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

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Abstract
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.

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