{U}$ results in the questionable range. Conflicting results consisted of two negative $\text{UdR}\text{U}$ tests in mouse cell cultures infected with M. orale. In separate studies, 3T-6 cultures freshly infected with M. orale yielded negative $\text{UdR}\text{U}$ results 3 days after infection, questionable results after 10 days and a positive $\text{UdR}\text{U}$ 17 days after infection. $\text{UdR}\text{U}$ detected infection in fibroblast, epithelial, and lymphocyte cell cultures. Highest $\text{UdR}\text{U}$ ratios were detected in human skin fibroblasts at early population doubling levels (PDLs), 4064 in one culture at PDL4. $\text{UdR}\text{U}$ was determined for IMR-90, a human diploid fibroblast at 12 different PDLs using the same lot of media. $\text{UdR}\text{U}$ gradually decreased throughout the life of the culture, from 2 125 at PDL6 to 340 at PDL36. Cultures in phase III and others exhibiting poor growth frequently yielded questionable or false-positive $\text{UdR}\text{U}$ results and were not included in tabulations of these results. $\text{UdR}\text{U}$ determined in endothelial cell cultures decreased as population density increased. In a representative experiment performed over a 4-day period, the $\text{UdR}\text{U}$ values were 1 808, 955 and 356 when the number of endothelial cells in culture were 5.3 × 10⁵, 6.6 × 10⁵ and 1.1 × 10⁶, respectively.     

Association between the cytotoxicity of thymidine and tumorigenicity of clones derived from C3H10T12 mouse embryo fibroblasts

We have measured the cytotoxicity of thymidine to $\text{C3H}\text{10T}\text{1}\text{2}$ mouse embryo fibroblasts derived from morphologically transformed foci of cells from cultures exposed to chemical carcinogens. Four of these cell lines have previously been shown to be tumorigenic in irradiated syngeneic hosts and were all more sensitive to the lethal effects of thymidine than were the non-transformed cells. Strikingly, the most tumorigenic of the cell lines were most sensitive to thymidine. Differences in plating efficiencies or growth rates of the various cell lines were not associated with differences in thymidine sensitivity.     

Trypsin inhibitory activity of a polypeptide isolated from red kidney beans, that also enhances lymphocyte stimulation.

A polypeptide isolated from red kidney beans, $\text{Phaseolus}\text{vulgaris}$, which has previously been shown to stimulate RNA synthesis in cultures of mouse spleen lymphocytes and plasmolyzed $\text{E.}\text{coli}$, is here shown to be a potent inhibitor of trypsin and α-chymotrypsin. This polypeptide is compared with commercially available trypsin inhibitors with regard to their capacity to inhibit some proteolytic enzymes and to stimulate $\text{in}\text{vitro}$ cultures of lymphocytes. Similar to FV the lima been trypsin inhibitor was found to possess a stimulating effect on the RNA as well as the DNA synthesis in lymphocyte cultures.     

Studies on the specificity of in vitro-induced lymphocytotoxicity to methylcholanthrene-induced fibrosarcoma cell lines

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from CS7B16(H2^b) and Balb/c(H2^d) mice to syngeneic or allogeneic methylcholanthrene-induced fibrosarcoma (MCAF) cell lines. The T cell-dependent cytotoxicity was specific to target cell lines to which the lymphocytes were immunized in vitro. Normal fibroblasts as stimulator cells did not induce lymphocytotoxicity to syngeneic MCAF cells or to normal syngeneic fibroblasts. The results indicate that the in vitro-immunized lymphocytes recognize individual specific tumor-associated antigens of the MCAF cells. In experiments in which the lymphocytes were immunized in vitro to allogeneic MCAF cells, cytotoxic reactions to alloantigens, but not to tumor-associated antigens, were detected. Incubation with phytohemagglutinin (PHA) during the sensitization period modified the specificity of the cell-mediated lysis of MCAF cells: Allogeneic as well as syngeneic target cells were destroyed by these effector cells. PHA induced a nonspecific cytotoxic effect which increased the specific lysis of target cells. The cytotoxicity of the in vitro-immunized lymphocytes was inhibited by incubation with membrane protein preparations from the syngeneic MCAF cell lines. In contrast to the specificity of the cytotoxic effect to the different syngeneic cell lines, the membrane extract of one individual syngeneic MCAF cell line was able to inhibit the lymphocytotoxicity to all other syngeneic cell lines. Membrane protein preparations from allogeneic MCAF cells or from normal syngeneic fibroblasts were not inhibitory. The in vitro-immunized cytotoxic lymphocytes did not impair the tumor growth in vivo as could be demonstrated by passive transfer of the lymphocytes in a Winn assay.     

