Cotton, Sarah, Final Thesis.

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THE ROLE OF SYK IN NATURAL KILLER CELL LYTIC SYNAPSE FORMATION AND
FUNCTION
BY
SARAH COTTON
AN HONORS THESIS
FOR THE
UNIVERSITY HONORS COLLEGE
Submitted to the
University Honors College
at Texas Tech University in
partial fulfillment of the requirement for
the degree designation of
HIGHEST HONORS
May 2015
Approved by:
__________________________________
Date _____________
Faculty Mentor: Boyd Butler
Department of Biological Sciences
__________________________________
Date______________
Second Reader: Ruth Serra-Moreno
Department of Biological Sciences
__________________________________
Date______________
Thesis Instructor: Keira Williams
University Honors College
__________________________________
Date______________
Dean: Michael San Francisco
University Honors College
The author approves the photocopying of this document for educational purposes.
Acknowledgements
Dr. Boyd Butler: for allowing me the opportunity to join his lab and teaching me a variety of
basic research skills that will allow me to succeed in the medical field.
Erin Kitten: for training me and guiding my thesis project.
Dr. Ruth Serra-Moreno: for her suggestions and comments about my thesis.
Undergraduate Research Scholars: for giving me the opportunity to participate in undergraduate
research through their financial support.
University Honors College: for the guidance and support to write an undergraduate thesis.
Life Sciences Imaging Facility: for the use of BD FACS Aria II SORP Instrument.
Table of Contents
ACKNOWLEDGEMENTS ............................................................................................................ ii
TABLE OF CONTENTS ............................................................................................................... iii
INTRODUCTION .......................................................................................................................... 1
CHAPTER 1: Literature Review .................................................................................................... 1
CHAPTER 2: Materials and Methods ........................................................................................... 5
CHAPTER 3: Results .................................................................................................................... 7
CHAPTER 4: Discussion ............................................................................................................. 10
CHATPER 5: Future Directions ................................................................................................... 12
APPENDIX ................................................................................................................................... 14
BIBLIOGRAPHY ......................................................................................................................... 15
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Sarah Cotton
8 April, 2015
Introduction
Spleen tyrosine kinase (Syk) is a signaling molecule in virtually every immune pathway
that plays a role in various functions throughout the body.17, 18 The purpose of this proposal is to
investigate the role of Syk signaling in natural killer (NK) cells. NK cells are a part of the innate
immune system that focuses on finding and eliminating virally infected cells.14 Syk is involved
in the immune and integrin pathways in NK cells. Integrin signaling in NK cells is mediated by
signaling through leukocyte function-associated antigen 1, LFA-1. Syk signaling through LFA-1
has been fully elucidated.5, 11 The main focus of this thesis is to elucidate the role of Syk in the
immune Natural Killer, Group 2, member D (NKG2D) receptor pathway. Therefore, this
proposal hypothesizes that without Syk signaling through NKG2D, the NK cell will still be
able to adhere to its target cell but will not be able to lysis the target cell.
Chapter 1: Literature Review
Natural Killer Cells
NK cells are lymphocytes that are a part of the innate immune system that target and lyse
virally infected cells or cancerous cells in the body.15 Unlike other immune cells, NK cells do
not need to be previously activated in order to lyse the cells which provide the body with a firstline of defense against viruses and tumorigenic cells.15 NK cytotoxicity is a two-step process:
actin polarization and degranulation. First, the NK recognizes the target cell, adheres and forms
the immunological synapse. Activation of NK cells occurs by the down-regulation of major
histocompatibility complex I (MHC-I) molecule on the cell surface of the target cell. MHC-I is a
peptide-presenting molecule that primarily presents self-peptides but may also present viral
peptides that will activate the immune system. Viruses can down-regulate MHC-I to avoid
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detection, but the absence of MHC-I will trigger a response from a NK cell. Identification of the
target cell causes the formation of the immunological, or lytic synapse.
The lytic synapse is the junction between the NK cell and the target cell requires the actin
cytoskeleton rearrangement and adhesion receptor clustering.15 The synapse allows the transport
of cytotoxic granules such as perforin and granzyme B to the target cell without affecting
bystander cells..15 After the lytic synapse is established, the polarization of the lytic granules
towards the immunological synapse occurs. The microtubule organizing center (MTOC) will
then move and polarize towards the lytic synapse while the lytic granules travel to the MTOC
where they aggregate.15 Microtubules serve as tracks for the transportation of cytotoxic granules
from the MTOC towards the lytic synapse. While the mechanism of granule release remains to
be fully elucidated, the vesicle fuses with the NK cell membrane and perforin pokes holes in the
target cell membrane, which allows for granzyme B to enter the cell and begins to signal for
apoptosis.15
Cytotoxicity of NK cells is highly dependent on the cytoskeleton and the rearrangement
that controls this process. Signaling pathways through cell surface receptors , NKG2D and LFA1, activate rearrangement. Syk is an important signaling node in both of these receptor-signaling
cascades and will be discussed in detail below. Various roles for Syk that have been identified,
but because of the ubiquitous nature of the signaling molecule, the specific function for Syk in
the NKG2D pathway is still under investigation.
