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