Effect of tritiated thymidine and 5-bromodeoxyuridine on development of immunologically competent lymphocytes

Results of the present experiments suggest that dividing cells in rat bone marrow can generate lymphocytes capable of initiating a systemic graft-versus-host reaction. In these experiments, Lewis bone marrow cells were incubated in vitro with either tritiated thymidine ([³H]thymidine) or 5-bromodeoxyuridine (BUDR)—agents which inhibit cell proliferation. Bone marrow cells prepared in this way and injected into an intermediate (Lewis×DA)F₁ host were unable to proliferate and mature into circulating immunologically competent lymphocytes.

Concentrations of [³H]thymidine and BUDR which inhibited the development of putative lymphocyte precursors in bone marrow had no obvious effect on the immunological performance of thoracic duct lymphocytes. Lewis lymphocytes, cultivated in medium containing these agents, caused a vicious graft-versus-host reaction in X-irradiated (Lewis×BN)F₁ hybrid rats. The results strengthen the view that [³H]thymidine and BUDR exert their inhibitory effect on replicating lymphocyte precursors and not on long-lived members of the circulating lymphocyte pool.

     

Migration of newly formed small lymphocytes from bone marrow to lymph nodes during primary immune response

Selective DNA labelling of bone marrow cells in vivo was used to determine the effect of antigenic stimulation on the migration of small lymphocytes from bone marrow to popliteal lymph nodes. Following footpad injection of keyhole limpet haemocyanin (KLH) in guinea-pigs the regional nodes showed an early increase in weight and cellularity together with a progressive increase in cell proliferation. When [³H]thymidine was injected into tibial and femoral marrow 2 days before KLH administration the DNA radioactivity of the KLH-stimulated nodes increased rapidly and always exceeded that of contralateral nodes. Simultaneously, in radioautographic sections of lymph nodes labelled small lymphocytes, indicative of an origin from marrow precursors, appeared throughout the cortex, post-capillary venules, subcapsular sinus, medullary cords and sinuses. In KLH-stimulated nodes the number of labelled small lymphocytes per section was higher than in contralateral nodes, especially in the cortex, and some of these cells appeared in germinal centres. Labelled large blast cells and macrophages were also increased in numbers. Similar changes were observed in lymph nodes of parental strain rats following intramyeloid [³H]thymidine administration and footpad injection of lymphoid cells from F1 hybrid rats. The results demonstrate that, during the early response of lymph nodes to various antigens, local changes in cell traffic include an enhanced accumulation of newly formed small lymphocytes, putative virgin B lymphocytes, generated in the bone marrow prior to the antigenic stimulation.     

The demonstration in vitro of the mitogenic effects of trypanosomal antigen on the spleen cells of normal, athymic and cyclophosphamide-treated mice

When the spleen cells of normal NIH mice were cultured with pokeweed mitogen (PWM), staphylococcal filtrate (SF) and trypanosomal antigen (TAg), and tritiated thymidine ([³H]Tdr) incorporation was used as a measure of mitogenic activity, the TAg (at a level of 25 μg/ml of spleen cell suspensions containing 2–3 × 10⁶ cells/ ml) was found to be a better mitogen than SF. PWM, however, was more effective than either of the two. [³H]Tdr incorporation by the spleen cells of Nu/nu (`athymic') mice was greater than that by the spleen cells of normal NIH mice when equal numbers of both cells were cultured with TAg. Pretreatment of NIH mice with cyclophosphamide suppressed [³H]Tdr incorporation by their cells when TAg was added to the cultures. The TAg used was derived from T. brucei TREU 226 obtained from Edinburgh University.     

Mitogenic activity of staphylococcal peptidoglycan.

Staphylococcus aureus peptidoglycan displayed a marked dose-dependent mitogenic activity for mouse splenocytes and human peripheral blood lymphocytes in vitro, as measured by increased [3H]thymidine incorporation. Similarly it was mitogenic for athymic nude mouse spleen cells, whereas no blastogenic effect was observed in T cell-enriched and B cell-depleted mouse lymphocyte cultures. These data demonstrate that peptidoglycan-responding cells in mouse spleen cell cultures are B lymphocytes.     