The properties of the signaling molecule Syk
Syk is a tyrosine kinase that is 72kD and functions in a wide variety of immune pathways.
In NK cells, Syk plays a role in both NKG2D (a C-type lectin) and LFA-1 (an integrin).19 Syk
has two Src homology (SH2) domains that allow it to dock to adapter molecules.
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Phosphorylation of Syk on its tyrosine residues activates Syk to phosphorylate downstream
effectors. 19 Auto-inhibition of Syk occurs in the absence of immunoreceptor tyrosine activating
motif (ITAM) phosphorylation by the folding of the domains into an inhibited conformation that
blocks the catalytic domains.19 The specific roles of Syk in specific signaling pathways will be
discussed in later sections.
Characteristics of Syk in the LFA-1 Integrin Pathway
Syk is in multiple immune pathways including the integrin pathway that signal through
LFA-1. Integrins are cell surface receptors that regulate adhesion, migration, cytokine
production, and slow rolling along endothelial cells.2 Endothelial cells line blood vessels and
express intercellular adhesion molecule 1, ICAM-1, a ligand for LFA-1.2 While LFA-1 is on
different leukocytes, LFA-1 signaling in NK cells is the focus of this review.
Natural killer cells primarily use LFA-1 for cell-to-cell adhesion to form a lytic synapse
with a target cell. Rearrangement of LFA-1 around the peripheral supramolecular activation
cluster (pSMAC) of the synapse allows secretion of cytotoxic granules into a cleft to destroy the
target cell while protecting neighboring cells. Integrins exhibit bi-directional signaling, insideout and outside-in. Signal propagating inside the cell and acting on the cytoplasmic tail of
integrins can regulate the affinity to ligands by conformational changes in the extracellular
domain. On the other hand, outside-in signaling starts with the ligand binding to the receptor to
cause a signaling cascade within the cell.2 This review will focus on outside-in signaling
responsible for adhesion and actin rearrangement. Activation of LFA-1 leads to the association
of the adaptor molecule DAP12 and the phosphorylation of tyrosine residues within DAP12
ITAMs. Phosphorylation then leads to recruitment of kinases and scaffolding molecules. Syk
kinase associates with the cytoplasmic tail of ITAMs by its SH2 domain in NK cells and allows
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for amplification of the signal by phosphorylating downstream effectors.5 Auto-phosphorylation
of Syk leads to its activation and allows it to phosphorylate phospholipase C-γ (PLCγ) and
SLP76.11 Downstream of SLP76 regulates cytoskeleton rearrangement through Vav, a rho-GEF.
Vav activates the rho-GTPases, Rac, Cdc42 and Rho, which results in actin cytoskeleton
rearrangement. The rearrangement of adhesion molecules and actin, as well as, polarization of
cytotoxic granules by activating LFA-1 is an important initial step in NK cytotoxicity.12
Downstream of SLP76 is a very important molecule in regulating cytoskeleton
rearrangement, Vav, a rho-GEF. A GEF molecule, of any type, activates GTPases which will
have large effects within the cell. This case is no different; Vav activates rho-GTPases that
cause the actin cytoskeleton to rearrange. The results of this rearrangement result in adhesion
and actin polarization of the cytotoxic granules, although they are not released.13 This
rearrangement is one of the effects of integrin activation, and it is an extremely important one in
Natural Killer cell cytotoxicity.12
Characteristics of Syk in the NKG2D pathway
NKG2D is a C-type lectin receptor that NK cells constitutively express to recognize the
expression of MHC-I receptors. Down-regulation of MHC-1 receptors in cancer or virusinfection will activate NK cells. Unlike CD8+ T cells and other lymphocytes, which only
associate with Dap10, the NK cell receptor, NKG2D, can associate with either Dap10 or Dap12
allowing a versatile response 10. The activation NKG2D recruits Dap10 or Dap12 as an adaptor
molecule and further recruits phosphatidylinositol-3-kinase (PI3K) or signals through Syk using
ITAMs.10,17 The versatility of NKG2D in NK cells versus T cells suggests an important
distinction in function that is related to Syk.10 Syk signaling in the NKG2D pathway is similar to
Page |5
LFA-1 signaling in that it is downstream of PI3K and upstream of Vav. Investigating the
downstream effectors of Syk is important in understanding the role of NKG2D-Syk signaling.
