REMOVAL OF MONOCYTES FROM CELL-SUSPENSIONS WITH ANTI-CD14 ANTIBODY AND CARBONYL-IRON, USING FC-GAMMA-R-DEPENDENT ACCESSORY FUNCTION AS A SENSITIVE MEASURE OF MONOCYTE PRESENCE

Human (FcgammaRI-positive) monocytes are required as accessory cells when T cell proliferation is induced by murine IgG2a anti-CD3 monoclonal antibodies (mAbs). This T cell proliferation assay provides a sensitive method for detecting the presence of monocytes (less than 1% of monocytes can be detected), and we have used it to monitor the effectiveness of different procedures for the removal of monocytes from peripheral blood mononuclear cells. Counterflow centrifugation, phagocytosis of carbonyl-iron, adherence to plastic, monocyte depletion with magnetic beads (Dynabeads M450), and panning with anti-CD14 antibodies each strongly reduced the number of monocytes. However, none of these methods, when used on their own, were capable of completely abolishing the mitogenic response to murine IgG2a anti-CD3 mAb. A virtually complete depletion of monocytes was obtained when the panning procedure using anti-CD14 antibodies was combined with phagocytosis of carbonyl-iron. Importantly, this method could also be used with cryopreserved cells. We have applied this improved method for the removal of monocytes, to study T cell proliferation induced by murine IgG2b anti-CD3 mAb. We were able to demonstrate with this model that cells other than monocytes were able to provide accessory function.