Long Term Growth in Vitro of Human T Cell Blasts with Maintenance of Specificity and Function

Human T lymphoblasts obtained via antigenic stimulation and gradient centrifugation purification can be maintained as perpetual dividing cells in vitro, retaining specificity and function. By repeated addition of supernatants obtained from in vitro lymphocyte cultures activated with T cell mitogens, one can avoid the need of having antigen continually present, while maintaining proliferation. We have utilized PHA to stimulate tonsil lymphocytes and harvested supernatants after 48-hr. Such supernatants contain growth-supporting factors for T blasts, and these factors are clearly distinguishable from the lectin used to induce the growth factors. Thus, the PHA can be removed from the supernatants via an anti-lectin immunosorbent coupled to Sepharose beads, without having any detectable negative impact on the T blast supporting ability of such supernatants. Conversely, although the lectin is capable of stimulating freshly isolated populations of lymphocytes, the supernatant after lectin removal will not activate detectable numbers of small lymphocytes. In the absence of supernatant factors, antigen-specific cells can also be maintained either by reexposure of the MLR blasts to the original stimulator cell population, or by the addition of the soluble antigen (PPD) to blasts in the company of irradiated syngeneic cells. Alternatively, syngeneic or allogeneic irradiated cells together with mitogen can generate growth-supporting factors and thus sustain blast proliferation.