Conclusions
Thus, research on Syk is still underway and the gaps in the pathway such as downstream
effectors and function are still unknown. Natural killer cells are an ideal model system for this
study because of the unique role of NKG2D through Syk signaling. This thesis aims to fill in the
specific role that Syk plays in NKG2D signaling by examining the effects of Syk on NK cell
function.
Chapter 2: Materials and Methods
Cell Culture
The cell lines NK92MI (ATCC) and K562 (ATCC) were cultured in MEMα media and
RPMI media supplemented with fetal bovine sera, respectively. A Syk knockdown (KD) was
generated in NK92MI cells using a tet-inducible shRNA(pSingle) vector system (Clontech). To
induce the knockdown, the cells were cultured in Doxycycline for 24-48 hours. A mCherry Syk
plasmid was transfected into NK92MI cells to demonstrate Syk overexpression and for
microscopy live cell imaging. Wild type NK cells, Syk KD cells, and mCherry Syk cells were
tested for various functions against the K562 target cells. K562 cell line was transfected with a
GFP-Actin plasmid.
Western Blot
Cells were lysed with 1% NP40 containing protease inhibitors and sodium orthovanidate
and mixed with a 1X loading dye. The samples were run on a standard 10% polyacrylamide gel
at 70V for 3 hours. The gel was transferred onto a PVDF membrane at 100V for 1 hour and
stained with Coomassie to visualize the proteins. The primary antibodies used were a Syk-
Page |6
Mouse (Sigma) and Actin-Rabbit. After exposure to Chemiluminescence, the image was
generated.
Assays
Cytotoxicity Assay: NK92MI cells were co-cultured with K562 in a round-bottom 96
well plate for 3 hours at 37°C. The NK92MI:K562 ration varied from 5:1, 10:1, and 15:1. The
LDH Cytotoxicity kit (Clontech) was used to measure cell death of the target cell. This assay
was performed using WT, KD and overexpressed cell lines. In addition, the 10:1 ratio was used
with varying levels of Syk inhibitor, Piceatannol (2.5, 5, and 10μg). Values were normalized to
the value for total release from 105 K562 cells using 10% Trition-100X. This normalized ratio
was plotted as the killing index.
Adhesion Assay: 96-well microtiter plates (Immulon II) were coated with poly-L-lysine
(50ug/ml) and fibronectin (10ug/ml) in PBS overnight. Wells were washed twice with in PBS
and coated with 1.0% casein in PBS for 30 min at room tempature. 1 x 105 NK92MI cells were
added per well for 30 minutes at 37°C in Hank’s buffered saline solution containing 1.0mM Ca2+
and Mg2+ with PMA (10ng/ml). Residual, unattached cells were gently washed off of the plate
and the remaining cells were then fixed in 3.7% formaldehyde for 1 hour and stained with 0.5%
Crystal violet. Unbound crystal violet was washed away and methanol added to dissolve the
crystal violet. Absorbance was measured at 540nm. Piceattanol was added at concentrations
mentioned above.
Conjugate Assay: mCherry Lifeact NK92MI cells were incubated with SDF1-α for 30
min at 37°C and GFP-Actin K562 cells were incubated with PMA (10ng/ml) for 30 min at 37°C.
After the incubation, the mCherry NK cells and GFP-Actin K562 cells were added to 2.5mL
tubes together with 10μg/mL of Piceatannol and incubated at 37°C for the indicated time points.
Page |7
The cells were treated with SYTOX Blue Dead Cell Stain before analysis (Life Technologies;
1uM). 10,000 cells per sample were analyzed by two color flow cytometry for red, green, and
red-green events at each time point (0, 5, 10, 15, 20, 25, 30, 45, and 60 min) by a BD FACS Aria.
Chapter 3: Results
Cytotoxicity Assay:
In Figure 1, NK92MI cells were incubated with K562 cells for four hours and the cell
lysis was measured at five minute intervals for 35 minutes. There were five treatment groups:
the DMSO (control) group, untreated (WT) group, and increasing dosages of Piceatannol
(2.5μg/mL, 5μg/mL, and /10μg mL). The 10μg mL shows a dramatic decrease in cell lysis
throughout the experiment (See Figure 1).
Piceatannol Killing Index
0.5
0.45
0.4
Cell Lysis
0.35
DMSO
0.3
Untreated
0.25
0.2
2.5
0.15
5
0.1
10
0.05
0
0'
10'
15'
20'
Time (min)
Figure 1: Piceatannol Cytotoxicity Assay.
