DONOR NATURAL KILLER CELLS AS VETO CELLS
A Project
Presented to the faculty of the Department of Biological Sciences
California State University, Sacramento
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF ARTS
in
Biological Sciences
(Stem Cell)
by
Ridhima Naidu
SPRING
2012
i
DONOR NATURAL KILLER CELLS AS VETO CELLS
A Project
by
Ridhima Naidu
Approved by:
_____________________________________, Committee Chair
Thomas Landerholm
_____________________________________, Second Reader
Christine Kirvan
_____________________________________, Third Reader
Jan Nolta
____________________
Date
ii
Student: Ridhima Naidu
I certify that this student has met the requirements for format contained in the
University format manual, and that this project is suitable for shelving in the Library
and credit is to be awarded for this project.
____________________________, Graduate Coordinator _________________
Ronald M. Coleman
Date
Department of Biological Sciences
iii
Abstract
of
DONOR NATURAL KILLER CELLS AS VETO CELLS
by
Ridhima Naidu
Stem cell therapies have a great potential for the treatment of diseases.
However, overcoming the host immune response to allogeneic stem cell grafts without
the need of a strong immunosuppression remains an elusive goal. The current method
for tolerance of grafts involves the use of systemic immunosuppression for the
remaining life of the patient. This method can potentially make the patient more
vulnerable to opportunistic infections and/or cancer. The removal of the
immunosuppression has been shown to place the transplanted graft at risk. Therefore, it
is essential for the recipient’s immune system to be insensitive to the donor’s Major
Histocompatibility Complex (MHC).
The immune system, specifically T cells play a major role in the rejection of the
donor stem cells. MHC class I are also the antigens that cause rejection of allogeneic
stem cells by host T cells. Previous studies have shown that activated NK cells could
be useful in donor-specific tolerance, such as that using donor bone marrow. Different
NK subsets could be used as clinical tools and may offer additional advantages when
used as potential veto cells when compared to whole NK cell populations. We have
applied this method in examining the tolerance of mouse embryonic stem cells
iv
(mESCs). It is seen that ES cells at different stages have different levels of MHC. ES
cells seem to be naturally immunosuppressive, giving it an added advantage. Since ES
cells have such great potential, it is important to understand its immunogenicity before
it can be applied as treatments. In this study we hypothesized that the activated donor
NK cells will act as “veto” cells by suppressing or deleting the host NK cells and
Cytotoxic T Lymphocytes (CTLs) to prevent reactivity against the mESCs. This can
potentially provide a strong immunosuppression post-transplant that is not limiting as
the current method of systemic immunosuppression.
B6 ALAKs were sorted for the separation of the NK subsets. Their functions
were examined using a chromium release assay. An inhibition assay showed noninhibitory Ly49 C/I+ subset function as more effective BALB/c suppressors compared
to the Ly49 G2/A+ and whole ALAK populations. The long term killing assay showed
at 72 hours, both NK and CD4+ T cells were susceptible to allogeneic killing by
activated NK cells. Imaging allowed for visual detection of the mESC in vivo. The
teratoma prevention further showed differential growth patterns between the subsets and
between the administration routes of NK cells.
________________________________, Committee Chair
Thomas Landerholm
________________________
Date
v
ACKNOWLEDGEMENTS
I would like to thank Dr. Thomas Peavy, Dr. Thomas Landerholm, and Dr.
Christine Kirvan for their support. I would also like to thank Dr. William Murphy for
his support and for opening up his lab to our program. This work was supported by the
California Institute for Regenerative Medicine (CIRM), the UC Davis Institute for
Regenerative Cures, and the CSUS Biological Sciences Department. I would also like
to say a special thank you to my parents for their limitless support throughout this time
that make all my efforts possible. Lastly, I would like to thank all of those who
supported me during this project.
