The ability to grow normal T lymphocytes in long term culture has advanced our understanding of T cell biology. The growth of CD4+ cell lines allowed a further evaluation and appreciation of functional subtypes within this group. Cytotoxic CD8+ T cells have been characterized as well. The routine and continuous culture of Ag-nonspecific CD8+ Ts cells has been difficult to achieve. We have found that CD8+ T cells that suppress T cell proliferation and lack cytotoxic activity against T cells can be routinely obtained from PWM or PHA-stimulated PBMC. Continuous culture of T cell blasts from PWM or PHA-stimulated PBMC resulted in the growth of CD4+ and CD8+ T cells. These lines developed suppressor cell activity within 7 days after stimulation with PWM and 3 to 4 wk after stimulation with PHA. Concomitant with the development of suppressor activity was the loss of CD4+ T cells resulting in homogeneous lines of CD8+ suppressor cells. These cell lines have been maintained in continuous culture for greater than 6 mo by addition of rIL-2 twice weekly and restimulation with feeder cells and PHA every 2 wk. Activity of these cell lines was relatively resistant to irradiation or treatment with mitomycin C. Both cell lines suppressed proliferation of autologous or heterologous CD4+ T cells stimulated with PWM, OKT3, or tetanus toxoid but failed to suppress proliferation of CD4+ T cells in a mixed lymphocyte reaction. CD4+ T cells stimulated with PWM produced equivalent amounts of IL-2 in the presence or absence of Ts cells but failed to express the IL-2R (TAC) on their surface in the presence of Ts cells. By contrast, CD4+ T cell lines or cytotoxic CD8+ T cell lines failed to suppress proliferation of CD4+ T cells. With these results we describe methods for the generation and continuous culture of Ag-nonspecific CD8+ Ts cells and define some of their properties. These cells lines should be helpful in further elucidating the functional and phenotypic repertoire of CD8+ Ts cells.

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