Tritiated-thymidine uptake in mixed leucocyte cultures: effect of specific activity and exposure time

The degree of in vitro transformation of lymphocytes to blast cells can be estimated by measuring the uptake of radioactive precursors into DNA. We have used ³H-thymidine uptake to quantitate blastogenesis in mixed leucocyte cultures. In experiments designed to standardize this procedure, the kinetics of thymidine uptake were studied by adding 4 μCi of ³H-thymidine of varying specific activities to 5-day mixed cultures. With high specific activity (0·194 μg total thymidine/culture), the rate of uptake was constant for only about 6 hr, then declined. There was no further cellular uptake between 8 and 24 hr, even though the total radioactivity of the supernatant medium did not diminish appreciably. This decreasing rate of uptake was at least partly the result of thymidine degradation to thymine and dihydrothymine by cellular enzymes. It is also possible that eventual radiation damage to blast cell nuclei may have retarded DNA synthesis when ³H-thymidine specific activity was high. With decreasing specific activity (up to 19·4 μg thymidine/culture), the rate of uptake became more nearly linear throughout 24 hr exposure to the isotope. The possible effects of thymidine degradation and radiation damage should be considered when measuring radioactive thymidine uptake in vitro, and short labelling times should be used whenever feasible.     

Membrane dynamics of HL-A–anti-HL-A complexes of human normal and leukaemic lymphocytes

Membrane dynamics of human lymphocytes treated with anti-HL-A antibodies were studied by means of indirect membrane fluorescence. When cells were incubated at 37°C for several hours, a striking diminution of the labelling index was observed. Furthermore, the pattern of the membrane fluorescence changed from a ring-shaped or patchy fluorescence to a polar concentration of the label (`cap formation'). `Cap formation' could be inhibited by incubation at 0°C and by 3 × 10⁻² M sodium azide. Part of the labelled HL-A–anti-HL-A complexes could be detected within intracellular vesicles, which suggests uptake by endocytosis.

Lymphocytes of patients with chronic lymphocytic leukaemia (CLL) did not show a decrease of the labelling index. `Cap formation' was almost completely lacking, and uptake of HL-A–anti-HL-A complexes by endocytosis was not observed.

These findings indicate an impairment of membrane dynamics of CLL lymphocytes.

The importance of these membrane dynamics for the activation of lymphocytes is discussed.

     

The response of human leucocyte cultures to stimulation by tuberculin and phytohaemagglutinin measured by the uptake of radioactive thymidine

A method is described for assessing the lymphocyte transformation of human leucocyte cultures induced by phytohaemagglutinin (PHA) and tuberculin purified protein derivative (PPD) by the measurement of the uptake of [¹⁴C]thymidine. The thymidine uptake of the cultures has been expressed as percentage of the total activity added. The mean basal uptake of twenty-two unstimulated cultures was 0·96%. The mean uptake of twenty-four PHA stimulated cultures was 19·5%, whilst the mean uptake of eleven PPD stimulated cultures from seven subjects, who were strongly positive on skin testing to tuberculin, was 20·1%. A rise of thymidine uptake was detected in five out of six tuberculin negative subjects (mean uptake 3·8%). Hydrocortisone hemisuccinate in final concentrations of 5 and 50 μg/ml usually reduced the thymidine uptake of unstimulated and PHA and PPD stimulated cultures, though the total cell count was reduced in only four out of ten cultures. An increase of thymidine from 1 to 64 μg/culture resulted in twoto three-fold increase in the uptake of thymidine; further increase of thymidine added to the cultures to 256 μg/culture resulted in a decrease of thymidine uptake.     

