Download What are NK Cells - Biology Department

Natural Killer Cells:
Their role in Cancer
Immunotherapy, And the
Significance of the Molding of
the KIR Repertoire
Jerome R. Trembley
Senior Seminar
February 19th, 2015
Topical Objectives
Introduction to work at MD Anderson
 What are Natural Killer Cells
 NK Cell Immunotherapy
 NK Cells and the Killer ImmunoglobulinLike Receptor
 Molding of the KIR Repertoire
 Future Work…

What did I do?
• Also Shadowed Dr. Lee weekly during clinic periods
• Able to observe the majority of his patients, including a Bone Marrow
Extraction/Transplant
Img Source: Self
What did I do?
Conducted bench-top research at MD
Anderson Cancer Center, Houston
Texas, from May 19th, to August 14th,
2013.
 14, 3 week long experimental batteries
performed.
 Abstract submitted, and accepted at
ASH – Presented on Dec 14th, 2013.

What are NK Cells –
What are they?

Part of the Innate Immune System
 Lymphocyte.

GVL, with minimal GVHD. (Lee, 2015)

Selective
 Two methods of cytotoxicity (Cheng., et.al.
2013)
○ Granzymes
○ Perforin

Highly Cytotoxic to Cancer
 Enhanced efficacy on a range of cancers.
(Cheng et.al., 2013)
NK Cell Utilization –
Why do we need NK Cells?
Img Source: Cancer Research UK, 2014
NK Cell Utilization –
Why do we need NK Cells?
Img Source: Cancer Research UK, 2014
What are NK Cells? –
NK Cell suppression in compromised systems
NK Cells are needed to maintain innate
immune defense.(Lion et.al., 2012)
 NK Cells are severely compromised via
current immunotherapy methods. (Lion et.al.,
2012)
 When compromised, cancer cells “escape”
detection. (Lion et.al., 2012)

Sources: Lion et.al., 2012
What are NK Cells? –
NK Cell suppression in compromised systems
NK Cells are needed to maintain innate
immune defense.(Lion et.al., 2012)
 NK Cells are severely compromised via
current immunotherapy methods. (Lion et.al.,
2012)
 When compromised, cancer cells “escape”
detection. (Lion et.al., 2012)

Sources: Lion et.al., 2012
What are NK Cells? –
NK Cell suppression in compromised systems
Img Source: Lion et.al., 2012
What are NK Cells? –
GVHD vs. GVL
“NK cells have shown to mediate graft-versus-leukemia immunity
towards recipient tumor cells without attacking normal tissues,
leading to graft-versus-host disease.” - Glycostem, 2014
Img Source: Glycostem, 2014
What are NK Cells? –
Why Are they Effective?
Video Source: Somanchi, et.al., 2011
What are NK Cells? –
Why Are they Effective?
Img Source: Cheent, K., & Khakoo, S., 2009
What are NK Cells? –
The Killerimmunoglobulin-Like Receptor
Passport “780-C1”
Passport “780-C2”
Passport “Bw4”
KIR-2DL2/3 Utilizes same
Passport,
Same Crosslinked Antibody
Img Source: Lanier, 2005
Point of Study
“NK Cell-based immunotherapy holds great
promise for treatment... Only moderate
clinical success so far…
“Progress in the field of understanding NK
cell biology and function is therefore
needed to assist in developing novel
approaches to effectively manipulate NK
cells for the ultimate benefit of treating
cancer patients.”
– Cheng et.al., 2013
Hypothesis
1 – The desired KIR Repertoire could be
molded through inhibition of undesirable
KIR populations through the crosslinking of
relevant Anti-KIR antibodies during
expansion.
2- The KIR Repertoire molding would have
no effect on expanded NK Cell cytotoxicity
Experiment – Methodology
Early Difficulties
1. Initial outlook was poor
1. Attempts to stimulate growth via antibodies
proved ineffective.
2. Determined inhibition protocol in early July.
3. Was able to isolate most efficient means by
July 30th.
1. Most efficient means determined by
stimulation on Day 3 by loading and
stimulating with K562.
Experiment – Methodology
Ideal Method
1. Initiate Experiment In accordance with Expansion Protocol.
A. Extract Cells from Buffy Coat
B. Determine Phenotype (KIR Receptors) of NK Cells
• Done using antibody identifiers.
C. Culture in accordance with Expansion Protocol
• FARG Solution (Fetal Bovine Serum + Culturing Media)
2. D=3, Load NK Cells with Anti-KIR aB.
3. Crosslink with Goat Anti-Mouse aB.
4. Suspend cells with 1:1 ratio of K562 Cells in accordance with
Expansion Protocol
Results
Population results for
Unloaded vs. KIRCrosslinked cell lines.
KIR2DL1±19.3%
70.4%
KIR3DL1±16.3%
53.5%
KIR2DL2/3– 56% ±17.5%
Gfx Source: Self
Results
Anti KIR
Percent Reduction
Margin of Error (±%)
2DL1
70.4
19.3
3DL1
56.0
17.5
2DL2/3
53.5
16.3
Gfx Source: Self
Results
Flow Cytometry Data:
NK Cell
subpopulations
Vs. Isotype control.
Crosslinking with
individual CD158e
(KIR3DL1) and Bulk
KIR populations.
Marked molding of KIR
Repertoire is noted in
undesired populations.
Gfx Source: Self
Results
Degranulation
response
demonstrated
efficacy against
tumor cells
expressing relevant
HLA Ligand for
targeted KIR
Additionally, Bulk NK
Cell cytotoxicity
against HLANegative targets
remained robust.
Gfx Source: Dr. Vladimir Senyukov, unpublished
Hypothesis Confirmed
A robust reduction of targeted KIR-Positive
populations was achieved.
 Cytotoxicity of bulk NK cell population
remained intact

