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Impact of Hypoxic Conditions on Natural Killer Cell Cytokine Secretion
Paul McClure MS2, Upasana Arvindam, Dr. Jeffrey Miller MDDepartment of Medicine, Division of Hematology, Oncology, and TransplantationUniversity of Minnesota, Minneapolis, MN 55455, USA
NK Cells - Background
• Main duties: killing virally infected cells, immune surveillance for cancer– NK Cells kill cells that do not express MHC-I or that do
express other markers of stress• No priming or prior exposure required, but NK cell
“education” plays a role in increasing function
Mechanisms of NK Cell Cytotoxicity
• Antibody-Dependent Cell-mediated Cytotoxicity (ADCC)
• Induction of apoptosis• Lytic granule release: Perforin and
Granzyme B• Cytokine release/lymphocyte
recruitment: IFN-gamma, constitutive chemokine secretion
Nature Rev. Immunology (2010)
Challenges to NK Cell Immunotherapy
• Tumor infiltration and survival
• Natural Killer Cell Exhaustion: Metabolic? Cytokine-
mediated? Continuous exposure to antigen?
• Cost: how to develop better, more affordable “off-the-
shelf” options for cancer treatments w/ a manageable
dosing schedule.
Cytokine Release in Normoxic (21%) Conditions
NK cell production of IFN-gamma, TNF-alpha both increase significantly when exposed to target cells
Constitutive (baseline) secretion of some chemokines (CCL5 e.g.)
Blood. Bryceson et al (2010).
The AVATAR Incubator
• Very precise control of O2, CO2 and pressure. • Better model of the TME for in vitro studies of immune
cell function. • 3 Conditions:
– AV1: 12% O2 2 psi (Peripheral blood)– AV2: 5% O2 0.6 psi (Bone marrow)– AV4: 1% O2 2 psi (Solid tumor)
Experiment Timeline
Day 1: Enrich NK cells
and plate on 96 well
plate in RPMI-10 w/1
ng/ml IL-15 (3x105/well)
Add plates to AVATAR
incubators
Day 4: Change
media, add fresh R10
w/ 1 ng/ml IL-15
Day 6: Harvest
and count NK
Cells, re-plate at
3x105 cells/well
w/ 1.5x105 K562
Target cells (or
”empty” media)
Day 7: Collect
supernatants
and freeze at
-80 C for
analysis by the
Cytokine
Reference Lab.
Analysis done
on Luminex,
using standard
human cytokine
panel
Results: NK Cell Secretome in Hypoxia
NK cells were cultured with K562 cells for 24 hours following 1 week incubation in AVATAR
NK Cell Phenotype Heterogeneity• CD56: the canonical marker
used for differentiating NK cell subsets
• Other considerations: proliferative capacity vs. cytotoxic function, NK cell ‘licensing’, unique lineages of NK Cells (NK92, NK101, etc.)
Ki-67 expression in 12% O2
Ki-67 expression in 1% O2
NK Cell Marker Expression in Hypoxia
Increased expression of
CD69 is significant due to
relationship with HIF-1a
Greater % of CD56dim
vs. CD56bright NK cells
in hypoxic conditions.
12% O2 (Blood) 1% O2 (Solid Tumor)
Other markers of
interest: Ki-67,
CD96, TIGIT etc.
Cytotoxicity is Impaired in HypoxiaNK Cell mediated killing of K562 target cells is dramatically impaired in conditions mirroring a solid tumor relative to peripheral blood or bone marrow
Discussion: Looking Beyond NK Cells
• The role of TGF-beta in immunosuppression is still largely unknown in vivo
• How to sustain proliferation and function of NK cells in solid tumors
Conclusion
• NK-cell cytokine production is highest in conditions mirroring those found in bone marrow, lowest in those representative of a solid tumor
• NK Cells do not produce significant amounts of TGFbin any of the studied conditions but K562 cells significantly increase their secretion in hypoxic conditions
Future Directions/Implications• Ongoing co-culture experiments with Regulatory T-
Cells and Myeloid Derived Suppressor Cells• Developing induced NK (iNK) cells that preserve
cytokine secretion and function in hypoxia• What is the role of cytokines in NK cell exhaustion?• Microscopic characterization of NK cell degranulation
and cytotoxicity in various incubation conditions
AcknowledgementsMiller LabUpasana ArvindamDr. Pippa KennedyBri EttestadPeter HowardDr. Martin FelicesDr. Jeffrey MillerCytokine Reference Lab for Luminex analysis
NIH Summer T35 ProgramDr. Dan MuellerStephanie Krischuk
All the faculty who came to present their work over the course of the summer
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