Purification of a lymphokine: Osteoclast activating factor from human tonsil lymphocytes

Osteoclast activating factor is a lymphokine produced by mitogen-stimulated human lymphocytes. The current studies describe purification to essential homogeneity of the major form of osteoclast activating factor present in supernatants of phytohemagglutinin stimulated lymphocyte cultures. Preliminary chemical and biological characterization of the purified material was carried out. The active factor is a peptide which migrates in polyacrylamide gel electrophoresis as an α-2 fraction in native gels and as a 9,000-dalton species in sodium dodecyl sulfate-urea gels. The purified fraction stimulates bone resorption $\text{in}\text{vitro}$ at doses between 0.1 and 500 ng/ml, with half-maximal stimulation at approximately 1 ng/ml.     

Action of insulin analogues on cultured human fibroblasts reflects biological potency.

The biological sensitivity of cultured human skin-derived fibroblasts was examined in terms of the ability of insulin and insulin analogues to stimulate the uptake of alpha-aminoisobutyrate in these cells. The relative biological activity measured: insulin=desalanine insulin > proinsulin > desoctapeptide insulin parallels exactly the relative activity of these compounds on fat and muscle preparations both $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$. Inactive insulin analogues do not stimulate alpha-aminoisobutyrate uptake. It is concluded that the chemical specificity of human fibroblast insulin receptors is retained in culture and that the biological responsiveness (alpha-aminoisobutyrate uptake) of such cells to insulin should prove useful for comparative studies of receptors obtained from different individuals.     

Characterization of in vitro differentiated secondary lymphocytes : Quantitative and ultrastructural study

Blast cells derived from rat lymphocytes by stimulation with concanavalin A (ConA) or pokeweed mitogen (PWM), or by sensitization on xenogeneic fibroblast monolayers, transformed into secondary small lymphocytes following their transfer to syngeneic monolayers devoid of mitogen or sensitizing antigen. This transformation resulted in the disappearance of morphological blast characteristics such as euchromatic nuclei, prominent nucleoli and the aggregation of ribosomes into polysomes. Secondary lymphocytes resembled non-stimulated cells, but differed from them in possessing a slightly larger cytoplasm containing large numbers of lysosomal bodies, interchromatin granules within the nuclei, nucleoli containing homogeneous fine granulo-fibrillar material and a relatively developed Golgi apparatus. Upon re-exposure to the stimulating mitogen or the sensitizing phenotype, the secondary lymphocytes rapidly transformed into blast cells with cytotoxic activity.     

The intracellular location of hepatic fructose 1,6-biphosphate aldolase.

Foemmel $\text{et}\text{al.}$ (J. Biol. Chem. $\text{250}$, 1892–1897, 1975) have presented evidence that rat liver aldolase exists $\text{in}\text{situ}$ as a specific complex with the endoplasmic reticulum. In the present study we have examined the alternative possibility that binding of enzyme to the particulate element represents an artifact of the dilution of the ionic constituents of the cytoplasmic milieu. To this end, procedures were developed for homogenization and subfractionation which effected less than a 3-fold dilution of the intracellular content. Using these procedures, virtually all of the liver aldolase was recovered in the soluble supernatant fraction. We conclude that aldolase is not associated with the endoplasmic reticulum $\text{in}\text{situ}$.     

