A cytoplasmic activator of DNA replication is involved in signal transduction in antigen-specific T cell lines.

Cytoplasmic extracts prepared from T cell lines undergoing antigen-specific, interleukin-2 (IL-2)-dependent proliferation were tested for their ability to induce DNA synthesis in isolated, quiescent nuclei. A tetanus toxoid (TET)-specific T cell line, established from peripheral blood of a normal human volunteer, was stimulated in the presence of relevant antigen and 1 unit/ml IL-2. Cytoplasmic extracts prepared from these cells were capable of inducing DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated, quiescent nuclei. The ability of cytoplasmic extracts to induce DNA synthesis in isolated nuclei correlated positively with the degree of proliferation induced in these cells. In contrast, incubation of this T cell line in the absence of antigen failed to induce proliferation and cytoplasmic extracts prepared from these cells induced little to no DNA synthesis in isolated, quiescent nuclei. The factor present in the cytoplasm of T cells stimulated with relevant antigen in the presence of IL-2 is similar, if not identical, to a factor which we have previously demonstrated in cytoplasmic extracts prepared from transformed lymphoblastoid cell lines and from mitogenically stimulated normal human peripheral blood mononuclear cells. This factor, which we have called activator of DNA replication (ADR) is a heat-labile protein, and is inactivated by treatment with protease inhibitors, including aprotinin. The ability of cytoplasmic extracts from T cells undergoing antigen-specific, IL-2-dependent proliferation to induce DNA synthesis in isolated, quiescent nuclei was markedly inhibited in the presence of aprotinin, providing strong evidence that a cytoplasmic activator of DNA replication, ADR, is involved in the signal transduction process for antigen-specific, IL-2-dependent T cell proliferation. ADR may represent a common intracellular mediator of DNA synthesis in activated and transformed lymphocytes.

Suppression of DNA synthesis in normal, but not neoplastic, nuclei by cytoplasmic extracts from resting cells.

We have previously shown that a heat-stable protein in cytoplasmic extracts from human quiescent peripheral blood lymphocytes (PBL) is capable of inhibiting the induction of DNA synthesis in isolated resting nuclei. We now report that these cytoplasmic extracts are also capable of suppressing DNA synthetic activity in replicative nuclei isolated from mitogen-activated PBL. PBL extracts had little or no inhibitory effect, however, on replicative nuclei derived from several transformed lymphoblastoid cell lines. These results suggest that the growth of normal lymphocytes may be negatively controlled by cytoplasmic inhibitory factors. Furthermore, the relative resistance of tumor cell nuclei to these inhibitory signals provides a possible explanation for the loss of growth control in neoplastic cells.

Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors.

Cytoplasmic extracts from spontaneously proliferating and mitogen-activated lymphoid cells contain a protein factor called ADR (activator of DNA replication) that induces DNA synthesis in isolated quiescent nuclei. ADR-containing preparations have proteolytic activity, as indicated by their ability to degrade fibrin in a plasminogen-independent and plasminogen-dependent manner. In addition, aprotinin, a nonspecific protease inhibitor, abrogates ADR-induced DNA synthesis in a dose-dependent fashion. Preincubation studies demonstrated that the effect of aprotinin is not due to its suppressive effects on the nuclei themselves. Other protease inhibitors such as leupeptin, p-aminobenzamidine, and N-alpha-tosyllysine chloromethyl ketone are also inhibitory, but soybean trypsin inhibitor is without effect. ADR activity can be removed from active extracts by adsorption with aprotinin-conjugated agarose beads and can be recovered by elution with an acetate buffer (pH 5). These findings are consistent with the interpretation that the initiation of DNA synthesis in resting nuclei may be protease dependent and, further, that the cytoplasmic stimulatory factor we have called ADR may be a protease itself.

Impaired nuclear responsiveness to cytoplasmic signals in lymphocytes from elderly humans with depressed proliferative responses.

