Previous studies established that the high density (resting) natural killer (NK) cell subset (R-NK) of peripheral blood NK cells is unresponsive to interleukin-2 (IL-2) but can be induced to proliferate when cultured with gamma-irradiated malignant melanoma (MM-170) cells or mitomycin-C-treated activated T cells in the presence of an IL-2-conditioned medium (IL-2-CM). This study has examined additional requirements of this activation process. The induction of proliferation was dependent on cell to cell contact with metabolically active stimulator cells, although no evidence was obtained that stimulation was effected by soluble factors produced by the stimulator cells. Compared with IL-2-CM, rIL-2 was an inefficient costimulator for the induction of NK cell proliferation, suggesting that factors in IL-2-CM were required in addition to IL-2, but rIL-2 was as efficient as IL-2-CM in maintaining the proliferation of activated NK cells. Under optimum culture conditions, NK growth of up to 3200-fold occurred during a proliferation cycle of 18 days. Phenotypic analysis of the culture-generated quiescent NK cells revealed novel heterogeneity in CD16 (Fc gamma RIII) and CD56 (N-CAM) expression. Some NK cells lacked expression of both CD16 and CD56 (as identified using currently available monoclonal antibodies), while other NK cells showed differential CD16 epitope expression. Since quiescent NK cells can be obtained in large numbers and high purity, they will be a convenient source of NK cells to study the molecular processes involved in initiating NK cell proliferation.
Summary NK cells from three donors with a NK (CD3 -CD56 " CD 16 ") lymphoeytosis of unknown aetiology showed differential expression of CD45R0, an isoform of CD45 not expressed by NK cells from normal donors unless stimulated to proliferate in vitro. For donor FC, 60% of NK cells expressed CD45R0 over a 16 month period during which there was a partial resolution of the NK lymphoeytosis. For donor SW, 37% of NK cells expressed CD45R0. increasing to 87% over a 14 month period during which the NK lymphoeytosis increased. For donor RN few if any NK cells expressed CD45R0. After in vitro proliferation, 100% of NK cells generated from all donors expressed CD45R0. For donors FC and SW, CD45R0 remained expressed on more than 90% of cells at 3-4 weeks following eessation of proliferation. By contrast CD45R0 expression was gradually lost during long-term culture of NK cells from donor RN, with 58% of NK cells regaining the pre-culture CD45R0-phenotype. NK cells from normal donors also varied in the extent to which activation acquired CD45R0 was lost during long-term culture. The results obtained are consistent with the notion that NK eells from the NK lymphoeytosis donors studied have previously undergone proliferation in vivo.
SUMMARYT lymphocytes spend much of the time as small non-cycling cells. To determine the pattern of cytokine expression in such resting cells, they were purified from human peripheral blood mononuclear cells (PBMC) on the basis of high buoyant density. The cells were stimulated and cytokine mRNA expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Expression of interleukin-2 (IL-2), IL-3 and interferon-g (IFN-g) was similar in high-density lymphocytes and in unfractionated PBMC. In contrast, the high-density lymphocytes expressed less IL-4 than PBMC, and little or no IL-5. Because a substantial minority of the high-density lymphocytes was CD45RO, the presence of this marker was not an indicator of the ability to express IL-4 and IL-5. In the high-density lymphocytes, IFN-g expression was confined to the CD45RO fraction, whereas IL-2 was expressed by both CD45RO and CD45RO ÿ subsets. To assess whether high-density lymphocytes could give rise to cells with a broader range of inducible cytokine expression, they were activated and then restimulated between 10 and 22 days of culture. Cells derived from both the CD45RO and CD45RO ÿ fractions of high-density lymphocytes expressed IL-5 after restimulation. Thus the high-density lymphocyte population has the potential to acquire a broader range of inducible cytokine expression.
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