In vivo receptor binding: attempts to improve specific/non-specific ratios.

$\text{In vivo}$ receptor binding was examined using ³H-spiperone and ³H-pimozide for dopamine receptors and ³H-LSD for serotonin receptors. Two strategies for improving total: nonspecific binding ratios were tested. The first was to deplete endogenous ligands by various pharmacological treatments prior to ³H-ligand administration in an attempt to increase specific receptor binding; the second was to perfuse the brain with ice-cold saline after ³H-ligand administration in an attempt to reduce nonspecific binding. Alteration of dopamine and serotonin by administering d-amphetamine, reserpine, alpha-methyl-paratyrosine or parachlorophenylalanine did not significantly elevate striatal: cerebellar or cortical: cerebellar (measures of total: nonspecific) bonding ratios. However, perfusion with ice-cold saline significantly improved the ratios for both dopamine and serotonin receptors. Thus, cold saline perfusion may be of value in reducing blank values in autoradiographic and other studies requiring $\text{in}$$\text{vivo}$ labelling of receptors.     

Dexamethasone selectively increases monoamine oxidase type a in human skin fibroblasts

The effects of several hormones known to affect monoamine oxidase activity $\text{in}\text{vivo}$ have been studied in living human skin fibroblasts grown in culture. Of the hormones tested, the synthetic glucocorticoid dexamethasone caused the greatest increases in activity at physiologic concentrations. Increases of 10–12 fold were observed after 8–9 days of exposure to 5 × 10^?8 M dexamethasone. This increase in activity was accompanied by a change in the relative proportion of the A and B types of activity in fibroblasts, from about 35% A:65% B in control cultures to 90% A:10% B in cultures exposed to dexamethasone. The increase in activity and the shift in the proportion of A and B activities could be accounted for almost exclusively by a specific increase in the number of Type A molecules.     

Glucose transport and metabolism in cultured human skin fibroblasts

Human skin fibroblast cultures, seeded at $\text{10}{}^5\text{cells}\text{5}\text{cm}$ plate and allowed to grow to confluence at approx. $\text{10}{}^6\text{cells}\text{5}\text{cm}$ plate, utilized a glycolytic mode of metabolism where the ratio of glucose utilized to lactate produced wa 0.62±0.05 (Zielke, R.H., Ozand, P.T., Tyldon, J.I., Sevdalian, D.A. and Cornblath, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4110–4114) (mean±S.E.). When the glucose in the medium was exhausted, the lactate produced during the highly glycolytic phase was then reutilized. In monolayer cultures that had been washed with phosphate-buffered saline, rates of glucose utilization were measured at 0.25 and 2 mM glucose by monitoring the appearance of ³H₂O from [5-³H]glucose. Rate of utilization for each concentration of glucose decreased markedly as the cultures became more confluent. This decrease also correlated with a reduced ability to transport glucose as measured by 2-deoxy-[³H]glucose uptake in washed monolayer cultures. In washed confluent culture of fibroblasts, glucose utilization was markedly decreased by the presence of pyruvate and lactate but not by glutamine. The respiratory inhibitors, rotenone and antimycin, did not increase the rate of glucose utilization except when added in combination with pyruvate. We conclude that cultured skin fibroblasts posses a highly glycolytic mode of metabolism but that this mode can become more oxidative in the presence of sufficient quantities of pyruvate and lactate.     

