Surface receptors and immune activity of purified human circulating mononuclear cells. I. The effects of lysis of the sheep red blood cells (SRBC) in the T-cell rosettes on the receptors and cytotoxic activities of the T cells. T cells with receptors for complement

Abstract

Human circulating T lymphocytes were isolated by rosetting with sheep erythrocytes (SRBC) followed by centrifugation in a discontinuous Ficoll-hypaque gradient. The SRBC in the rosettes were lysed according to one of four different conditions: 1) buffered ammonium chloride at 37°C; 2) buffered ammonium chloride at 4°C; 3) water at 4°C; and 4) antibody-complement-mediated lysis at 37°C. The T cells were subsequently washed a number of times in Hank's balanced salt solution and aliquots of the cells were incubated at 37°C for 24 hr in Medium 199 supplemented with fetal calf serum (final concentration 10%). Both the unincubated and incubated T cells were investigated in terms of their ability to rosette with E, EA (IgG), EA (IgM), and EAC indicator erythrocytes and for the capacity to mediate ADCC, NOCC, and MICC cytolysis of target cells. It was consistently observed that unicubated T cells formed more rosettes with EA(IgG) than did the incubated T cells. No EA(IgM) rosettes were seen with any of the freshly isolated (unincubated) T lymphocytes. However, T cells incubated for 24 hr following ammonium chloride lysis of the adherent SRBC formed the greatest numbers of EA (IgM) rosettes. The number of EA(IgM) rosettes was markedly decreased following lysis of the SRBC with water. All of the T lymphocytes gave much higher numbers of EAC rosettes following 24 hr of incubation. T cells exposed to complement-mediated lysis of the SRBC lost the capacity to mediate ADCC lysis and did not regain this activity even after 24 hr of incubation. Similarly, MICC cytotoxic activity was also lost subsequent to complement-mediated lysis of the SRBC, but it was essentially regained after 24 hr of culture. NOCC activity was maximally lost following lysis of the SRBC with ammonium chloride. Lysis of the SRBC with water resulted in the least loss of ADCC, MICC, and NOCC activity of the T cells. It is concluded that different methods of lysis of the SRBC components of the T cell rosettes induce different nonparallel effects on the receptors and cytotoxic activities of the T cells. This fact must be taken into consideration when analyzing cells for receptors and cytotoxic activity following their isolation by rosetting and lysis of the adherent indicator erythrocytes.