Adhesion Assay:
25'
30'
35'
Page |8
NK cells not only use adhesion receptors to adhere to their target cells, but also use them
to migrate towards a target cell. Therefore, it was important to look at Syk’s role in adhesion
(Figure 2).
Piceatannol Adhesion Assay
1.4
1.2
1
NK Untreated
0.8
DMSO
0.6
2.5
0.4
5
0.2
0
Fibronectin
Poly-L-Lysine
Uncoated
Figure 2: Piceatannol Adhesion Assay. NK92MI cells treated with varying dosages of
Piceatannol (2.5μg/mL and 5μg/mL) and incubated on fibronectin, poly-L-lysine, or untreated
wells. These results were inconclusive and suggest that Piceatannol does not affect NK92MI cell
adhesion.
Conjugate Assay:
The Conjugate assay is a different way of visualizing the NK cell cytotoxicity. The
SYTOX Blue Dead Cell Stain measured the amount of dead and living cells (Figure 3).
Page |9
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
Dead
15
20
Time (min)
25
NK Alone
DMSO
10ug/ml
NK Alone
DMSO
10ug/ml
10
NK Alone
DMSO
10ug/ml
NK Alone
DMSO
10ug/ml
5
NK Alone
DMSO
10ug/ml
NK Alone
DMSO
10ug/ml
0
NK Alone
DMSO
10ug/ml
NK Alone
DMSO
10ug/ml
Alive
NK Alone
DMSO
10ug/ml
Cell Count
Piceatannol Conjugate Assay
30
45
60
Figure 3: Piceatannol Conjugate Assay. It is important to note that there are two meaningful
pieces of information in this graph. The overall number of conjugates does not change
significantly between the untreated cells and the Piceatannol treated cells, which indicates that
the NK cells retain the function to find and attack target cells. In contrast, the ratio of dead
conjugates versus alive conjugates is drastically different in Piceatannol treated cells. This leads
to the assumption that the role of Syk is not for attachment but has a role in signaling for lysing
the target cell. If the NK cell cannot lyse the target, they will remain attached indefinitely.
Syk shRNA Knockdown in NK92MI cells:
To further confirm the role of Syk in NK signaling and verify the inhibitor results, a
knockdown construct was created. Knockdowns provide away to investigate the role of a
specific protein without the off-target effects of drug treatments. Even though Piceatannol is a
very specific Syk inhibitor, it is important to generate knockdowns to verify these results.
Unfortunately, these knockdowns proved difficult to express in NK92MI cells and a stable cell
P a g e | 10
line was not created. Two different sequence specific shRNA Syk oligos were ligated into a
pSingle plasmid (Figure 4).
Uncut
Syk
1.5
Figure 4: Syk Ligation Verification. The pSingle plasmid was cut with Xho1 (Thermo fisher)
and HindIII (Thermo fisher) and ligated with two different Syk oligo sequences. To test the
ligation accuracy, a Mlu1 cut site is in the oligo sequence. The ligated plasmids were cut
overnight at 37°C with Mlu1 (Thermo fisher). One sequence results on a 0.8% Agarose gel are
shown. The pSingle uncut plasmid is shown in the middle and the Syk 1.5 ligated pSingle is
shown on the right. Supercoiling of the larger, cut plasmid caused it to run slower than the uncut
plasmid.
Chapter 4: Discussion
While the project is still ongoing, these data suggest a mechanism for the role of Syk.
After examining Figure 1, it is clear that there as the concentration of Piceatannol increases, the
cytotoxicity of the NK cells decreases. The largest dosage that resulted in the lowest cell lysis
(0.2) was 10g/ml. This indicates that the inhibition of Syk causes a significant decrease in the
NK cell’s ability to lyse target cells.
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In Figure 2, the adhesion assay is inconclusive. There was a wide variety of values
between the coating of the wells with fibronectin, poly-L-lysine, or uncoated and the different
dosages that seem reproducible. While it might seem like a technical error in the assay, further
data from a conjugate assay in figure 3 reveals a defined role for Syk signaling in NK cells. The
number of conjugates throughout the time points are consistent across all treatments. Therefore,
NK cells can locate and adhere to the target cells even without Syk signaling. This confirms the
results of the adhesion assay. If the NK cell still retains the ability to adhere to the target cell,
then the adhesion to fibronectin or poly-L-lysine in the adhesion assay would probably not show
any differences as well.
