Download NK cell function and education in Hypoxic Environment

NK cell function and education in Hypoxic Environment
Kyung-Mi Lee
Professor, Department of biochemistry, Korea University Medical School
Anam-Dong 5-Ga, Sungbukku, Seoul, 136-705, Korea
NK cells provide the first line of defense against tumor and viruses, without the
requirement for prolonged pre-activation. However, anti-tumor function of NK cells is
progressively dampened in the terminal stages of cancer patients, largely associated with the
tumor-induced immune evasion mechanism. Hypoxic tumor environment provides once such
mechanism providing NK cell tolerance, and become a potential therapeutic problem to
overcome in the patients of solid cancer for NK immunotherapy. To better understand the
effect of hypoxia on NK cells, we assessed the anti-tumor function of NK cells in response to
decreasing concentrations of O2 pressure. As expected, NK cells exposed to severe hypoxia,
0.5% O2, demonstrated diminished cytotoxicity and IFNg against A375 melanoma targets
and failed to expand to a larger number ex vivo. However, NK cells incubated in mild
hypoxia of 1.5% O2 showed slightly better CTL activity than normoxic condition, but still
failed to proliferate. These data explain why there are so few NK cells within resected solid
tumor tissues. Since CTL killing activity is elevated in NK cells cultured in 1.5% hypoxia, we
hypothesized that if we pre-activate NK cells in normoxia prior to switching to hypoxia, we
could educate (or license) NK cells to function and proliferate within hypoxic tumor
microenvironment. To test this hypothesis, we started culturing NK cells in normoxia then
transferred them into 1.5% hypoxia chamber at day 3, 5, 9. Our results showed that NK cells
switched to day 5 or 9 were expanded to a much higher number and mounted increased antitumor effector function, when CTL reaction was performed both in normoxic and hypoxic
conditions. But those switched to 1.5% hypoxia chamber after 3 days did not proliferate,
indicating that NK cells need O2 for initial priming. The hypoxia-exposed NK cells
demonstrate upregulation of major activating NK cell receptors, NCRs and NKG2D receptors
including STAT3, while inhibiting apoptosis during ex-vivo expansion. Microarray analysis
results revealed that AK3L1, PFKFB4, MT1G, BNIP3 genes were elevated and BTLA,
KLRG1 genes were downregulated in hypoxia-exposed NK cells, providing the basis for the
increased NK effector function and survival. These data highlight the need for ex-vivo
activated NK cell therapy, and further suggest that NK cell education within hypoxic
environment during expansion can indeed facilitate the proliferation and function of NK cells
that can overcome NK cell intolerance in the patients undergoing NK cell immunotherapy.
We hypothesized that tumor targets sensitive to NK lysis would drive vigorous expansion of
NK cells from human peripheral blood mononuclear cells (PBMC). Here, we provide the
basis for developing a novel ex vivo expansion process. By screening class I-negative or mismatched tumor cell lines we identified a Jurkat T-lymphoblast subline termed KL-1,
which was highly effective in specifically expanding NK cells. KL-1 addition to PBMC
cultures achieved approximately 100-fold expansion of NK cells with nearly 90% purity,
accompanied by reciprocal inhibition of T-cell growth. Marked elevations in expression of
activation receptors, natural cytotoxicity receptors (NKp30, NKp44), and adhesion molecules
(CD11a, ICAM-1) were associated with high tumor-lytic capacity, in both in vitro and in vivo
models. KL-1-mediated expansion of NK cells was contact dependent and required
interactions with CD16, the Fcγ receptor on NK cells, with ligands that are expressed on B
cells. Indeed, B-cell depletion during culture abrogated selective NK cell expansion, while
addition of EBV-transformed B cells further augmented NK expansion to approximately 740fold. Together, our studies define a novel method for efficient activation of human NK cells
that employs KL-1-lysed tumor cells and cocultured B cells, which drive a robust expansion
of potent antitumor effector cells that will be useful for clinical evaluation.