MOLECULAR ANALYSIS OF HIGHLY ENRICHED POPULATIONS OF T-CELL-DEPLETED MONOCYTES

CD4(+) T lymphocytes and monocytes/macrophages are important component s of the immune system. Blood monocytes are usually targeted for studi es of the human macrophage lineage cells because of their accessibilit y through blood sampling. Most separation techniques currently availab le to obtain human monocytes either require large volumes of blood or do not yield a monocyte fraction sufficiently depleted of other cell t ypes. We have developed a simple strategy to isolate a highly enriched population of monocytes from small volumes (<6 ml) of peripheral bloo d by using an anti-CD14 monoclonal antibody and magnetic microspheres. Yields of monocytes ranged from 75 to 80% of CD14(+) cells in periphe ral blood. CD4(+) T cells were subsequently selected from the monocyte -depleted peripheral blood by using an anti-CD4 monoclonal antibody an d immunomagnetic beads. The effectiveness of immunomagnetic selection to yield a monocyte population highly depleted of T tells was analyzed by using a sensitive molecular strategy based on PCR amplification an d detection of T-cell receptor (TCR) gene rearrangements. The relative frequency of rearranged TCRs within the monocyte population was compa red with the frequency of rearranged TCRs within the CD4(+) T-cell fra ction from the same individual. Molecular analysis indicated that a vi able monocyte population which contains fewer than 2% residual T lymph ocytes can be consistently selected from small aliquots of blood.