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TABLE OF CONTENTS
Page
Acknowledgements……….………...………………………………………………...vi
List of Figures…….....……………………………………………………………….viii
INTRODUCTION……………………………………….……………………………1
METHODS...………………………………………………………………………...10
Mice.………………………………………………………………………….10
mESC Culture…..……………………………………………………………10
ALAK Cell Culture…..………………………………………………………11
Cell Sorting…………………..……………………………………………….11
Chromium Release Assay…………………………………………………….12
Inhibition Assay………………………………………………………………13
Flow-based Killing Assay…………………………………………………….13
Veto Mixed Leukocyte Reaction……………………………………………..14
IVIS Imaging………………………………………………………………....14
Teratoma Model…………………………………………………………… 15
RESULTS…………………..………………………………………………………...16
DISCUSSION………………………………………………………….……………..34
Literature Cited………………………….…………………………………………....38
vii
LIST OF FIGURES
Figures
Page
1.
Whole ALAKs and sorted NK subsets purity check.……..……………………..17
2.
51
Cr release assay of YAC-1 killing by B6 sorted subsets vs.
whole ALAKs cultured for 3 days post-sort.……………………………………18
3.
B6 Ly49 subset inhibition of BALB/c splenocytes.……….…………………….20
4.
Flow-based killing assay showing B6 ALAK vs. resting BALBc
NK cells and CD4+T cells...…………………………………………………….21
5.
NK veto T cells in MLR assay…….………….….……..……………………….23
6.
GFP expression check of Luc-GFP vector in mESCs.…….…………………….24
7.
IVIS imaging timeline…..………………………………...…………………......25
8.
IVIS imaging of mESCs in B6 mice.………..…………………………….…….26
9.
IVIS imaging of mESCs in BALBc mice…….………………………………….29
10.
Teratoma prevention model……..………..……………………………………..30
11.
Teratoma prevention….……………..…………………………………………..31
12.
mESCs, teratoma, and YAC-1 killing by B6 ALAKs……..…………………….33
viii
1
INTRODUCTION
The field of bone marrow transplantation arose from blood transfusions. The first
successful transplant was performed in the 1950s. It was then that research into the
physiology of the blood forming system led to the discovery that bone marrow, the origin
of all blood cells, must have a very special composition. From this, a unique cell was
discovered called a bone marrow or hematopoietic stem cell. Hematopoietic stem cell
transplantation (HSCT) was initially developed to treat individuals suffering from
radiation exposure (Welniak et al. 2007). With this procedure, it became possible to treat
patients with cancers such as multiple myeloma or leukemia (Hu et al., 2010, Hallet et al.,
2006). However, transplantation with bone marrow was shown to cause a secondary
complication now known as graft-versus-host-disease (GVHD) which develops when the
immune-competent cells of the graft, which are the incoming donor cells, recognize
major histocompatibility antigens and mount an immune attack against the cells in the
recipient (Hallet et al., 2006). The current method used to induce tolerance of a
transplant, including stem cells, from a non-identical, allogeneic donor is through the use
of systemic immunosuppression.
According to the Center for International Blood and Marrow Transplant Research,
there are approximately 25,000 allogeneic HSCT performed annually worldwide
(CIBMTR, 2012). Additionally, more than 28,000 solid organ transplants are performed
in the United States per year (Engels et al., 2011). There are two sources of stem cells
that can be applied in stem cell based therapies. Autologous stem cells are those that
come directly from the patient. There can also be the application of stem cells from a
2
donor, in which the Major Histocompatibility Complex (MHC) is matched
(haploidentical) or what is seen in majority of cases where the MHC is unmatched
(allogeneic). Unless the donor is an identical twin, perfect HLA-matching is difficult
with close relatives. Allogeneic bone marrow transplantation has transformed the
treatment of leukemia, lymphoma, and other hematologic malignancies (Hu et al., 2010).
Although the donor is closely matched with the recipient, there are still issues associated
with graft rejection. In order to prevent any sort of rejection, many patients are placed on
life-long immunosuppressant therapy, which can predispose the individual to infections
and cancer (Ruggeri et al., 2002, Trapani et al., 2002). Removing the
immunosuppressive therapy can place the graft at risk for rejection. It has been seen that
transplant followed by immunosuppressive therapy has been consistently associated with
reduced quality of life, poorer general health, and a reduced ability to function in the
workplace (Pidala et al., 2009). Thus, there is a definite need to overcome the
immunological hurdles associated with the current method of tolerance (Hu et al., 2010,
Reich-Zeliger et al., 2004).