Sensitivity of SARS‐CoV‐2 to different temperatures

This study was designed to investigate the sensitivity of SARS‐CoV‐2 to different temperatures, to provide basic data and a scientific basis for the control of COVID‐19 epidemic. The virus was dispersed in 1 mL basal DMEM medium at a final concentration of 10^3.2 TCID₅₀/mL and then incubated at 4, 22, 30, 35, 37, 38, 39 and 40°C for up to 5 days. The infectivity of residual virus was titrated using the Vero E6 cell line. The results showed that the virus remained viable for 5 days at 4°C, and for 1 day only at 22 and 30°C. We found that the infectivity of the virus was completely lost after less than 12 hours at 37, 38 and 39°C, while at 40°C, the inactivation time of the virus was rapidly reduced to 6 hours. We show that SARS‐CoV‐2 is sensitive to heat, is more stable at lower temperatures than higher temperature, remains viable for longer at lower temperatures, and loses viability rapidly at higher temperatures.     

Microtechnique for quantitative evaluation of in vitro lymphocyte transformation

A microtechnique is described to quantitate in vitro transformation of lymphocytes from man and marmosets following stimulation by PHA and different antigens. The ¹⁴C-thymidine uptake of lymphocytes grown in cultures of 0·06–0·2 ml of whole blood was measured by liquid scintillation. The magnitude, consistency and reproducibility of the results were similar to those achieved using cultures of purified lymphocytes or cell-rich plasma but fewer technical procedures were required.     

Lymphocyte transformation using whole blood cultures: an analysis of responses

The use of whole blood cultures as a simplified replacement for separated lymphocyte cultures was investigated. Washed whole blood cells, in optimum volumes, gave higher substrate uptake per lymphocyte than did separated lymphocytes. ³H-uridine and ³H-thymidine labelling after PPD stimulation both gave peak uptakes after 5 days of culture. Uridine uptake increased early in stimulated cultures but the thymidine labelled response on day 5 was of much greater magnitude. A comparison of the peak responses to PPD at 5, 25 and 125 μg/ml over a 3-day period showed that not all antigen concentrations produced maximum responses on the same day. The lower concentrations peaked earlier. The delayed response with 125 μg/ml was still increasing when the weaker responses began to decline. This suggests that quantitation of sensitivity to antigens in vitro requires assay of response to several different antigen concentrations over several days.     

The uptake of tritiated thymidine by human spleen cell cultures: a comparison between fresh and stored cells

Individual cell suspensions were made from four human spleens, and part of each suspension was stored at—196° for up to 50 days. After being thawed the cells were placed in tissue culture and `flash' labelled with tritiated thymidine at successive intervals in the culture period. The pattern of thymidine uptake was similar when comparison was made between cultures of stored cells and similar cultures of fresh cells from the corresponding spleens. It is concluded that individual stored spleen cells retain normal viability, following storage for at least 50 days.     

Rapid cytotoxicity of human B lymphocytes induced by VH4-34 (VH4.21) gene-encoded monoclonal antibodies

We have previously described two human cold agglutinin MoAbs 216 and A6(H4C5), that are derived from the VH4-34 (VH4.21) gene that bind specifically to a cell surface ligand on human B lymphocytes. In this study, we report that binding of 216 and A6(H4C5) leads to rapid killing of target B cells. This complement-independent cytotoxicity was measured by three independent assays, cell viability dye uptake on FACS, ³H-thymidine uptake, and the 3(4,5)-dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxicity was specific for CD20⁺ mononuclear cells in human spleen and peripheral blood. The MoAbs were also cytotoxic to human B cell lines Nalm-6, OCI-LY8, Arent and SUP-B8, but not to T cell lines HuT 78 and PEER. As observed by scanning electron microscopy, membrane pores were formed within 15 min of exposure to the MoAbs. Cytotoxic activity was dependent on MoAb concentration and temperature of exposure. Killing was greater at 4°C than 37°C. Sodium azide and EDTA did not block the cytotoxic activity. No DNA fragmentation typical of apoptosis was observed. This rapid cytotoxic activity, independent of physiologic cellular processes and independent of complement, suggests a novel mechanism of cell death via membrane perturbations.     