In Vitro Proliferation of T Lymphocytes from Listeria-Infected Rodents: Assay Conditions for Rat Peritoneal Exudate Cells and Characterization of an Inhibitor

Conditions favorable to [³H]thymidine incorporation into antigen-stimulated T lymphocytes from Listeria-infected rats have been established. In cultures of peritoneal exudate (T) lymphocytes purified twice with nylon-wool vigorous antigen-specific proliferation was observed within 2 days. Cultures of lymphocytes from nodes draining a subcutaneous Listeria-infection site differed in that back-ground proliferation was higher than for peritoneal exudate lymphocytes, and [³H]thymidine incorporation was maximal at day 3. A critical factor for the rate of proliferation was the lymphocyte-to-macrophage ratio; optimal cultures of peritoneal exudate lymphocytes contained 2 to 5% macrophages. Macrophages exceeding a proportion of 10% strongly, if not completely, inhibited [³H]thymidine incorporation into antigen-stimulated lymphocytes. Inhibition was associated with mononuclear cells, adherent to plastic or nylon-wool, of the stimulated or unstimulated peritoneal cavity. It was neither attributable to release of cold thymidine from macrophages nor to rapid degradation of particulate antigen by macrophages. The degree of inhibition reflected the metabolic activity of macrophages; on a cell-for-cell basis, heat-killed and glutaraldehyde-fixed macrophages were less inhibitory, and stimulated macrophages were more inhibitory than macrophages from the unstimulated peritoneal cavity.     

Immunological interaction between allogeneic and xenogeneic rodent spleen cells in culture: stimulation of DNA synthesis

An early proliferative response measured by [³H] thymidine uptake occurred following mixing and culture in vitro of spleen cell suspensions derived from genetically unrelated rodents. This response reached a maximum value at 40–44 hr. When either component of the mixture in equivalent amounts was cultured alone, only a limited response occurred.

This marked stimulation of DNA synthesis occurred when spleen cells derived from LAF₁ mice and a pool of spleen cells from three non-inbred rats were mixed and cultured. No such response occurred in the mouse spleen cultures either in the absence of rat spleen cells, or in the presence of disrupted rat spleen cells. An equally marked response occurred when spleen cells from one rat were mixed and cultured either with C57BL10J or DBA/2J mouse spleen cells, or to a lesser extent with spleen cells from a second unrelated non-inbred rat. A similar marked response occurred with cells derived from C57BL10J and DBA/2J and from BALB/c and C3H/HeJ mice.

The existing data suggest that the response of these rodent spleen cells in the mixed lymphocyte culture (MLC) test is dependent on certain genetic differences inherent in the two cell populations.

     

A rapid micro-method for the phytohaemagglutinin-induced human lymphocyte transformation test

A quantitative in vitro method for phytohaemagglutinin (PHA) induced lymphocyte transformation is described. This method has the following advantages.

Cultures require as few as 10⁵ lymphocytes and are performed in microtrays, over a short incubation period (48 hr).

A short pulse time of 4 hr with [³H]thymidine is utilized. This enables high levels of [³H]thymidine to be maintained throughout the labelling period, ensures maximum incorporation of thymidine into cellular DNA, and diminishes cellular damage by internal irradiation.

The extraction process has been simplified by using a communal washing procedure after drying the cultures on glass-fibre filter discs. The procedure is both quicker and more reproducible (coefficient of variation 6%) than extraction and drying on filtration manifolds (coefficient of variation 23%).

The effect of adjusting variables such as the number of cells, incubation time, concentration of [³H]thymidine, concentration of PHA, specific activity of [³H]-thymidine and duration of [³H]thymidine pulse has been studied in order to approach optimal labelling conditions for the assay system.

The reproducibility of the method has been investigated by repeated testing of normal individuals on a day-to-day basis and over extended periods. The mean coefficient of variation for samples repeated daily and over longer time intervals (weeks or months between samples) was 15%.