Both innate and adaptive immune systems contribute to immediate and long term
rejection, respectively. Specifically, T cells play a major role in the rejection of the donor
stem cells (Ruggeri et al., 2002, Trapani et al., 2002, Reisner et al., 2007). T cells are
lymphocytes which are part of the adaptive immune system. The T cells function by
recognizing targets that present foreign antigen that is bound to the major
histocompatibility complex (MHC) molecule. Naïve T cells are activated when their Tcell receptor (TCR) interacts with a peptide-bound MHC molecule (Trapani et al., 2002).
3
The cytotoxic T lymphocytes (CTLs) destroy foreign tissues, which are the antigenic
source. The CTLs are responsible for graft rejection in allogeneic transplants. These
particular cells are known as CD8+, due to the presence of this unique receptor on the T
cells. Helper T cells, also known as CD4+, assist in the activation of other immune cells
including CTLs. The primary cause of GVHD is allo-reactive CD8+ T cells (Hallet et al.,
2006). The MHC molecules act as antigens in transplants and can initiate an immune
response which leads to the difficulties seen in transplant engraftment.
NK cells are large, granular lymphocytes that are part of the innate immune
system (Hu et al., 2010, Hallet et al., 2006, Trapani et al., 2002, Lanier, 2005). The
innate immune system provides immediate defense against foreign agents (Trapani et al.,
2002, Lanier, 2005). NK cells were first identified when lethally irradiated mice were
able to reject allogeneic bone marrow (Hallet et al., 2006). NK cells play a role in the
recognition and removal of tumors and viruses. They are called natural killers because
they are not antigen specific and they target cells by recognizing those that do not express
MHC class I, and also from the expression of stress signals through inhibitory and
activating receptors (Hu et al., 2010, Trapani et al., 2002, Lanier, 2005). In humans, all
cells express MHC class I with the exception of red blood cells. Cancer and virallyinfected cells have been shown to down-regulate the expression of MHC class I to avoid
detection by the adaptive immune system and represents a strategy to avoid recognition
by cytotoxic T cells. Thus, NK cells provide immunological protection against infectious
agents or cancer cells that evade adaptive immune system destruction (Koch et al., 2008,
Raulet et al., 2006). The main mechanism that NK cells utilized to eliminate target cells
4
is through the release of granzyme and perforin (Colucci et al., 2003). NK cells can also
mediate apoptosis through cell surface and soluble immune effector molecules such as
Fas Ligand (FasL) and TNF-related apoptosis inducing ligand (TRAIL) and NK cells
have shown to play a major role in antibody-dependent cellular cytotoxicity along with
macrophages (Welniak et al., 2007, Hu et al., 2010, Trapani et al., 2002).
NK cells are regulated by inhibitory and activating receptors recognizing MHC-I
and MHC-I-like molecules to prevent NK activation in the presence of self-cells. It has
been proposed that in order to prevent self-lysis, NK cells initially must undergo a
maturation process in the bone marrow termed “licensing” (Yokoyama et al., 2006).
During development, if an NK cell’s inhibitory receptors are able to bind self-MHC-I on
bone marrow stromal cells, the NK cell becomes “licensed to kill” so that in the event it
encounters cells with low or no self-MHC-I, such as during a viral infection, it can
recognize and destroy these cells. NK cells which lack inhibitory receptors for self-MHCI remain unlicensed and hypo-responsive (Colucci et al., 2003, Yokoyama et al., 2006).
Hence, they never become a danger to self.