A mammalian cell mutant with temperature-sensitive thymidine kinase

We have isolated a mutant clone from mouse FM3A cells with temperature-sensitive defects both in cytokinesis and in thymidine kinase enzyme activity. The clone, designatedtsCl.B59, was isolated after mutagenesis at 33°C followed by exposure to cytosine arabinoside at 39°C. It was derived from a thymidine kinase deficient, 5-bromodeoxyuridine-resistant clone (S-BUCl.42) which was originally derived from wild-type clone H-5 of FM3A cells. The temperature-sensitive mutant clone grows normally at 33°C, but not at 39°C, where it exhibits an increased frequency of multinucleate cells due to defective cytokinesis. Unlike the parental S-BUCl.42 cells, which have negligible thymidine kinase activity and are unable to incorporate ³ H-thymidine, the mutant incorporates substantial amounts of ³ H-thymidine at 33°C, although its thymidine kinase activity remains lower than that of wild-type H-5 cells. When cultures oftsCl.B59 cells are transferred to 39°C, incorporation of ³ H-thymidine decreases markedly. The decrease has been shown to be due to thermolability of the thymidine kinase in tsCl.B59 cells.     

Characterization of the cellular immune defect in lepromatous leprosy: a specific lack of circulating mycobacterium leprae-reactive lymphocytes

The blastogenic response of leucocyte cultures from patients with tuberculoid and lepromatous leprosy has been studied. The leucocytes from the two groups were studied simultaneously and cultivated in the same pool of normal human serum. While the leucocytes from twenty-eight tuberculoid patients responded quite strongly to Mycobacterium leprae after 7 days of culture (average lymphocyte transformation 11·1%), there was a complete lack of response in similar cultures from twenty-seven lepromatous patients (average 0·1% transformed cells). These results were confirmed by studies on cellular incorporation of ³H-thymidine in the cultures from four tuberculoid and four lepromatous patients.

This lack of response was quite specific as leucocytes from several lepromatous patients responded to BCG. Furthermore, four patients with both lepromatous leprosy and tuberculosis responded as strongly to BCG and PPD as tuberculous patients without leprosy. In the mixed leucocyte reaction, between two lepromatous or two tuberculoid patients respectively, the lepromatous cells responded well (average 15·0%) and comparably to tuberculoid cells (average 12·1%).

The blastogenic response of purified lymphocytes to M. leprae revealed a similar pattern, i.e. the tuberculoid cells responded well, while again there was a lack of response in the lepromatous group.

It is concluded that the lepromatous patients lack circulating lymphocytes responding to M. leprae, indicating that their immunological defect as observed in the present study has features in common with immunological tolerance.

     

Validation of autoradiography for recognition of antigen-binding lymphocytes in blood and lymphoid tissues: quantitation and specificity of binding

Antigen-binding lymphocytes were recognized by their reaction with radioiodine labelled antigens such as flagellin and haemocyanin. Counts varied according to the antigen and species studied. For flagellin, counts in human blood of antigen-binding lymphocytes (mean ± 1 SD per 1000 lymphocytes) were 19·0±3·0, and in foetal thymus 18·2±5·0 and spleen 3·5±0·5. Results depended on contact time of cells with antigen, concentration of antigen, autoradiographic exposure, presence of natural antibody and antibody levels after immunization. Antigen-binding lymphocytes in blood were not antibody-producing cells. The specificity of the antigen-binding reaction was shown by exposing lymphocytes to 0·5 μg of two antigenically distinct flagellins; there was a 67–100% increase in the counts in contrast to the 20–45% increase on doubling the dose (0·5 μg to 1 μg) of flagellin from Salmonella adelaide. Cytophilic antibody as the cause of antigen binding was excluded.

The binding of flagellin to lymphocytes was prevented by anti-human IgM and light chain antisera, but not anti-human IgG sera. The binding of labelled flagellin was prevented by unlabelled flagellin but 100 times more was needed for blood lymphocytes than thymocytes. It is inferred that thymocytes, T cells, have considerably fewer receptors than most β lymphocytes detectable in blood.

Using standardized conditions, radiolabelled antigen binding provides a reproducible, immunologically specific and flexible technique allowing study of the nature and role of antigen-binding cells and cell surface receptors.

