Isolation of Ia-like antigen-bearing cells from human peripheral lymphocytes through the use of a monoclonal antibody to framework determinants of Ia-like antigens

Abstract

A non-complement fixing monoclonal antibody (Q2/70) to framework determinants of human Ia-like antigens was used to develop a method for isolating Ia-like antigen bearing, i.e., Ia-like (+) cells and cells lacking these antigens, i.e. Ia-like (−), from human peripheral blood lymphocytes (PBL). The method was based on sensitization of PBL with the antibody Q2/70, followed by rosetting with sheep (ShE) coated with purified rabbit anti-mouse Ig antibodies, differential centrifugation on a Ficoll-Hypaque gradient, and finally recovery of Ia-like (+) and Ia-like (−) cells from the bottom and the interface of the gradient respectively. Marker analysis of the two cell subpopulations showed that the majority of the bottom cell fraction were Ia-like (+) and carried B cell markers such as membrane bound immunoglobulins (MbIg) and C3 receptors. On the other hand, the majority of the interface cell fraction were Ia-like (−) and carried T cell markers such as receptors for 2-aminoethylisothiouronium treated sheep erythrocytes (AETShE) and goat erythrocytes (GoE). Serological and functional studies showed that the Ia-like (+) cells (1) were useful targets in complement mediated cytotoxicity assays for HLA-DR typing; (2) served as stimulator but not as responder in unidirectional mixed lymphocyte reactions (MLRs); (3) did not display lytic activity in natural killer (NK) cell cytotoxicity and in antibody dependent cellular cytotoxicity (ADCC); and (4) proliferated in response to pokeweed mitogen (PWM) stimulation in the presence of helper T cells. On the other hand, the Ia-like (−) cells (1) responded to but failed to stimulate allogeneic lymphocytes in the MLRs; (2) were highly active in NK and ADCC assays; and (3) provided helper activity in PWM stimulation of purified B cells.