Search

We have used limiting-dilution clonal analysis (LDA) in microwells to study the inhibitory effects of T lymphocytes (T-cells) or natural killer (NK) cells on human marrow progenitor cell growth. In four subjects with normal hematopoiesis, the growth of progenitors showed single-hit kinetics both before and after T-cell removal, indicating that, in the presence of colony-stimulating activity, T-cell have no effect on progenitor growth. In a patient with marrow hypoplasia associated with thymoma, hypogammaglobulinemia, and an increased number of suppressor T-cells (Good's syndrome), the progenitor growth deviated from linearity, demonstrating the presence of cells with suppressor activity. After T-cells were removed from this sample, the progenitor growth showed single-hit kinetics. The suppressive action of E-rosette-positive cells with NK or cytotoxic activities was also suggested in a patient with severe combined immune deficiency and in a patient with T gamma lymphocytosis. Poor progenitor-cell growth in three other patients with aplastic anemia was not significantly altered by T-cell removal. Thus, LDA of human hematopoietic progenitors is useful for evaluating cell-mediated interactions affecting hematopoiesis. This method may facilitate elucidation of mechanisms of myelosuppression in clinical settings.

Normal donor bone marrow buffy coat (BMBC) cells were fractionated into three subsets by counterflow elutriation centrifugation. Fraction 1 (Fr-1) was lymphocyte-enriched, 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.