     

Lymphocyte surface components. I. Stimulation of enzyme-treated rabbit lymphocytes by non-specific mitogens

Rabbit lymphocytes from various locations were treated briefly with proteases and the rate of incorporation of thymidine following stimulation by `non-specific' stimulants (Staphylococcus filtrate, SF, and Phaseolus vulgaris phytohaemagglutinin, PHA) measured. The proteases themselves, especially at concentrations exceeding 0·005%, sometimes caused cells to incorporate more thymidine than untreated cells. Stimulation with SF or PHA was much greater when lymphocytes were first treated with protease at low concentrations (when enzyme alone failed to stimulate). This effect was shown particularly by lymphocytes from gut-associated lymphoid organs. Peripheral blood lymphocytes, while unaffected by proteases at a concentration of 0·005%, incorporated increased amounts of thymidine after treatment with proteases at higher concentrations: pronase is especially potent. Further evidence is presented, distinguishing between the cytoagglutinating and mitogenic characters of PHA.     

Lymphocytotoxins in infectious mononucleosis: a non-cytotoxic effect on their cytotoxicity in vitro

The lymphocytotoxic activity (LCA) of sera from patients with infectious mononucleosis (IM) was tested against lymphocytes under various experimental conditions. Firstly, lymphocytes from 11 healthy donors were preincubated with pools of normal human sera (NHS) or IM sera at 37°C and then tested for (a) reactivity with the same IM sera in a standard lymphocytotoxin assay at 15°C; (b) rosetting with various sheep erythrocyte (E) preparations (E, EA and EAC) and (c) stimulation by non-specific activators (phytohaemagglutinin, pokeweed mitogen and concanavalin A). These experiments showed that preincubation of normal cells with IM sera caused significant reduction in subsequent lymphocyte killing at 15°C (P<0·01) compared to unincubated cells or those preincubated with pooled NHS. There was no change in the binding of E, EA and EAC or mitogen stimulation following incubation. Culture of preincubated lymphocytes in lymphocytotoxin-free medium for 24 hr did not restore LCA at 15°C. Secondly, a pool of normal lymphocytes was incorporated into media containing either 2,4-dinitrophenol or sodium azide and tested for LCA against 11 acute IM sera and two NHS at both 15 and 37°C. No significant change in cell killing was observed at 15°C in the presence of these inhibitors, but there was a significant return of LCA at 37°C. Finally, normal lymphocytes and cells from two patients with IM were cultured at 37°C in lymphocytotoxin-free medium to determine the role of down-regulation of lymphocyte surface receptors in reducing autolymphocytotoxicity during the acute phase of the illness. There was no change in cell killing by IM sera after culture for 24 hr. These experiments show that lymphocytotoxic sera from patients with infectious mononucleosis interact with normal lymphocytes at 37°C without causing cell killing. This interaction caused a change in surface-binding characteristics that was not reversed by culture in ligand-free medium for 24 hr. Studies using metabolic inhibitors suggested that the failed lymphocytotoxicity at 37°C resulted, at least in part, from lymphocyte metabolism, although this did not inhibit the reaction between cytotoxic material and the lymphocyte surface.     

Specific inactivation of sensitized lymphocytes in vitro using antigens labelled with astatine-211

This is a study of the use of astatine-211 as an alternative to iodine isotopes in radioactive antigen suicide experiments. The theoretical advantages of ²¹¹At are that it is a high energy (5·87 MeV), short half-life (7·2 hr)α-emitter with a short path length of 60 μm. Its destructive effect, measured in terms of degree of ionization per micron is 300 times that of ¹²⁵I and ¹³¹I. Streptokinase (SK), tuberculin (PPD) and phytohaemagglutinin (PHA) were labelled electrolytically with ²¹¹At (3–9% incorporation). These antigens were not destroyed by the labelling technique or by self-irradiation during radioactive decay. Autoradiography showed that only a small fraction of lymphocytes bound ²¹¹At-labelled SK and PPD whilst most lymphocytes bound ²¹¹At-labelled PHA. Lymphocytes exposed for 40 min to 0·35–1·40 μCi of labelled antigens lost 35–83% of their ability to transform upon subsequent exposure to the unlabelled antigens. The inhibition was specific in that responsiveness to unrelated antigens was retained. These results extend our previous findings with the use of ²¹¹At in in vivo antigen suicide experiments.     