     

Radiosensitivity of T and B lymphocytes. II. Effect of irradiation on response of T cells to alloantigens

The radiosensitivity of T cells was investigated by studying the effect of irradiation in vitro in suppressing the capacity of parental strain thoracic duct lymphocytes (a) to induce splenomegaly in newborn F₁ mice, and (b) to proliferate in adult irradiated F₁ mice as measured by incorporation of tritiated thymidine ([³H]dThd) 4 days after transfer. By these parameters, small T lymphocytes were found to be highly radiosensitive. It was calculated that, of cells with the reactivity to the alloantigens studied, 0.3 % were capable of a proliferative response after exposure to 500 r. Radiosensitivity was considered to be a reflection of lymphocyte death in interphase. The radiosensitivity of H-2-activated T cells (T. TDL) differed from that of small T cells. Thus, [³H]dThd incorporation by T. TDL measured 1 day after transfer to irradiated F₁ hosts was not abolished, although lowered, by exposure to doses as high as 5000 r; [³H]dThd incorporation measured at 2 days, however, was greatly reduced by much smaller doses of irradiation. In view of evidence obtained elsewhere that the response of T. TDL to alloantigens involves DNA synthesis but not cell division, the present studies were interpreted in terms of irradiation causing death of T. TDL in interphase before entry into DNA synthesis. It was concluded that T. TDL were far more resistant to irradiation-induced interphase death than were small T cells. The small numbers of lymphocytes obtained from thoracic duct lymph of mice exposed to whole body irradiation 4 days before consisted almost entirely of T cells; these cells, although viable, were found incapable of mounting a proliferate response when exposed to alloantigens on transfer.     

Immunosuppressive factors released by transforming lymphocytes in the delayed hypersensitivity skin response to tuberculin

Experiments were carried out in BCG-sensitized cattle to see if factors produced by transforming blood lymphocytes could modify the tuberculin skin reaction or the uptake of [³H]thymidine by sensitized bovine lymphocytes. It was found that the subcutaneous injection on one side of the neck of tuberculin, tuberculin-stimulated autologous blood lymphocytes or culture supernates from tuberculin-stimulated lymphocytes depressed the response to intradermal injection of tuberculin on the other side. The suppression of the response appeared to be antigenically specific since the delayed skin reaction to the intradermal injection of brucallergen was not suppressed by the injection of the same lymphocyte culture supernates.

In vitro studies of [³H]thymidine uptake by autologous lymphocytes showed that the culture supernates from tuberculin-stimulated lymphocytes had a mitogenic activity, but at higher concentrations of supernate, this mitogenic activity was depressed. The higher concentration of supernate also non-specifically suppressed [³H]thymidine uptake by autologous lymphocytes stimulated with phytohaemagglutinin or brucallergen.

Since an immunosuppressive α-globulin fraction had been demonstrated in bovine serum by other workers, it was thought that this may have been the factor released in vitro by the transforming lymphocytes. The α-globulin fraction was therefore isolated from sera of four tuberculous cattle, obtained 10 days after tuberculin skin testing, and from four uninfected and untested control cows. This serum α-globulin fraction from both groups of cattle, suppressed [³H]thymidine uptake by homologous lymphocytes stimulated with tuberculin or PHA but levels of this factor in sera of tuberculous cattle were not raised above those in control cattle.

It was concluded that the antigenically-specific suppression of tuberculin skin-reactions was most likely mediated by antigen—antibody complexes. On the other hand results with cultured lymphocytes may have been due to a non-specific immunosuppressant released from lymphocytes and which had properties in common with a serum α-globulin fraction.

     

Incorporation of [3H]thymidine into the spleen of intact mice during the immune response to sheep erythrocytes (SRC)

The incorporation of [³H]thymidine, administered shortly before killing, into the spleens of intact mice during the primary immune response to SRC has been studied, using autoradiography with exposure periods of 184 days before development.