Lymphocytes from many elderly individuals exhibit depressed proliferative responses to the plant phytohemagglutinin (PHA). We have previously reported that this proliferative defect is not due to a failure to generate a cytoplasmic activator of DNA replication (ADR). In the present study, we tested the DNA synthetic response of nuclei derived from aged cells to an exogenous source of ADR. We found that nuclei from aged lymphocytes exhibiting low PHA responses were impaired in their ability to synthesize DNA in responses to ADR, compared with nuclei from younger adult donors. In contrast, those aged cells maintaining intact PHA responses provided nuclei that were also unimpaired in their response to ADR. The relationship between PHA responsiveness of the intact cells and ADR responsiveness of nuclei derived from these was a linear one. These results suggest that the depressed cellular reactivity of aged lymphocytes to PHA (when seen) may be due to a failure of these nuclei to respond to cytoplasmic stimulatory signals induced by the mitogen.

The regulation of DNA synthesis in quiescent lymphocytes by cytoplasmic inhibitors.

We previously have shown that proliferating lymphoid cells contain an extractable cytoplasmic protein that is capable of inducing DNA synthesis in isolated quiescent nuclei (activator of DNA replication, ADR). ADR is present in continuously proliferating (transformed) lymphoblastoid cell lines and in mitogen- or interleukin 2-stimulated human peripheral blood leukocytes (PBL) but is not detectable in extracts from resting (unstimulated) PBL. In the present study, we investigated the possibility that quiescent PBL may contain a factor than can inhibit ADR activity. We found that unstimulated human PBL contain a heat-stable protein greater than or equal to 50,000 Da that is capable of suppressing the induction of DNA synthesis in isolated nuclei by ADR derived from both normal and neoplastic sources. The inhibitor was not detectable in freshly isolated PBL but appeared within the cells after a brief (2-6 hr) culture period. Depletion experiments revealed that the inhibitor was neither derived from nor dependent upon the macrophage component of the PBL cultures. These results suggest that one mechanism by which resting lymphocytes may maintain quiescence may involve the suppression of cytoplasmic mitogenic signals by intracellular inhibitors. In addition, the implications of our findings provide a possible explanation for the loss of growth control in abnormal proliferative states, such as neoplasia.

The role of cytoplasmic intermediates in IL 2-induced T cell growth.

In previous studies we showed that PHA-stimulated human peripheral blood lymphocytes contain an extractable cytoplasmic protein that is capable of inducing DNA synthesis in isolated quiescent nuclei. This factor, which we have called ADR (for activator of DNA replication), appears to represent an endogenous intracellular mitogen, or cytoplasmic transducer of extracellular mitogenic signals. In light of the known role of IL 2 in PHA-induced T cell mitogenesis, we continued these studies to investigate whether ADR may be involved in the mechanism of action of IL 2. Results from both dose-response and time-response studies strongly suggest a functional role for ADR in IL 2 mediated T cell growth. Furthermore, the absence of ADR in dexamethasone-treated, PHA-stimulated cells suggests that the induction of ADR is not an early event in T cell activation associated with the expression of IL 2 receptors; rather, the induction of ADR appears to be a later event resulting directly or indirectly from the binding of IL 2 to IL 2-responsive cells. In sum, our findings suggest a pathway for T cell growth in which extracellular exogenous mitogenic signals are transmitted to the nucleus first through an extracellular endogenous growth factor (IL 2) and finally through an intracellular, endogenous molecule (ADR) that acts at the level of the nucleus itself.

Intracytoplasmic signals for DNA replication in lymphocyte proliferation.

Cytoplasmic extracts prepared from continuously proliferating lymphoblastoid cells, as well as mitogen-activated normal lymphocytes, contain a protein or proteins (ADR) capable of inducing DNA synthesis in isolated quiescent nuclei. Lymphocytes obtained from aged donors with depressed proliferative responses have normal levels of ADR, but depressed nuclear responses to exogenous ADR, suggesting a nuclear defect in these cells. In no instance could an effect of ADR on intact cells be demonstrated. Moreover, it is not released into the extracellular milieu by cells that contain it. These observations suggest that it serves as an intracellular mitogenic signal in replicating cells. Thus, it may be an important intermediate in the pathway leading from cell membrane activation to nuclear activation.