The mechanism of action of soluble lymphocytic mediators: I. A pulse exposure test for the measurement of macrophage migration inhibitory factor

Culture supernatants containing macrophage migration inhibitory factor (MIF) were obtained by incubating lymphocytes of guinea-pigs, immunized with Freund's complete adjuvant (FCA), with tuberculin PPD in vitro. Exposure of normal peritoneal macrophages to MIF-containing supernatants for 2 hours at 37° (pulse exposure), followed by suspension in culture medium and transfer to capillaries, resulted in inhibition of migration in vitro for the next 24 hours. No inhibition was seen when macrophages were incubated with MIF at 4°. On the other hand when exposure to MIF at 4° was followed by incubation of the cells for 2 hours at 37° in culture medium, in the absence of MIF, inhibition of migration was obtained. These results indicate that: (a) macrophages possess a specific receptor able to bind MIF at either 4° or 37°, and (b) inhibition of migration by receptor bound MIF requires a temperature-dependent active process, the nature of which remains unknown.

Passage of lymphocytes through columns of glass beads resulted in a population of cells with intact or heightened MIF-forming ability, as assessed by both conventional and pulse exposure techniques.

     

Disseminated Intravascular Coagulation Induced with Leukocyte Procoagulant

The procoagulant activity of rabbit peritoneal leukocytes significantly increased when the leukocytes were incubated in suspension cultures at 37 C for 24 hours. Intravenous infusions of Iysates of 232 × 10⁶ rabbit leukocytes which had been incubated in cultures at 37 C for 24 hours produced disseminated intravascular coagulation and vasculitis involving the pulmonary arteries in normal rabbits. Intraaortic infusions of lysates of 230 × 10⁶ similarly incubated leukocytes produced renal thrombosis and renal cortical necrosis in normal rabbits. These observations suggest that the procoagulant of granulocytic leukocytes could play a role in the generalized Shwartzman reaction and other syndromes of disseminated intravascular coagulation.     

Synthesis,storage and release of [14C]acetylcholine in isolated rat diaphragm muscles

1. Segments of rat diaphragms were kept in choline-free media for 4 hr and were then exposed to a physiological concentration of [¹⁴C]-choline (30 μM) at 37° C. The synthesis, storage and subsequent release of [¹⁴C]acetylcholine by the muscles was assessed by isotopic- and bio-assays after isolation of the transmitter by paper electrophoresis.2. Replacement of endogenous acetylcholine (0·92 μ-mole/kg) with labelled acetylcholine proceeded slowly at rest, but rapidly during nerve stimulation. [¹⁴C]Acetylcholine accumulated most rapidly when hydrolysis of the released transmitter, and thus the re-use of endogenous choline, was prevented by an esterase inhibitor. Fully replaced stores were maintained during nerve stimulation by synthesis rates sufficient to replenish at least 35% of the store size in 5 min.3 In the presence of hemicholinium-3, which inhibits choline uptake, acetylcholine stores declined rapidly during stimulation, and residual synthesis was slight, indicating little intraneural choline. Net choline uptake into nerve terminals was estimated from the highest observed synthesis rate and from previous measurements of the number and size of terminals, as 3-6 p-mole/cm² sec.4. Transmitter synthesis was localized in the region of end-plates, and was reduced to a few per cent of normal 6 weeks after phrenic nerve section. Release experiments suggested that at least half of the acetylcholine in phrenic nerves is in their terminals; from this content and the morphology of the terminals, the average concentration of transmitter in the whole endings would appear to be about 50 m-mole/l. Homogenization of the muscles freed choline acetyltransferase into solution, but left some [¹⁴C]acetylcholine associated with small particles, presumably synaptic vesicles.5. Resting transmitter release was about 0·013% of stores/sec. With 360 nerve impulses at 1-20/sec, release increased up to 0·43% of stores/sec, and amounted to 3·5-7 × 10^-18 moles per end-plate per impulse. The release rate was unaffected by the doubling of store size which occurred with eserine, but the extra transmitter did help to maintain releasable stores during prolonged stimulation. Experiments with fractional store labelling indicated that newly synthesized acetylcholine was preferentially released.6. Preformed [³H]acetylcholine was not taken up and retained by muscle or nerve cells in the absence of an esterase inhibitor. With eserine present, labelled acetylcholine was taken up uniformly by muscle segments; when eserine was then removed, radioactive acetylcholine remained only near neuromuscular junctions.     

