Lymphocyte Commitment Research Articles

Production of a suppressor of lymphocyte proliferation by two human oral carcinoma cell lines.

This study examined the production of an immunosuppressive factor by the KB and H191 human oral squamous carcinoma cell lines. Conditioned media (CM) from both cell lines markedly inhibited mitogen- and alloantigen-induced proliferation of normal human and rat peripheral blood lymphocytes. By contrast, the proliferation of an exponentially-growing fibroblast cell line remained unchanged by CM. The immunosuppressive factor appeared to act after lymphocyte commitment as indicated by continued blast cell formation, the failure of CM to suppress resting lymphocytes and the fact that CM caused maximum inhibition of lymphocyte proliferation 72 h after the addition of PHA. The addition of exogenous IL-2 did not counteract lymphocyte suppression. Inclusion of indomethacin and isoniazid during cell culture did not significantly alter the degree of suppressive activity. Mycoplasma contamination was absent and CM did not act directly with the thymidine or mitogen. The factor was heat stable at 50 degrees C, acid labile and had a molecular weight in excess of 300 kDa. The results demonstrate that human oral squamous carcinoma cell lines produce an immunosuppressive factor that may have a role in tumour evasion of the host immune response.

Transfer ribonucleic acid populations in concanavalin-A-stimulated bovine lymphocytes

Transfer RNA isolated from lymphocytes stimulated by concanavalin A and that from resting cells were compared with respect to amino-acid acceptance, integrity of the CCA-terminus, extent of modification and isoacceptor distribution. Following growth stimulation the overall amino-acid acceptance of the tRNA is elevated, in particular the relative acceptor activities for threonine and arginine are increased. The reduced acceptor activity of the tRNA from the quiescent cells is not due to a preferential degradation of the CCA-end, since it persits even in the presence of ATP(CTP):tRNA nucleotidyltransferase. We therefore conclude that this reduced activity is caused by structural differences of the tRNAs. The content of modified nucleotides in newly synthesized tRNA from lymphocytes cultured in the presence and absence of concanavalin A was determined. tRNA from resting cells was found to be undermodified with respect to pseudouridine and dihydrouridine. Upon monitoring the tRNA isoacceptor distribution by affinity chromatography on immobilized elongation factor Tu and subsequent two-dimensional gel electrophoresis, a preferential synthesis of particular lysine- and threonine-accepting tRNAs was observed upon mitogenic stimulation. Evidently, a specific tRNA population is needed by the proliferating cells. These results are discussed in view of the hypothesis that the commitment of lymphocytes to proliferation is at least in part under translational control.

Inhibition of mitogen-induced proliferation of mouse T and B lymphocytes by bloodstream forms of Trypanosoma cruzi.

The role of virulent forms of Trypanosoma cruzi in modulating mitogen-induced lymphocyte responses was investigated in this work. Bloodstream forms of T. cruzi inhibited normal mouse spleen cell responses to Con A and LPS in a dose-dependent manner. Reduced responses were observed over relatively large ranges of concentration of Con A (50-fold) and LPS (160-fold). The inhibitory action of the parasites could not be overcome by increasing the mitogen dose beyond optimal levels. Furthermore, absorption of mitogen solutions with four times as many parasites as used in the proliferation assays revealed that sufficient mitogen activity remained to produce optimal lymphocyte responses. Therefore, reduced lymphocyte responsiveness was not due to absorption of mitogen by the parasite. Inhibited responses were also seen when a sonicated T. cruzi preparation was used, indicating that parasite viability was not required to produce suppression. Inhibition of Con A- or LPS-induced responses by the parasites occurred only when the trypanosomes were incorporated into the system during the first 24 hr of culture. These results show that virulent forms of T. cruzi can induce suppression of T and B cell responses in vitro, and suggest that the parasite affects lymphocyte commitment to blastogenesis during the early stages of lymphocyte activation.

The Role of Macrophages in the Activation of T-Lymphocytes by Concanavalin A I. Macrophages Support Proliferation after Commitment of Lymphocytes