In mice, the Ly49 receptor family plays a key role in the regulation of mouse NK
cell activation. The Ly49 proteins are expressed as disulfide linked homodimers, and
individual Ly49s are found on subsets of the total NK cell population (Sun et al., 2012,
Raziuddin et al., 1996, Raziuddin et al., 1998, Raziuddin et al., 2000). Most Ly49 are
inhibitory and recognize MHC-I H2 molecules. Ly49I was first described in the B6
mouse strain, and it possesses 93% amino acid homology to the B6 Ly49C protein. Ly49I
and Ly49C are collectively referred to as Ly49 C/I for the purposes of functional studies
5
(Lanier, 2005, Yokoyama et al., 2006). It has been reported that NK subsets with Ly49
molecules that bind to self MHC-I were the dominant subset responsible for bone marrow
rejection (Sun et al., 2012). Having highly activated NK cells can provide an added
advantage in attacking the host effector CTLs and NK cells. IL-2 is a cytokine that has
been used in previous studies to activate NK cells (Hu et al., 2010, Ruggeri et al., 2002,
Reich-Zeliger et al., 2004). Evidence from bone marrow transplant suggests that
activated donor NK cells can suppress the recipient’s immune response. Previous studies
have shown that activated NK cells could be useful to induce donor-specific tolerance of
donor bone marrow (Hu et al., 2010, Reich-Zeliger et al., 2004). We had proposed to sort
NK cells for subsets with receptors that will not be inhibited by the host MHC-I and
therefore have maximal killing potential. Different subsets could be used as clinical tools
and may offer additional advantages when used as potential veto cells when compared to
whole NK cell populations.
The regular functions of CTLs make them problematic in a transplant situation.
During GVHD, donor T cells recognize the recipient as foreign by the major
histocompatibility differences and then attack the recipient tissues (Hallet et al., 2006).
For this reason, T cells in the past have been removed from the transplant to prevent
GVHD, but this also results in a higher chance of graft rejection (Hallet et al., 2006). The
advantages of NK cells are being easily in-vitro expanded, highly lytic, and they
preferentially attack cells of hematopoietic origin (Reich-Zeliger et al., 2004, Asai et al.,
1998, Miller et al., 1980). The CTLs are the classical veto cells (Miller et al., 1980).
Veto activity was stated as the capacity to specifically suppress CTL precursors directed
6
against antigens of the veto cells themselves but not against third party antigens (Miller et
al., 1980). Previous studies have shown that suppression by CTLs and NK cells is
mediated through apoptosis (Reich-Zeliger et al., 2004). Studies have shown veto CTLs
target not only recipient CTL effectors but also other recipient mature and memory T
cells and NK cells, both in vitro and in vivo (Hu et al., 2010, Reich-Zeliger et al., 2004).
Furthermore, cells other than CTL were shown to act as veto cells, with cytokineactivated NK cells exhibiting the second strongest veto activity after CTL in vitro (ReichZeliger et al., 2004). NK cells show promise in acceptance of stem cell transplants in
recipients. NK cells can possibly be a novel targeted immunosuppression that can
maintain the integrity of the transplant and improve the quality of life.
NK cells may hold the key for cancer therapy that may develop following a
transplant. During follow-up, more than 10,000 cases of cancer occurred among
transplant recipients, roughly twice what would be expected in the general population
(Engels et al., 2011). Teratomas are tumors that contain tissues of ectodermal,
mesodermal, and endodermal origin (Dressel et al., 2010). Pluripotent stem cells, which
have the potential to differentiate into all tissue types, also have been known to form
teratomas (Koestenbauer et al., 2006). This indicates the close relationship of
pluripotency and tumorigenicity in pluripotent stem cells. Therefore, it is not surprising
that the risk of tumor formation is among the major hurdles that must be overcome before
implementation of pluripotent stem cells into clinical practice (Dressel et al., 2010).
Pluripotent cells are unlikely to be used directly in regenerative medicine. However, all
grafts that are derived from pluripotent stem cells are in principle at risk of containing
7
teratoma forming cells. The cytotoxic activity of NK cells is not only controlled by
inhibitory receptors recognizing MHC class I molecules, but also by a diverse set of
activating receptors that recognize specific ligands on targets (Dressel, 2011). The
recognition of these ligands by NK cells is known to trigger cytotoxicity against such
target cells.
