Long-term cultured IL-2-dependent T cell lines demonstrate p16(INK4a) overexpression, normal pRb/p53, and upregulation of cyclins E or D2.

Acquisition of an immortal phenotype by circumvention of the normal senescence program can be an important step in tumor development and progression. The regulation of life-span checkpoints is complex and abrogation of these processes can occur at different levels. To better understand these mechanisms in long-term cultured lymphocytes we have characterized two human long-term cultured IL-2-dependent T cell lines regarding telomere length, telomerase activity, and the expression of selected cell cycle regulators (pRb, p53, cyclin E, cyclin D1, cyclin D2, cyclin D3, cdk4, p16(INK4a), p21(WAF1), p27(KIP1), c-myc, bcl-2, and NPAT). We compared these cell lines with a primary T lymphoblast population with a limited life span from the same donor. Both T cell lines with extraordinary growth capacity showed telomere length stabilization, high telomerase activity and demonstrated wild-type pattern of pRb and p53 but strong p16(INK4a) protein expression. The growth inhibitory activity of p16(INK4a) seemed to be abrogated by enhanced expression of cyclin D2, cdk4, and c-myc in one T cell line and overexpression of cyclin E in the second T cell line.

Early supernatants of mitogen-stimulated mononuclear cells promote long-term IL-2-dependent growth of human T lymphocytes.

Human wheat germ agglutinin (WGA)- or PHA-activated T lymphocytes from either peripheral blood or normal spleens can be propagated in vitro for several months in the presence of IL-2 but not IL-4 when periodically restimulated with supernatants collected from 24 h cultures of mitogen-stimulated human blood or spleen mononuclear cells. Following several rounds of stimulation with these supernatants some of the spleen-derived T lymphoblast lines acquire the ability of continuous growth (over 100 population doublings) in the presence of IL-2 alone. We postulate that the ability of 24 h supernatants from mitogen stimulated mononuclear cells to maintain T lymphoblast IL-2-dependent growth depends upon yet undefined cytokine(s) released early after activation.

TG and TnonG lymphocyte involvement in the proliferative response to PHA. II. The role of TG and TnonG lymphocytes in the regulation of the PHA-induced proliferative response of non-T cells.

The TnonG subpopulation from human peripheral blood provides help for PHA induced proliferation of non-T cells, while the TG subpopulation does not. Thus the inducer cells in the proliferative response of T and non-T cells to PHA are at least in part different.

Surface phenotypes of human T cell lines generated after WGA- and PHA-stimulation.

We have reported earlier that human T lymphocytes from blood or spleens of normal donors can be propagated in long-term culture in the presence of IL-2, after stimulation either with wheat germ agglutinin (WGA) or phytohemagglutinin (PHA) and with the use of repeated restimulation with supernatants collected after 24 h of such cultures. In the present study we demonstrate that lymphoblast lines generated with this method contain predominantly the cells expressing CD3 molecule and alpha beta heterodimer of T cell receptor (TcR alpha beta). In PHA-initiated lymphoblast lines CD4-CD8+ cells prevailed, whereas in lymphoblast lines generated after WGA-stimulation various proportion of CD4+CD8-, CD+CD8- and CD4-CD8- cells were found. In some cell lines a gradual selective outgrowth of TcR alpha beta CD4+CD8- or CD4-CD8- cells was observed. These data indicate that the long-term growth potential of human normal T lymphoblasts is not restricted to a single subset and that the surface phenotypes acquired during differentiation of the cells can be retained even after more than 100 population doublings in culture.

TG and TnonG lymphocyte involvement in the proliferative response to PHA. I. Reactivity and regulatory functions of TG and TnonG subsets in the proliferative response of T lymphocytes.

Both TG and TnonG subpopulations from human peripheral blood were found to secrete helper and suppressor factors for the PHA-induced T cell proliferative response. The TnonG subpopulation contained cells which were both able to secrete helper factor and to respond by proliferation, whereas in the TG subpopulation cells with a proliferative potential could not be conclusively demonstrated. Suppressor and helper activities in TG and TnonG culture supernatants displayed a different time kinetics and were influenced by the dose of mitogen in test cultures.

Long-term growth of human WGA-activated T-lymphocytes without feeder cells.

Human wheat germ agglutinin (WGA)-activated T-lymphocytes from either peripheral blood or spleen could be propagated for several weeks in the presence of culture supernatants from human mononuclear cells obtained after a 1-day stimulation with WGA and after a subsequent 3-day culture without mitogen. The continuous T-cell growth in this culture system required alternate exposition of the cells to the above supernatants, both with and without WGA, but proceeded in the absence of feeder cells. After 3 weeks of propagation most cells displayed the CD4+ phenotype, expressed IL2 receptors, and responded to PHA, Con A, and WGA. It has been shown that WGA-activated T-cells could be cloned by limiting dilution and propagated using the above culture system.