A mixture of weakly and heavily labelled nuclei were situated in well-defined areas of the follicles. A marked increase of heavily labelled nuclei coincided with an increased uptake of [³H]thymidine into the spleen DNA during the first 3 days of the immune response. At this time the number of lightly labelled nuclei in the follicles was reduced. The heavily labelled nuclei were first apparent in the periarteriolar zone, then in germinal centres spreading out into the red pulp. After the peak of [³H]thymidine incorporation was over (day 3–4) weakly-labelled nuclei accumulated in the red pulp and persisted until day 9 after the injection of SRC.

It was concluded that many non-dividing cells in mouse spleen were incorporating small amounts of [³H]thymidine into their nuclear DNA. In view of the accumulation of such cells in the red pulp during the course of the immune response to SRC, it was considered that this evidence of DNA synthesis was a manifestation of metabolic turnover of this molecule and relevant to the immune process. From the data presented it was also concluded that only a small proportion of the total spleen population engaged in DNA synthesis and proliferation were actually induced to produce specific antibodies. This preliminary investigation showed the complex nature of the immune process leading to antibody synthesis, which requires much further detailed study.

     

The effect of colchicine and colcemid on the mitogeninduced blastogenesis of lymphocytes

The effect of colchicine and colcemid (1 × 10^?6 M) on the blastogenic response of human lymphocytes to concanavalin A was studied in vitro by three different methods. (a) Measurement of [³H]thymidine ([³H]dThd) incorporation which was strongly suppressed by the drugs. (b) Volume spectroscopy showing that the growth of cellular and nuclear volume was only moderately affected by the drugs during the first two days of culture: in drug-treated cultures, 25% of the cells responded by measureable growth of their nuclear volume as compared to 30% in untreated cultures. After two days, however, growth stagnated in drug-treated cultures, and cell division never occurred. (c) Flow cytofluorometry, showing that in drug-treated cultures the number of cells measurably increasing their DNA content, i.e. entering S-phase, was about 60% of that in untreated cultures. However, the drugs caused a majority of the responding cells to stop DNA synthesis before completing S-phase. This effect could not fully account for the strong suppression of the [³H]dThd incorporation indicating that colchicine and colcemid caused a malfunction of the [³H]dThd assay. It is concluded that colchicine and colcemid do not significantly inhibit initiation of a blastogenic response indicating that microtubuli, which are known to be affected by these drugs, are not essential for the triggering of blastogenesis.     

Effects of staphylococcal protein A-treated human leukemic serum on autologous leukemic blast growth and mitogenesis of lymphocytes

Sera and leukemic blasts of 14 patients with acute myelogenous leukemia were stored at –70°C. In eight patients in whom a remission was achieved, peripheral blood lymphocytes were cultured together with irradiated autologous leukemic cells in treated serum (serum adsorbed with protein A Sepharose) or control serum (Sepharose-treated). Lymphocyte activation was determined after 7 days in culture by [³H]thymidine incorporation. In the absence of stored leukemic blasts, significantly more [³H]thymidine incorporation occurred in six of the eight patients' lymphocytes cultured in treated serum compared to control. Enhanced activity was observed in all eight patients when irradiated leukemic blasts were cocultured with autologous lymphocytes in treated serum. In five patients, the addition of 10% or more of control serum to treated serum inhibited lymphocyte mitogenesis. Protein A immunoadsorption may allow increased stimulation of acute myelogenous leukemia remission peripheral blood lymphocytes, which is further enhanced in some patients by the presence of autologous leukemic cells. This change in lymphocyte activation may contribute to the antitumor effects of treating serum with protein A.     

