PREPARATION OF CD8(BRIGHT) AND CD8(DIM) LYMPHOCYTE POPULATIONS USING 2 POSITIVE SELECTION METHODS IN TANDEM

Two positive selection methods were compared for the ability to captur e both the bright and dim subsets of CD8 lymphocytes in mononuclear ce ll (MC) preparations from ten healthy individuals. The first method ut ilized anti-CD8-coated magnetic beads; captured cells were then recove red using a polyclonal sheep anti-mouse Fab reagent. At all bead: CD8 cell ratios tested (4: 1, 8: 1, 16: 1), the selected cells were > 94% CD8+, and these CD8 cells were enriched for CD8bright cells (77-85%) w hen compared to CD8 cells in the starting MC preparation (68%). The se cond method utilized anti-CD8-coated culture flasks; captured cells we re recovered by physical dislodgement. The recovered cells were > 90% CD8+, and these CD8 cells were modestly enriched for CD8dim cells (52% ) compared to starting CD8 cells (32%). To further enrich for CD8dim c ells, we used these two methods in tandem (n = 10). MC were first incu bated with anti-CD8-coated magnetic beads (4:1 ratio) to obtain a CD8b right-enriched population (97% of all cells CD8+, 83% of all cells CD8 bright). Uncaptured cells were incubated with anti-CD4-coated magnetic beads, and the uncaptured cells from this step were then placed in an anti-CD8-coated flask. The recovered flask-selected cell population w as highly enriched for CD8dim cells (87% of all cells CD8+, 85% of all cells CD8dim). CD8 cells in the CD8bright population were 94% CD3+ an d 6% CD16+, whereas those in the CD8dim population were 29% CD3+ and 6 6% CD16+. In proliferative studies, CD8bright cells were preferentiall y activated by immobilized anti-CD3, whereas CD8dim cells were prefere ntially activated by exogenous IL-2. In assays of natural killer activ ity, CD8dim cells were markedly more active than CD8bright cells. This method provides an alternative to cell sorting for obtaining enriched populations of CD8bright and CD8dim lymphocytes.