Molecular analysis of highly enriched populations of T-cell-depleted monocytes

Abstract

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