Antigen handling in aging II. The role of Kupffer and endothelial cells in antigen processing in fischer 344 rats

A method has been developed for the purification of Kupffer and endothelial cells from rat liver by collagenase enzyme perfusion followed by centrifugal elutriation. After intravenous injection of a soluble antigen, [³H] azoaniline bovine serum albumin ([³H] BSA), its distribution was studied in isolated cell populations from liver and spleen tissue of two aged groups of male F-344 rats. In young adult rats (6–8 months) both sinusoidal cell types contained the same amount of [³H] BSA; however, in older rats (22–24 months) the amount of antigen in the endothelial cells was significantly decreased. In comparison to the liver, the spleen retained only a small fraction of the injected dose. In order to assess the catabolic properties of both Kupffer and endothelial cells, supernatants obtained from in vitro cell culture were evaluated for both biological and physiochemical properties. Antigen was almost completely degraded by both cell types as determined by gel filtration and did not directly stimulate BSA-primed lymphocytes in vitro; however, these supernatants were shown to enhance the lymphoproliferative response of primed lymphocytes to additional antigen exposure. Kupffer cell receptors, F_c and C₃, assayed by direct rosetting, did not vary with age; endothelial cells also possessed F_c receptors that were found to be unchanged with age. These studies are an initial attempt to better define our previous finding of defective antigen handling with aging by use of isolated pure cell populations.     

Dual pathway of B lymphocyte differentiation in vitro

Direct visualization of the events resulting from LPS stimulation of mouse spleen cells in vitro was achieved by characterizing the cells during four days of culture for morphology, Ig and Θ surface markers and autoradiography after [³H] thymidine uptake. The changes observed were related to biochemical parameters such as incorporation of [³H] thymidine into DNA, Ig biosynthesis and secretion. Two pathways of B lymphocyte differentiation were observed: a) the generation of a large number of small B lymphocytes with high density of surface Ig but no internal pool detectable by immunofluorescence, and b) the maturation of a very small proportion of cells with a large intracellular pool and the ability to secrete Ig. Both cell types arise from dividing blast cells, either physically separated or traced by pulse chase experiments with [³H] thymidine. We discuss whether this duality is caused by the triggering of different B cell subpopulations at different developmental stages, preprogramed to one or the other pathway or whether the final direction of development depends on the microenvironment of individual dividing cells.     

Migration inhibition factor and blast transformation in human lymphoid cultures stimulated by different agents

Macrophage inhibition factor (MIF) like substances were found in the supernatants of human lymphoid cultures stimulated by concanavalin A, tuberculin and allogeneic lymphocytes. These agents are usually regarded as elective stimulators of T lymphocytes. No such a phenomenon was shown when lymphocytes were stimulated by lipopolysaccharide or by goat anti-human F(ab')₂ serum, which are known as preferential B-cell stimulators.

The kinetics of MIF production and [³H]thymidine incorporation show a dissociation between these two phenomena; the MIF production precedes the DNA synthesis.

     

The relationship between antibody formation and deoxyribonucleic acid (DNA) synthesis in mouse spleen during primary and secondary response to sheep erythrocytes (SRC)

Incorporation of [³H]thymidine into the spleens of intact mice during the primary and secondary responses to SRC has been studied. In both responses, auto-radiographs show a well marked increased incorporation of [³H]thymidine by cells in the peri-arteriolar zone during days 1 and 2. The degree of labelling indicated that many of these cells were in S phase in preparation for mitosis. A similar response occurred in cells situated in the mantle layer on day 3 to day 4 of the primary response, but was much less evident in the secondary response. This was in keeping with the results of scintillation counting which showed that increased uptake of [³H]thymidine was more sustained in the primary than in the secondary response to SRC.

Many lightly labelled cells indicating metabolic turnover of DNA developed in the red pulp of the spleen at times which coincided with the maximum development of antibody-producing cells (PFC) which occurred about 24 hours earlier in the secondary than in the primary response.

Germinal centres showed many lightly labelled nuclei. In both primary and secondary responses these areas enlarged, becoming maximal in size after the peaks of PFC were achieved. It was concluded that the enlargement of germinal centres was the result of trapping of cells in the areas rather than proliferation.

From these studies it appears that metabolic turnover of DNA occurred in areas of the spleen intimately associated with antibody formation. The possible role of this process in antibody synthesis is discussed in the light of these observations.

