This presentation was made for Journal club session about 2017 article from Journal of cancer immunotherapy. It reveals a new method of chemokine receptor gene modification on NK cells to improve their migration into tumor site. Such modification helps us to "cheat" tumor by changing the pattern of immune cells homing.

1. “Genetic engineering of human NK cells
to express CXCR2 improves migration to
renal cell carcinoma”
Kremer1 et al., 2017 Sep 19
Pathophysiology. Immunology. Oncology
Journal Club by Ihor Arkhypov

2. Institutions
1.Department of Oncology-Pathology, Karolinska Institutet, Stockholm,
Sweden.
2.Department of Molecular Oncology, The Netherlands Cancer Institute,
Amsterdam, Netherlands.
3.Department of Medicine Solna, Karolinska Institutet, Stockholm,
Sweden.
4.Department of Radiation Oncology, Weill Cornell Medicine, New York,
NY, USA.
5.Department of Oncology-Pathology, Stockholm South General Hospital,
Stockholm, Sweden.
6.Department of Woman and Child Health, Karolinska Institutet,
Stockholm, Sweden.
7.Department of Urology, Stockholm South General Hospital, Stockholm,
Sweden.
8.Department of Oncology-Pathology, Karolinska Institutet, Stockholm,
Sweden.
9.Cell Therapy Institute, Nova Southeastern University, Fort Lauderdale,
FL, USA.
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3. I. Background
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4. Natural killer cells
Receptors on NK cells:
1) Inhibitory
• killer Ig-like receptors (KIRs)
• CD94/NKG2A
2) Activatory
• “natural cytotoxisity receptors”
(NCR): NKp46, NKp30, NKp44;
• CD16, responsible for antibody-
dependent cytotoxicity;
• NKG2D and DNAM-1, that
recognize de novo expressed
ligands on transformed cells
Koch et al.,2013; Fionda et al.,2015; Rajalingam, 2016;
McWilliams et al., 2016
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5. • ClinTrial II phase – ovarian & breast cancer - 
• metastatic melanoma or renal cell carcinoma
• “Adoptive transfer of autologous natural killer
cells leads to high levels of circulating natural
killer cells but does not mediate tumor
regression”
Is NK transfer efficient?
Geller et al., 2011
Parkhurst et al., 2011
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6. Where to go???
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7. RCC infiltration by immune cells:
• More NKs – favourable prognosis
• More T-killers – worse prognosis
NKs in Renal cell carcinoma (RCC)
Geissler et al., 2015
Igarashi et al., 2002
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8. Chemokines in tumor environment
CXCR3
ligands
CXCR2
ligands
or tumor cell !!!
NK, T cell
infiltration
angiogenesis,
tumor growth,
metastasis Mestas et al., 2005
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9. • Neutrophils, monocytes +
• Peripheral blood NKs +
• In vitro cultured NKs – loss…
CXCR2 expression
Humoral
immunity!
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10. II. Methods
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11. 1. Patient samples
– Blood samples: centrifugated
(get plasma), stored -20°C
– Tumor samples: stored -80°C,
tissue lysates prepared
2. Cell lines
– Primary RCC cell lines (TINCA1, TINCA3, TINCA7,
MAR, ACHN, Caki-2, A498, Caki-1, 786-O),
myelogenous leukemia line K562
Primary materials
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12. • RCC patient plasma
• Lysates from tumor specimens
• RCC cell line supernatants
– CXCL1,8 – ELISA
Chemokine analisis
Bio-Plex Pro™
Human
Chemokine
Panel, 40-Plex
(immunoprecipitation,
magnetic beads)
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13. • Retroviral vector for CXCR2
• Retroviral vector for NGFR-N
(human nerve growth factor
receptor) – control
• Blood – PBMCs – NK – IL2
Expression and retroviral transduction of NK
cells
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14. • Chromium release assay (K562, ACHN, Caki-2)
• Calcium mobilisation assay
• Conjugate formation assay (with K562 by flow
cytometry)
Are they active after all?
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15. Chemotaxis assays
• IncuCyte ClearView
• CXCR2 inhibitor
• Cytoxicity after migration (51Cr-labeled K562)
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16. • CD56, CD16
• CXCR2
• CD11,18
• CD2
• …
Flow cytometry
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17. III. Results
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18. • Tumor > plasma
RCC tumors express CXCR2 ligands
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19. • Tumor < peripheral blood
• Ex vivo activation 
CXCR2 + NK cells
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20. Retroviral transduction
doesn’t alter the function
of NKs
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21. Increased adhesion properties
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22. Increased adhesion properties
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23. • CD11a blocking Ab!
Increased adhesion properties
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24. Increased Ca2+ mobilization
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25. Increased migration activity
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26. IV. Discussion
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27. • Correlated with NK infiltration in patients
• Epithelial-cell derived
• Constrain the ability to form metastasis
[Lopez-Lago et al.,2013]
• Low CXCL5 – aggressive tumor growth in
colorectal cancer model and poor prognosis
[Speetjens et al.,2008]
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Role of CXCL5

28. • Low level on peripheral blood NKs [Campbel
et al., 2001]
• No CXCR2 expression on fresh and in vitro
activated NKs [Inngjerdingen et al., 2001]
• In vitro cultured NKs – loss… [authors]
• Short-term CXCL8 exposure – recycled CXCR2,
long – degradation [Fan et al., 2003]
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CXCR2 on NKs

29. • Costimulatory receptor – CD2
• CD11 – a-subunit of LFA-1
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+CD2, +CD11

30. • Many controls
• Different tumor samples and cell lines
• Functioning controlls
• For further research – interplay with tumor
microenvironment
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Comments

31. Thank you for attention!
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