Release of soluble factors from lymph nodes containing mycobacterial granulomas and their effect on fibroblast function in vitro

A study has been made of the action of culture supernatants from guinea pig lymph nodes containing mycobacterial granulomas on protein and DNA synthesis of homologous fibroblast cultures. Supernatants from both the Bacillus Calmette-Guerin (BCG) and Mycobacterium leprae granulomas release soluble nondialysable factors in vitro which stimulate [¹⁴C]proline and [¹⁴C]leucine incorporation by fibroblasts and depress their [³H]thymidine uptake. These supernatants did not show any detectable migratory inhibitory activity in vitro. On the other hand, supernatants from sensitized lymphocytes incubated with purified protein derivative (positive migratory inhibitory activity) had no effect on fibroblast function. Thus, the effect of granuloma supernatants is unlikely to be due to lymphokines. However, supernatants from dinitrofluorobenzene-sensitized lymph nodes also showed a stimulation of [¹⁴C]proline incorporation into total protein synthesised by fibroblasts and depressed the [³H]thymidine uptake. Furthermore, supernatants from live BCG organisms in culture on addition to fibroblasts enhanced their [³H]thymidine uptake in vitro. It would appear therefore that fibroblast activation in lymph nodes containing mycobacterial granulomas could result from the release of soluble factors of lymphocyte origin rather than from cells of the mononuclear phagocyte system. These factors appear to be independent of classical lymphokines that act on macrophages in vitro. An additional factor may be derived from the mycobacteria themselves.     

Decreased rate of cytolysis of Colpidium striatum by cystic fibrosis serum. I. Bioassay and evidence for the possible involvement of a C/F factor--IgG complex

Treatment of the protozoan ciliate $\text{Colpidium}$$\text{striatum}$ with whole serum from cystic fibrosis (C/F) patients, their parents (obligate heterozygote carriers), and normal controls uniformly caused complete cytolysis of the protozoa. At a 1:10 dilution of serum, a distinct decrease in the rate of cytolysis was found for 16 out of 19 cystic fibrosis patients, and 12 out of 16 heterozygote samples. An investigation into the cause of the decreased cytolytic rate indicated that a serum component associated with the IgG fraction from homozygote and heterozygote serum was responsible for the decreased rate.     

Proteins with haemagglutinin activity in larvae of the Colorado beetle, Leptinotarsa decemlineata

The haemagglutinating activity of larval haemolymph of Leptinotarsa decemlineata against red blood cells of various origins has been examined. This activity appeared to be unspecific, since all the different types of erythrocytes were agglutinated by a haemolymph dilution of $\text{1}\text{128}$ to $\text{1}\text{512}$. Only horse erythrocytes were agglutinated to a greater degree ($\text{1}\text{3200}$. Red blood cells became much more sensitive after treatment with trypsin, while formol fixation also resulted in a better agglutinability. Sulphated polysacchrides (heparin, mucin, dextran sulphate) were good inhibitors of the haemagglutination reaction. A weaker inhibition was obtained with hexosamines. As demonstrated by immunoelectrophoresis, two haemagglutinins occur in larval haemolymph. One is specific for larvae and pupae, and is therefore called the larval-pupal haemagglutinin. It is absent in adults. The second haemagglutinin is the well-known chromoprotein 2, which is present in all developmental stages, including the egg, where it constitutes an important element of yolk proteins. The affinity of chromoprotein 2 toward dextran sulphate was confirmed by precipitation tests in agarose.     

Acquired cellular immunity: extracellular killing of Listeria monocytogenes by a product of immunologically activated macrophages

A listericidal factor was released by monolayers of peritoneal cells from BCG-immune guinea pigs following incubation with PPD in cell culture. Significantly less listericidal factor was released by monolayers of BCG-immune cells incubated without PPD, or by monolayers of nonimmune cells incubated in the presence or absence of antigen. Culture supernatants containing listericidal factor were active at a dilution of 1:10, but supernatants diluted 1:100 were not listericidal. Dialysis of supernatants against fresh tissue culture medium did not affect their ability to kill Listeria, although heating at 60 °C for 30 min destroyed all activity. Supernatants from cultures of guinea pig fibroblasts or ascites-form hepatoma were not listericidal.