     

Specific in vitro elimination of histocompatibility antigen reactive cells (HARC)

Human lymphocytes proliferating in response to allogeneic cells in mixed lymphocytes cultures (MLC), can be selectively eliminated by incubating the culteres initially with [³H] thymidine of high specific activity. After such treatment the remaining cells are vitually incapable of responding towards the initial stimulating cell donor. The response towards other allogeneic cells was unaltered, except for the non-specific effects of radiation damage. The addition of 0.25 μl/ml of PHA to the cultures during the period of [³H] thymidine incorporation, appeared to enhance the elimination of the proliferating cells.     

The early antibody-forming response to Salmonella antigens: A study of morphology and kinetics in vivo and in vitro

This paper describes a new method for the morphological study of individual antibody-forming cells (AFC) on cell smears of the quality of normal haematological preparations.

The early AFC response to polymerized flagellin of S. adelaide was studied in vivo using C57BL mice, which have very low background levels of AFC and in vitro using dispersed spleen cell cultures from CBA mice. AFC, arising as a result of in vivo or in vitro stimulation were found to comprise a heterogeneous population, including basophilic mononuclear cells, lymphocytes of most sizes, immature blast cells and occasional plasma cells. The earliest AFC detected comprised a high percentage (28 per cent in vivo, 31 per cent in vitro) of small lymphocyte-like cells. Studies of the incorporation of [³H]thymidine showed that most AFC arose by proliferation but that a proportion of AFC, the small lymphocyte-like cells, arose by differentiation of precursor cells not involving cell division.

The effects of antigen concentration on the kinetics of AFC were investigated in vitro. Subtolerogenic antigen doses caused a delayed and decreased AFC response.

     

Human lymphocyte proliferation. I. Correlation between activated and proliferating T-lymphocytes

The response of human peripheral blood lymphocytes to Con A and PHA has been analyzed by [³H]thymidine incorporation and cytofluorometry. Using the latter method, it is possible to quantitate the number of cells in the G₀ phase (normal RNA and DNA content) and in the G₁ phase (elevated RNA, but normal DNA content). A very high correlation is found between numbers of Con A or PHA-induced G₁ cells and [³H]thymidine incorporation in healthy donors. This high correlation is found when culture medium is enriched with 10% autologous plasma or 10% AB-serum. The use of a recently developed defined serum-free medium (RPMI 1640 with albumin, alanine, transferrin, sodium selenite and zinc chloride), however, suggest that donors can be divided into two groups according to different medium requirements for PHA-stimulated lymphocytes. Because several immunoregulatory mechanisms at the level of T-lymphocytes take place in the G₁ phase, it can therefore be expected that cytofluorometric analyses of lymphocytes in the various cell cycle phases may improve the interpretation of altered lymphocyte response to lectins and antigens.     

Deficient growth of C57BL marrow cells transplanted in F1 hybrid mice: Association with the histocompatibility-2 locus

The ability of C57BL, C3H, and A strain marrow cells to proliferate on transplantation into irradiated isogenic, F₁ hybrid, and backcross progeny mice has been investigated by the spleen-colony technique and by measuring the newly-formed DNA in the recipient spleen with [¹³¹I] 5-iododeoxyuridine. Transplants of C57BL cells grew poorly in (A×C57BL)F₁ and in (C57BL×C3H)F₁ and reciprocal hybrids, as compared with isogenic and allogeneic hosts, whereas C3H and A strain marrow grafts were successful in isogenic, F₁ hybrid and backcross recipients. In segregating backcross progeny, i.e. in offspring from F₁ hybrid females mated to C57BL males, the frequency of success or failure of the C57BL grafts suggested that the trait was controlled by a single pair of genetic determinants at an autosomal locus. The latter is apparently linked with, or part of, the H-2 region in the IXth linkage group. The experimental evidence suggested also that the failure of C57BL haemopoietic cell grafts in H-2 heterozygotes was not related to exhaustion of donor cells by excess of recipient isoantigen but rather to lack of expression in the heterozygotes of histocompatibility-related growth requirements yet undefined. These requirements are specific for haemopoietic cells, but not for skin grafts, and resulted most probably from the effect of genetic (inter-allelic) interaction involving H-2 or an H-2-linked locus.