Enhancement of human lymphocyte responses to phytomitogens in vitro by incubation at elevated temperatures.

Incubation of human lymphocytes with PHA or Con A at 39 degrees C for 3 days caused a consistent, statistically significant, increase in [3H]thymidine uptake, compared with cultures incubated at 37 degrees C. The responses were also significantly increased after 2 days of incubation, suggesting that hyperthermia not only enhanced, but also caused an earlier onset of the mitogen response.     

Kinetics of the proliferative response to antigen in vitro of rabbit lymph node cells taken at various times after immunization

The antigen stimulation of thymidine uptake by immune rabbit lymph node cells in vitro was studied. The kinetics of the response varied depending on the time between immunization and culture. Cultures set up early after immunization showed a peak response over day 1–2 of culture while those set up late after immunization showed a peak response over day 3–4.

Studies using the metabolic inhibitor BUDR suggested that this is due at least in part to a larger recruitment of cells into the response during the third day of culture when lymph node cells taken late after immunization were used.

Removal of antigen from cultures after brief exposure of cells at 4° reduced the magnitude but did not eliminate the proliferative response, suggesting that some antigen is specifically bound to the cells in the cold. Readdition of antigen restored normal reactivity.

Holding cells at 4° for 4 hours without antigen had no effect on their response to subsequent addition of antigen. However, if cells were held at 4° for 3 hours with antigen present a severe degree of depression of subsequent thymidine incorporation was observed in some but not all experiments. This depression of responsiveness was interpreted as an in vitro phenomenon comparable to immunologic tolerance.

     

Mitogenic response of marmoset lymphocytes: cytokinetics and identification of responsive cells

Isotope (tritiated thymidine, [³H]Tdr) incorporation into lymphocytes from two marmoset species, New World primates which are haemopoietic chimeras, was studied using peripheral blood lymphocyte (PBL) cultures incubated in vitro for 1–5 days with different concentrations of Concanavalin A (Con A), leucoagglutinin (LA), bacterial lipopolysaccharide (LPS) from gram-negative bacteria, rabbit anti-marmoset immunoglobulin (anti-Ig) serum, thymosin or irradiated histoincompatible (xenogeneic) lymphocytes. Only the plant lectins and xenogeneic lymphoid cells stimulated the uptake of [³H]Tdr. Lymphocyte enrichment experiments demonstrated that cells responsive to plant lectins and xenoantigens were primarily thymus-derived (T) lymphocytes. Bacterial endotoxin (LPS), however, enhanced the mitogenic response of PBL to Con A when LPS and plant lectin were added together at culture initiation. Thymosin caused either enhancement or suppression of the response to Con A depending on its time of addition relative to lectin stimulation. Addition of thymosin to lymphocyte cultures with Con A or 24 h later caused a decrease in isotope incorporation, while addition of thymosin 48 h later caused a 12–100% increase in [³H]Tdr uptake. Lymphocyte chimerism did not influence the mitogenic response since single-born, nonchimeric marmosets responded to plant lectins in a manner similar to marmosets with varying proportions of chimeric blood elements. Cytological analysis of stimulated lymphocytes from heterosexual marmosets revealed the percentage chimerism in the polyclonal mitogenic response and the clonal mixed lymphocyte reaction to be similar.