Implications of Work
•
•
Increased Anti-tumor affect from utilization of KIR
Mismatch
Easy to use Immunotherapy
• Cheap
• Easily understood
• Easily replicable for clinical grade application
Most Importantly:
It has the potential to enhance the efficacy of NK
Cell Immunotherapy!
Future Work
Further Experimentation
 GVHD vs GVL – A problem…

Issues with NK Cell ImmunotherapyGVHD vs. GVL – Possible Connection?
1. Direct effect via lysis or
indirect activation of
adaptive immunity
2. Cytokine-mediated
upregulation of HLA for
T-Cell recognition
3. Additional stimulation of
Cytotoxic T-Cells
4. Activation of Helper TCells
5. Maturation of dendritic
cells for antigen
presentation
Img Source: Lee, 2015
Future Work
Further Experimentation
 GVHD vs GVL – A problem…
 Signaling Pathway – STAT3 Confirmed!

“…data show that NKG2D
expression in NK cells is
regulated at the transcriptional
level by STAT3.”
-Zhu, et.al., 2014
Acknowledgements

Dr. Dean Lee, MD, PhD
 Associate Professor – MD Anderson, Pediatrics.

Dr. Vladimir Senyukov, PhD
 Research Scientist, MD Anderson, Pediatrics.
St. Baldrick’s Foundation
 MD Anderson Cancer Center
 Fresno Pacific University –

Biology Department
References
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Cancer Research UK. Childhood Cancers by Cancer Site: 1996-2005. (2014, March 11).
Retrieved February 15, 2015, from http://www.cancerresearchuk.org/cancerinfo/cancerstats/childhoodcancer/mortality/childhood-cancer-mortality-statistics
Cheent, K., & Khakoo, S. (2009). Natural killer cells: Integrating diversity with
function. Immunology, 126(4), 449-457. doi: 10.1111/j.1365-2567.2009.03045.x
Cheng, M., Chen, Y., Xiao, W., Sun, R., & Tian, Z. (2013). NK cell-based immunotherapy
for malignant diseases. Celllular & Molecular Immunology, 10, 230-252. doi:
10.1038/cmi.2013.10
Glycostem. Allogeneic NK cells for immunotherapy. (2014). Retrieved February 16, 2015,
from http://glycostem.com/technology/nk-cell-immunotherapy
Lanier, L. (2005). NK Cell Recognition. Annual Review of Immunology, (23), 225-274. doi:
10.1146/annurev.immunol.23.021704.115526
Lee, D. (2015). The off-target effects of nonspecific NK cells. Blood, 125(5), 774-775.
Lion, E., Willemen, Y., Berneman, Z., Van-Tendeloo, V., & Smits, E. (2012). Natural killer
cell immune escape in acute myeloid leukemia. Leukemia, (2012 Review), 1-8. doi:
10.1038/leu.2012.87
Somanchi, S., Senyukov, V., Denman, C., & Lee, D. (2011). Expansion, Purification, and
Functional Assessment of Human Peripheral Blood NK Cells. Journal of Visualized
Experiments, 48
Zhu, S., Phatarpekar, P., Denman, C., Senyukov, V., Somanchi, S., Nguyen-Jackson, H., &
Lee, D. (2014). Transcription of the activating receptor NKG2D in natural killer cells is
regulated by STAT3 tyrosine phosphorylation. Blood,124(3). doi: 10.1182/blood-2013-05499707