Another class of MHC class I-specific receptors expressed in both humans and
mice includes the C-type lectin molecule CD94, which is associated with a member of
the NKG2 family (Hallet et al., 2006). NKG2D receptor on NK cells has been shown to
exert activating signals (Hallet et al., 2006). Normally, NKG2D ligands are not
expressed on healthy cells but they can become induced by conditions such as heat shock,
virus infection, or toxic stress. The NKG2D ligands appear to signal the presence of
potentially dangerous cells to the immune system and they contribute to tumor immune
surveillance (Dressel, 2011). Embryonic stem cells (ESCs) and teratoma cells have been
shown to express high levels of NKG2D stress ligands and relatively low levels of MHCI and therefore are good targets for NK cells (Dressel, 2011). NK cells may be
advantageous if they can preferentially kill tumorigenic cells, which may contaminate the
grafts.
The potential of pluripotent stem cells to repair the damaged tissues holds great
promise in development of novel cell replacement therapeutics for treating various
degenerative diseases. However, previous reports show that stem cell therapy in
autologous and allogeneic settings, trigger immune response (Koch et al., 2008, Dressel
et al., 2010, Koestenbauer et al., 2006). Therefore, an important issue that still needs to
8
be addressed is how the recipient immune system responds to engrafted stem cells. It is
seen that ESCs at different stages have different level of MHC. ESCs are pluripotent
stem cells derived from the inner mass cells of blastocytes (Shiraki et al., 2008, Li et al.,
2010). They have the potential to differentiate into many different cell types, including
all three germ layers (Koestenbauer et al., 2006, Shiraki et al., 2008, Iwamuro et al.,
2010). There is evidence from previous studies that show that murine ESCs do not
produce MHC class I or MHC class II antigens (Koch et al., 2008). This characteristic
changes, as the tissue differentiates so does the expression of MHC. ESCs are also
naturally seen to be immunosuppressive, which gives it an added advantage (Koch et al.,
2008). The need for specific donor suppression may vary due to the degree of
differentiation in the donor graft stem cells. Mouse embryonic stem cells (mESCs) will
be tested for susceptibility to killing by syngeneic or allogeneic effector CTLs or NK
cells. Since ESCs still need further understanding, it is important to understand the
immunogenicity of them (Dressel et al., 2010). This relationship will be examined invitro and can be further applied to in-vivo studies using the mouse model. The murine
studies of ESCs can provide us with the groundwork to apply to human embryonic stem
cells (hESCs). The presence of activated donor NK cells will suppress the ESC killing by
directly suppressing the recipient NK cells and recipient CTLs. This can potentially
provide a strong immunosuppression post-transplant without the negative side effects
seen by the current method of systemic immunosuppression. By selectively only deleting
those immune cells of the recipient that will reject the graft, we will leave/maintain the
9
rest of the immune system intact and therefore reduce the chance for opportunistic
infections and cancer relapse.
Therefore, we hypothesize that the activated licensed donor-type NK cells will act
as “veto” cells by suppressing or deleting the recipient alloreactive cells against the
mESCs. This method may also be used to examine the tolerance of BMT with NK as veto
cells in examining the tolerance of mESCs. This study can not only be applied to stem
cell therapies but also to whole organ transplants and will help to expand our knowledge
of NK cells and tolerance induction. NK cells will play the role of attacking the attackers
to allow the proper grafting by the stem cells. It is still not clearly understood what
occurs to prevent the proper grafting of stem cells. Thus, it is necessary to investigate the
role of the immune cells and how they view the incoming donor stem cells. Given this
information, donor NK may be used to selectively suppress donor-specific recipient
immune cells and further become a useful therapy in transplantation medicine.
