Depletion of T lymphocytes from human bone marrow by the use of counterflow elutriation centrifugation

Normal donor bone marrow buffy coat (BMBC) cells were fractionated into three subsets by counterflow elutriation centrifugation. Fraction 1 (Fr‐1) was lymphocyteenriched, fraction 2 (Fr‐2) was a mixture of lymphocytes and myelomonocytic cells, and fraction 3 (Fr‐3) was enriched for myelomonocytic cells. The extent of T cell depletion was determined by limiting dilution analysis of the growth of T cells. The cells in each group were stimulated with phytohemagglutinin and cultured in the presence of interleukin‐2. These conditions allowed clonogenic growth of one of three T cells purified from peripheral blood. After a 3‐week culture period, replicate wells were scored for growth and precursor frequency was determined. The frequency of T cells in BMBC was 1/22 (n = 4), 1/16 in FR‐1 (n = 4), and 1/246 in Fr‐2 (n = 2), and in Fr‐3 it ranged from 1/3,000 to 1/10,000 (n = 4). For comparison, depletion of T cells from bone marrow by cytofluorographic sorting with OKT‐11 and twice E‐rosetting yielded 98.1% and 97.4% removal of T cells, respectively. Thus, the clonogenic assay assured that more than 99.3% of the original T cells were depleted by elutriation; however, hematopoietic progenitor assay (CFU‐GM, CFU‐GEMM, and BFU‐E) showed no enrichment in any particular fraction, implying that the progenitors measured in this assay are of diverse sizes. Elutriation represents a useful method for depletion of T cells from human bone marrow. Limiting dilution assay of T cell growth is a sensitive measure to monitor T cell depletion.