Human cytotoxic T lymphocytes. III. Large numbers of peripheral blood T cells clonally develop into allorestricted anti-viral cytotoxic T cell populations in vitro.

Abstract

Cytotoxic T lymphocytes (CTL) recognize antigen in the context of syngeneic MHC class I gene products. The "learning" of MHC restriction is thought to take place during the early intrathymic development of cytotoxic lymphocyte precursors (CLP). This view does not allow for any significant number of "allorestricted" (as opposed to selfrestricted) T cells to occur among mature, peripheral T cells. Recent evidence indicates, however, that large numbers of antigen-specific, allorestricted CLP can be readily detected among splenic T cell populations from several strains of unprimed normal mice. The frequencies of allorestricted CLP as determined under limiting dilution (LD) culture conditions are in fact in the same order of magnitude as frequencies of selfrestricted CLP. These findings were at the origin of the present study, which was designed to investigate whether antigen-specific, allorestricted CTL populations could also be detected among human peripheral blood T lymphocytes. To address this issue we studied the CTL response to virus-infected allogeneic stimulator cells in two different LD systems. In the first system, peripheral T cells from normal donors were cocultured under precisely defined LD conditions with Epstein-Barr virus (EBV)-transformed allogeneic lymphoblastoid cell lines (LCL). Frequencies of CLP that lysed the stimulating LCL ranged from one in 70 to one in 200, while frequencies of CLP that lysed the respective allogeneic ConA blast targets were 3-40-fold lower. The split-well analysis suggested that a large fraction of developing CTL colonies specifically lysed the stimulating LCL targets but neither the respective ConA blasts nor HLA-mismatched third party LCL targets. CTL generated in this culture system thus displayed allorestricted specificity for LCL membrane antigens. Comparable results were obtained in a second LD system where T cells from normal donors were cocultured with mumps virus-infected allogeneic mononuclear cells (MNC) or ConA blasts. One of 600 to one of 2,800 T cells gave rise to a cytotoxic colony that lysed mumps virus-infected stimulator-derived ConA blast target cells. To assess the lytic specificity of the in vitro expanding CTL populations, individual microcultures were split into three aliquots and tested for cytolytic activity against mumps virus-infected and noninfected specific targets as well as mumps virus-infected, HLA-mismatched third party targets. Clonal CTL populations from four of seven donors lysed virus-infected stimulator targets but did not lyse either noninfected stimulator targets or mumps virus-infected third party targets, i.e., they again showed an antigen-specific allorestricted lytic r