Depletion of human lymphocytes from peripheral blood and bone marrow by affinity ligands conjugated to agarose—polyacrolein microsphere beads

Abstract

Protein-A or goat anti-mouse-Ig (GAMIg) covalently bound to agarose-polyacrolein microsphere beads (APAMB) were employed for the removal of T cells from human peripheral blood leukocytes (PBL) and bone marrow (BM). The cell suspensions were treated with a monoclonal anti-T cell antibody (Leu-1) or monoclonal antilymphocyte antibody (CAMPATH-1) and passed through the conjugated APAMB columns.

Cell separation efficacy was determined by assaying the number and function of T cells in the final cell preparation in comparison with a sample of unseparated cells. The number of cells that form rosettes (E-RFC) with sheep red blood cells (SRBC) in a sample of PBL treated with anti-Leu-1 antibodies and subsequently passed once through GAMIg-conjugated APAMB dropped from a range of 41.5–86.0% to a range of 1.6–13.3%. The in vitro response to concanavalin-A (Con-A) dropped to a range of 0.7–27.2% (GAMIg) and a range of 1.2–21.8% (protein-A column) of the response of untreated PBL. Treatment with CAMPATH-1 antibody and passage through a protein-A-conjugated APAMB reduced E-RFC from a range of 55.6–57.4% to a range of 3.2–3.9% and abolished the Con-A induced proliferative responsiveness to background levels.

Treatment of BM cells with CAMPATH-1 and passage of the cells through either GAMIg or protein-A conjugated APAMB columns resulted in reduction of E-RFC from a range of 12.4–17.7% to a range of 0–1% and from a range of 17.7–19% to a range of 1.6–3.2%, respectively. Viability of BM precursors, determined by the CFU-GM assay in semisolid medium, was not affected by these cell separation procedures.

The data suggest that protein-A or GAMIg-conjugated APAMB columns may be a useful tool for separation of BM cell suspensions into specific cell subsets that can be defined by monoclonal antibodies.