The developmental biology of the syngeneic mixed lymphocyte reaction.

The syngeneic mixed lymphocyte reaction (SMLR) is the proliferative response of T lymphocytes cultured with non-T lymphocytes. Normal mouse serum supports proliferative activity in the SMLR comparable to levels observed when fetal calf serum is used. Thus, the SMLR does not appear to be a response to xenogeneic antigens. The ontogeny and senescence of the SMLR was also studied. The SMLR is impaired in mice less than 4 weeks of age but attains adult levels of activity at 4 weeks of age. The SMLR is also impaired in 24-month-old mice. These impairments were observed regardless of the source of serum used in the cultures. Finally, monoclonal antibodies have been developed which identify a subpopulation of spleen cells which respond in the SMLR. In indirect immunofluorescence these antibodies stain 50-80% of cells activated in the SMLR, but only 5-8% of spleen cells or cells activated in the allogeneic MLR. These antibodies block the proliferative response in the SMLR but do not interfere with the response to alloantigens or PHA.

Induction of DNA synthesis in isolated nuclei by cytoplasmic factors from spontaneously proliferating and mitogen-activated lymphoid cells.

Cytoplasmic extracts, prepared from continuously proliferating lymphoblastoid cells, as well as mitogen-activated normal lymphocytes, contain an extractable factor capable of inducing DNA synthesis in isolated quiescent nuclei. This factor is not detectable in resting cells. It is nondialyzable, precipitable by 30-50% saturated ammonium sulfate, and inactivated by trypsin. It is heat sensitive, but stable to cold and lyophilization. The molecular weight of the factor is greater than 100,000. This cytoplasmic activator of nuclear DNA replication is not released from the cell, and has no effect on intact cells. This suggests that it serves as an intracellular mitogenic signal in replicating cells.

Studies on the syngeneic mixed lymphocyte reaction. I. The ontogeny of the syngeneic mixed lymphocyte reaction in mice.

The syngeneic mixed lymphocyte reaction (SMLR) is the proliferative response of T lymphocytes cultured with syngeneic non-T lymphocytes. The ontogenic development of the SMLR has been studied in BALB/c mice. The SMLR is not demonstrable in BALB/c mice less than 3 weeks old. The SMLR attains an adult level of activity at 4 weeks of age. Splenic T cells from mice less than 3 weeks old do not respond and splenic non-T cells do not stimulate an SMLR. T-cell preparations acquire the capacity to respond in the SMLR between 2 and 3 weeks of age and non-T cells acquire the capacity to stimulate in SMLR between 3 and 4 weeks old. Spleen cells from 1 week old mice suppress the adult SMLR. Suppressor activity was eliminated by depleting either Thy-1.2 positive cells or adherent cells. When suppressor activity was eliminated, spleen cells from 1 week old donors were capable of stimulating syngeneic T cells. Spleen cells from 1 week old nude BALB/c mice do not suppress the SMLR and stimulate syngeneic T cells normally. These results suggest that the impaired SMLR in very young mice is, at least in part, due to cell-mediated suppression of the SMLR.

Studies on the syngeneic mixed lymphocyte reaction. II. Decline in he syngeneic mixed lymphocyte reaction with age.

The syngeneic mixed lymphocyte reaction (SMLR) is the proliferative response of T lymphocytes cultured with syngeneic non-T lymphocytes. In previous studies, we found that the SMLR reaches adult level of activity at 4 weeks of age in BALB/c mice. We now report that the SMLR declines with age in this strain. The decline was first documented at 12 months of age, when non-T spleen cells were less able to stimulate young adult T cells than were non-T cells from 2 to 3 month-old mice. Splenic T cells from 12 month old mice were as responsive as splenic T cells from 2 to 3 months old mice. By 24 months of age, mice had no significant SMLR activity. Splenic T cells from 24 month old mice did not respond and splenic non-T cells did not stimulate SMLR when cultured with cells from young adult mice. Finally, suppressor cells were demonstrated in spleen cells preparations from 24 month old mice and may explain or contribute to the impaired SMLR in these animals.