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Human natural killer cell expansion is regulated by thrombospondin-
mediated activation of transforming growth factor-beta 1 and independent
accessory cell-derived contact and soluble factors
BA Pierson, K Gupta, WS Hu and JS Miller
Department of Chemical Engineering and Materials Science, University of
Minnesota, Minneapolis, USA.
Natural killer cells (NK) were studied to determine factors important in
their expansion. Flourescence-activated cell sorter (FACS) purified
CD56+/CD3- NK cells cultured alone for 18 days in rIL-2 containing medium
(1,000 U/mL) showed enhanced cytotoxicity but only minimal expansion. NK
expansion was increased (12.5 +/- 1.6-fold) by coculturing NK with soluble
factors produced by irradiated peripheral blood mononuclear cells (PBMNC)
in which the two populations were separated by a microporous membrane.
However, maximal NK expansion was always observed when NK were cocultured
in direct contact with irradiated PBMNC (49.4 +/- 5.9-fold). To determine
if marrow stroma, which supports differentiation of primitive NK
progenitors, was a better accessory cell population than irradiated PBMNC,
NK were cocultured in direct contact with primary marrow stromal layers. NK
expansion with marrow stroma was similar to PBMNC. Fibroblast cell lines
(M2-10B4, NRK-49F, NIH-3T3) and human umbilical vein endothelial cells
(HUVEC), all homogeneous populations and devoid of monocytes, also
exhibited a similar contact-dependent increase in NK expansion. Experiments
were designed using fixed M2-10B4 stromal cells to separate the
contact-induced proliferative stimuli from soluble factors. NK plated
directly on ethanol/acetic acid-fixed M2-10B4, which leaves stromal ligands
(cell membrane components and ECM) intact, resulted in increased NK
expansion compared with medium alone. We further show that the combination
of independent contact and soluble factors is responsible for maximal late
NK expansion (days 28 through 40) but paradoxically inhibits early NK
expansion (day 7). The proliferation inhibitory effects were verified by
3H-thymidine uptake and could be detected at days 2 through 6 but no longer
14 days after the initiation of the culture. We show that both laminin and
thrombospondin inhibit early NK proliferation, whereas only thrombospondin
was capable of also stimulating late NK expansion. The effect of
thrombospondin on early NK proliferation is related to activation of
transforming growth factor- beta 1 (TGF-beta) because anti-TGF-beta
neutralizing antibody completely abrogated thrombospondin-mediated
inhibition of early NK proliferation. Although inhibitory early in culture,
active TGF-beta added only at culture initiation increases late NK
expansion similar to thrombospondin. TGF-beta was not present in the
thrombospondin preparation but latent TGF-beta in serum, or TGF-beta
transcripts identified in IL-2-activated NK could explain paracrine or
autocrine mechanisms for the regulation of NK proliferation. Finally,
anti-TGF- beta neutralizing antibody only minimally affects stroma-mediated
inhibition of early NK proliferation suggesting that aside from
thrombospondin/TGF-beta, additional contact factors are important for the
regulation of NK proliferation.
Volume 87,
Issue 1,
pp. 180-189,
01/01/1996
Copyright © 1996 by The American Society of Hematology
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