Simplified techniques for the isolation of alloreactive cell lines and clones with specific cytotoxic or proliferative activity

A rapid method is described for the isolation of alloantigen specific proliferative and cytotoxic clones from primary mixed lymphocyte cultures (MLC). To raise cell lines and clones with specific alloantigenic proliferative activity in vitro, responder splenocytes were depleted of Lyt-2⁺ cells by monoclonal antibody and complement prior to in vitro sensitization. This procedure resulted in cultures highly proliferative to alloantigen with little or no lytic activity after expansion in interleukin-2 (IL-2). Subsequent cloning of lymphocytes from Lyt-1⁺ enriched allosensitized cultures by limiting dilution led to proliferative clones in extremely high yield, while cloning from nondepleted allosensitized cultures led to cytotoxic clones in high yield. Furthermore, conditions of high antigen and low IL-2 concentration favor the growth of proliferative cells while high IL-2 concentrations favored the growth of cytotoxic cells. These experiments indicate that selection for cytotoxic or proliferative clones may be enhanced by specific depletion of T cell subpopulations and by alteration of culture conditions.