Synergistic and suppressive interactions among mouse T lymphocytes in the response to phytohemagglutinin.

A synergistic effect in the proliferative response to phytohemagglutinin (PHA) can be observed in cultures containing a mixture of mouse CBA/Ca lymph node cells (LNC) and syngeneic CBA/T6T6 thymocytes (ThC) when compared to cultures containing only one cell type. This effect was analyzed, at various days of culture and in LNC-ThC mixtures of different ratios, by comparing the origin of the cells in mitosis (detected by caryotypic analysis), the stimulation of DNA synthesis, the number of blasts, and the percentage of blasts labeled after pulses of [3H]thymidine (detected by autoradiography). The following conclusions were reached: (a) ThC are induced to proliferate by the presence of LNC, while they are almost unresponsive to PHA when cultured alone; and (b) the strongest "synergistic" effect is exerted on LNC, whose proliferation is markedly enhanced. Evidence is presented that this last effect is not specific to the presence of ThC, but results from a dilution of LNC which retards the time when the culture reaches a critical concentration of blasts, above which proliferation progressively stops. Thus, conditions of culture allowing the response to PHA of a low concentration of LNC leads to the most prolonged T-cell proliferation. These observations may be relevant to the types of T-cell interactions, "synergistic" or "suppressive," occurring during in vitro or in vivo immune responses.

Study of the cells proliferating in parent versus F hybrid mixed lymphocyte culture.

Caryotypic analysis of the cells dividing in mouse parent-hybrid MLC showed an F1 hybrid cell proliferation, which varied depending upon the source of lymphoid cells used: strong in spleen MLC (sometimes equal to that of the parental cells), less marked in lymph node cell MLC, and most often absent in MLC between cortisone-resistant (CR) thymocytes. MLC between parental spleen cells and F1 CR thymocytes showed, however, that in certain conditions of culture F thymocytes can also proliferate. Using parental or F1 spleen cells lacking T lymphocytes, it was found that F1 cell proliferation is entirely dependent upon the presence of parental T cells, but does not require the presence of T lymphocytes among the F1 cells. Immunofluorescence analysis of the blasts observed in one-way MLC showed that about 70% of the parental blasts were T blasts, and 25%B blasts (containing a high proportion of plasmablasts); among the F1 blasts, there was also the same percentage of B blasts and plasmablasts, but many of the T blasts bore only small amounts of T-cell antigen (MTLA), and there was also about 20%of unstained blasts, possibly T blasts bearing MTLA in amounts undetectable by immunofluorescence. The possibility is discussed that the F1 responding T cells belong to a subpopulation performing a suppressive function; MLC lacking F1 T cells showed increased [3H] thymidine incorporation. The proliferation and differentiation of parental and F1 B cells may result mainly from an unspecific, "polyclonal" triggering.

The prematurely evoked synthesis of liver tryptophan oxygenase.

Livers of normal rats are devoid of catalytically or immunochemically reactive tryptophan oxygenase (EC 1.13.1.12) up to the 10th postnatal day; the enzyme reaches adult concentrations on about the twentieth day. Premature tryptophan oxygenase synthesis can be evoked in 4-day-old rats: if an injection of glucocorticoid is followed, a day later, by an injection of tryptophan, adult levels of tryptophan oxygenase activity and antigen content can be attained within 5 hr. The prematurely evoked tryptophan oxygenase is degraded in about 2 days, but the preparatory action of the glucocorticoid is longlasting. Even 4 days later, an injection of tryptophan can evoke significant enzyme formation. Actinomycin D, injected together with or 12 hr before the tryptophan, is not inhibitory, but if injected with the glucocorticoid it prevents enzyme formation upon later injection of tryptophan. The observations suggest that appearance of the new enzyme in developing tissue results from the sequential action of more than one stimulus and that the potentiality for its transcription may develop long before its actual synthesis.

Induction or suppression of B cell proliferation and differentiation by phytohemagglutinin or concanavalin A in mouse spleen cell cultures.

Cultures of mouse spleen cells or various mixtures of mouse T and B cells were stimulated with PHA or Con A, and the T, B, or plasmablast nature of the transformed cells, was determined by immunofluorescence 2 to 4 days later. The lectins enhanced B cell proliferation and plasmablast differentiation ("helper" effect) provided one of the following conditions was fultilled: a) suboptimal doses of lectin were used, b) cultures were performed at low cell concentration, c) cultures were made of spleen cells containing a small percentage of T cells, d) the cultures contained a mixture of T-depleted spleen cells and T cells rendered unable to proliferate by irradiation. In contrast, cultures performed with 1.5 10(6) or more spleen cells/ml and optimal doses of lectin contained almost exclusively T blasts, as did cultures stimulated in the same conditions with both PHA and LPS. This last observation idicates the existence of a lectin-induced "suppressor" effect, since LPS, a B cell mitogen, induces, in the absence of PHA, a marked B cell proliferation and differentiation into plasmablasts. These helper and suppressor effects were entirely mediated by T cells, since they were not observed in spleen cell cultures depleted in T cells by anti-thets + C. Analysis of the cultures by immunofluorescence and radioautography after pulses of 3H-thymidine showed that these antagonistic effects could be related to the number of T blasts present in the culture and to their proliferative behavior. Heoper effect is observed in cultures containing a relatively low number of T blasts (or none in cultures made with irradiated T cells), whereas suppressive effect is observed in cultures containing a high number of T blasts, a large proportion of them having left the proliferative cell cycle. It is proposed that when a critical concentration of T blasts is reached ("saturation density"), further proliferation and differentiation is prevented, resulting in a suppressive effect on the generation of plasmablasts. The helper effect of lectin-activated T cells seems to be exerted on a subpopulation of B cells which was, at least in part, already proliferating in vivo, and to result in a polyclonal IgM plasmablast differentiation.