Also in Figure 3, the ratio of dead to living cells is interesting. This assay provides a
different way of viewing the killing assay. Instead of viewing the amount of dead cells at the end
of an incubation period, this assay looks at the conjugate formation and the ratio of dying cells to
living cells within the conjugate population (red-green events) by flow cytometry. SYTOX Blue
Dead Cell Stain (Life Technologies) is used to stain the dead or dying cells. The dye enters the
cell through perforations in the cell membrane and fluoresces on the BD FACS Aria. This
unique point of view shows the quantification and possible explanation of why the NK cell is not
able to lyse the target cell. In the untreated cells, the ratio begins with more alive than dead. As
time progresses, this changes to the majority of the conjugates are dead until it reaches a
threshold. In contrast, the Piceatannol treated cells increase the ratio of alive conjugates to dead
conjugates. This confirms our hypothesis that the NK cell needs Syk signaling in order to
activate and secrete lytic granules to the target cell, but does not require it for attachment. Until
the target cell is destroyed, the NK cell will remain adhered to the target cell. This creates the
increase in living conjugates that is illustrated in Figure 3.
P a g e | 12
Unfortunately, knockdowns could not be generated to test this hypothesis and explore the
mechanism further. Two stable constructs were created but they were difficult to express within
the cell line. There are several possible reasons for this happening. NK cells can be difficult to
transfect. The particular cells that tested did not handle the stress and eventually died. Another
reason is the conditions for transfection were not ideal. Therefore, we are continuing to optimize
the protocol by testing different parameters; such as, electroporation solution, nucleofector
programs, cell viability, time, and technique to create a stably transfected NK92MI cell line that
contains the plasmid. Also, if the plasmid was lost after transfection, then the cells would no
longer be resistant to the G418 selection media and the cell line would crash.
From these results, the role of Syk is still unknown. The results indicate that Syk does
not affect cell adhesion through LFA-1. Instead, Syk may function through NKG2D or LFA-1
for actin clearance and rearrangement of the cortex and microtubule recruitment and capture at
the lytic synapse. Interestingly, by microscopy, the lab has also observed the same phenotype of
NK attachment to the target cell without lysis when hDia1, a protein in the formin family, is
knocked down. hDia1 is necessary for microtubule capture at the lytic synapse for the
trafficking of lytic granules.7
Chapter 5: Future Directions
One of the main assays that I was unable to accomplish because of time constraints is the
immunoprecipitation (IP) assay. The main purpose behind this assay is to analyze the direct
interaction of certain proteins. One of the main mysteries in the NKG2D pathway is the
relationship of Syk to other downstream molecules and this assay might have provided insight
into this question. First, I would have precipitated Vav with Syk because they are known to
associate together. Next, I like to test a variety of other downstream molecules that are in the
P a g e | 13
LFA-1 pathway and NKG2D pathway to understand if actin rearrangement or microtubule
capture is being affected by Syk signaling in NK cytotoxicity. In order to investigate LFA-1 or
NKG2D alone, I would like to express the ligands, ICAM-1 or ULBP, individually on drosophila
S2 cells. Since drosophila-derived cells do not endogenously express any ligands for NK cells,
this would allow the engineering of a specific target cell. I would then be able to define whether
Syk signaling needs LFA-1, NKG2D or both.
To confirm the results of this paper, each assay performed in this thesis will need to be
repeated so that error bars can be generated. This will show that the mechanism suggested is in
this thesis is relevant and reproducible.
In addition to perfecting the current assays, these results should be tested using Syk
knockdowns. As stated previously, this data could be the result of an unknown target of the
inhibitor and could be not related to Syk at all. I would like to further trouble shoot the pSingle
vector system because it allows for stable cell lines to be generated without knocking down the
protein until it is induced. However, I would like to try using different methods of knockdowns
until the pSingle system is working like siRNA or the pSIREN-RetroQ-TetP system (Clontech).
Microscopy is another route to investigate the role of Syk. One of the first tasks is to
identify the localization of Syk, both phosphorylated and non-phosphorylated, at the lytic
synapse. Another method would be to visualize the knockdown cell line’s lytic synapse by Total
Internal Fluorescent Microscopy, TIRF, to look at actin rearrangement and microtubule capture.
It would be interesting to see if hDia1 is no longer able to capture microtubules without Syk as
they share similar phenotypes with inhibition or knockdowns.
P a g e | 14
Appendix
Figure 1: Piceatannol Cytotoxicity Assay....................................................................................... 7
Figure 2: Piceatannol Adhesion Assay ........................................................................................... 8
Figure 3: Piceatannol Conjugate Assay .......................................................................................... 9
Figure 4: Syk Ligation Verification .............................................................................................. 10
P a g e | 15
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