T lymphocytes require the presence of small numbers of macrophages to enter mitosis when activated by mitogens such as concanavalin A. The aim of this study was to decide when macrophages may operate in the course of activation. Lymphocytes from the mesenteric lymph nodes of Lewis rats were depleted of macrophages by adherence on 0.1 mm glass bead columns. Lymphocytes in the column effluent, between 40 and 50 percent of the cells originally placed on the column, contained less than 0.2 percent macrophages as determined by uptake of neutral red. Macrophage-depleted lymphocytes were completely unresponsive to stimulation with con A or PHA to enter DNA synthesis. The proliferative response to con A was completely restored by adding macrophages elicited intraperitoneally by various means. Addition of an optimal number of macrophages after 24 hrs of con A stimulation of macrophage-depleted lymphocytes also supported DNA synthesis, although to only 40-60% when compared to DNA synthesis in the continuous presence of macrophages, with a peak response temporarily identical to cultures where macrophages were present from the beginning. After 24 hrs of con A stimulation all lymphocytes were committed for mitogenesis, as removal of surface bound con A by a-methyl mannoside had no effect when added at this time. Substitution of macrophages after 24 hrs of con A stimulation in the presence of α-methyl mannoside resulted in an identical induction of DNA synthesis as in the absence of this hapten sugar. The data fit a hypothesis that initiation of lymphocyte activation with con A does not require the presence of macrophages. Macrophages may deliver a second signal, which is independent of the binding of the mitogen to these helper cells, and which supports induction of DNA replication after lymphocytes are committed for mitogenesis.

An inducible gene involved in commitment of lymphocytes to transform.

CONCANAVALIN A (Con A) stimulates nondividing lymphocytes (in G0) to transform and enter the division cycle1. Within 3 h a significant proportion of the cells become committed to transform and do so after the removal of Con A (ref. 2). When the translation of mRNA into protein is inhibited (by low concentrations of anisomycin) lymphocyte transformation is also blocked3. Anisomycin has proved particularly useful in studying lymphocyte transformation because its effect is reversible: in the presence of Con A it holds the cells in G0, apparently without adverse effects, and the cells transform following its removal3. Using anisomycin it has been shown that Con A induces the synthesis of factor(s) which commit lymphocytes to transform3. I have examined the stability and nature of the ‘transformation signal’, again exploiting the reversible effect of anisomycin, and report here that on binding to the surface of nondividing lymphocytes, Con A induces the transcription of a short-lived mRNA. This mRNA encodes a ‘transformation factor’ which, when translated, commits lymphocytes to enter the division cycle; extracellular Con A is no longer required.

Lymphocyte commitment to DNA replication induced by concanavalin A

Commitment of lymphocytes to DNA replication induced by ConA was investigated by adding α-MM (a saccharide that competes with the lectin's lymphocyte binding sites) at various times to lymphocyte cultures that had been treated with a wide range of ConA concentrations. After 24 h of culture, α-MM addition has essentially no effect on cells treated with optimal concentrations of ConA, results in a marked blastogenic response of cells treated with supra-optimal (non-mitogenic) concentrations of ConA, and severely inhibits blastogenesis of cells treated with suboptimal concentrations of the lectin. Furthermore, the time of commitment is progressively shortened as the concentration of mitogen is increased. Thus, cells were committed to DNA replication as early as 5 h after incubation with supra-optimal concentrations of ConA. Induction of commitment does not occur when cells are incubated with ConA at 4 °C. Extensive crosslinking of membrane sites appears to be associated with the induction of early commitment by ConA, since the bivalent succinyl ConA failed to exhibit this effect. These findings indicate the need for re-evaluation of previous studies utilizing competing saccharides to determine kinetics of cell commitment to DNA replication.

Correlation between temperature-stable E-rosette formation and lymphocyte commitment of activation

Human peripheral blood T lymphocytes acquire the capacity to form temperature-stable sheep erythrocyte (E) rosettes within 1 hr following phytohemagglutinin (PHA) stimulation. Whereas lymphocyte interaction with PHA or concanavalin A (Con A) induces early membrane-associated events allowing E-rosette formation at 37 as well as 4°C, wheat germ agglutinin, a nonmitogenic, erythroagglutinating lectin, is unable to effect a similar membrane change. Only mitogenic concentrations of PHA induce temperature-stable rosette-forming cells although this phenomenon occurs prior to lymphoblast transformation and is independent of DNA synthesis per se. The capacity to bind E at 37°C parallels cellular commitment to activation. Interference with Con A-mediated lymphocyte activation by addition of α-methyl- d-mannoside within several hours after culture initiation inhibits both rosette formation at 37°C and DNA synthesis to a comparable degree.

Divalent mitogens as tools in the study of commitment of lymphocytes to proliferation

We have reinvestigated the question of whether lymphocytes are committed to proliferation by an early, relatively brief, exposure to mitogens with conventional multivalent lectin, concanavalin A, and antigen, dinitrophenyl bovine serum albumin and with strictly divalent lectin, succinyl concanavalin A and antigen, di-dinitrophenyl polyethylene oxide. Whereas very brief incubation with multivalent mitogens leads to substantial irreversible stimulation, much longer exposure to divalent mitogens is required. It appears that the stimulatory signal to any given cell is reversible until the onset of DNA synthesis.