10
METHODS
Mice
All animal protocols were approved by the UC Davis Animal Care and Use
Committees. Female BALB/c (H2ᵈ) and C57BL/6 (B6, H2ᵇ) mice were obtained from
the Animal Production Area, National Cancer Institute (Frederick, MD). Female BALB/c
SCID mice were purchased from Jackson Laboratory (Sacramento, CA). All mice were
kept under specific pathogen-free conditions until use at least 8 weeks of age.
mESC Culture
Mouse embryonic stem cells (JM8.A) are derived from C57BL/6N mice. The
sub-lines derived from the JM8 parental line is considered to be feeder independent. The
JM8A3.N1 mES cells were transduced with GFP-luc construct pCCLc-MNDU3-LUCPGK-EGFP-WPRE kindly provided by the Nolta laboratory. The cells were cultured on
0.1% Gelatin (Sigma, St. Louis, MO) coated culture dish with JM8.A ES cell medium
used consisted of Knockout Dulbecco’s Modified Eagle Medium (KO DMEM, Gibco,
Carlsbad, CA), Fetal Bovine Serum (FBS) (Hyclone), GlutaMax (Gibco), Non-Essential
Amino Acids (Gibco), Leukemia Inhibitory Factor (LIF, Gibco), and 2-(β)
Mercaptoenthanol (Sigma). The mESCs were maintained at less than 80% confluency
and the medium was changed daily.
11
ALAK Cell Culture
Adherent lymphokine activated killer cells are a subpopulation of activated NK
cells. These were isolated from the spleens and bone marrow from the femur, tibia, and
spine of C57BL/6 or BALB/c mice. Single cell suspensions were prepared from the
spleens and bone marrow by crushing the organs either using the top portion of a 3 ml
syringe or mortar and pestle for the latter followed by filtering the suspension through a
sterile mesh to remove the debris. Red blood cells were lysed with Tris-Buffered
Ammonium Chloride (ACT) buffer. T cells were depleted using the anti-Thy1.2 (clone
30H12) and rabbit complement (Cedarlane Low Tox-M CL3051.) The remaining cells
were cultured in RF-10 complete media with 1000 international units (IU) /mL of
recombinant human Interleukin-2 (rh IL-2) to activate the cells. Cell activation was
determined by the presence of cell surface marker Thy1.2 (CD90). The culture media
(cRF-10) used consisted of Roswell Memorial Park Institute (RPMI) 1640 medium w/out
glutamine (Gibco), Hepes (HyClone), FBS (Gibco), Glutamine (Gibco), Non-essential
amino acids (Gibco), Sodium Pyruvate (Gibco), Penicillin/Streptomycin (Gibco), and 2mercaptoethanol (Gibco). On day 3 or 4, cells were split and recultured in 50%
conditioned media and 50% fresh media with a fresh dose of 1000 IU/mL rhIL-2. ALAK
cells were harvested on day 6 or 7.
Cell Sorting
ALAK cells were harvested either day 6 or 7. Cells were counted and washed
with staining buffer. Cells were incubated with Fc Block (BD Biosciences, San Jose,
12
CA) to prevent any non-specific binding for 10 minutes at 4oC. Cells were then stained
with CD45 (Pacific Blue) and CD3 (PC7) (BioLegend, San Diego, CA). CD122 (biotin),
Ly49 G2 (FITC), Ly49 A (FITC), Ly49 C/I (PE) (BD Bioscieces) and incubated for 20
minutes at 4o C. The cells were washed and were incubated with Streptavidan-APC (BD
Biosciences) as a secondary step for biotin for 10 minutes at 4 oC. The cells received a
final wash and were resuspended in staining buffer at 8x106 cells / mL. The cells were
filtered through meshed covered tubes and sorted on a FACS Aria II into non-inhibitory
C/I positive and inhibitory G2/A positive subsets. Each subset and whole ALAKs were
counted using a hemacytometer and were cultured in flasks with 1000 IU/mL rh-IL2 for 3
days for expansion of the populations. Purity checks for the populations were conducted
pre and post sort on the Fortessa Flow Cytometer.
Chromium Release Assay
NK cell activity was determined by using a chromium release assay with
responding cells. The effector cells were titrated onto a 96-well round-bottom plate.
Target cells were labeled with 51Cr at 100 µCi of 51Cr/106 cells for 1 hour. They were
then washed with PBS and incubated in cRF-10 medium for 20 minutes to reduce
nonspecific background. The target cells were washed again and plated at 104 cells with
titrated NK effectors starting at 105 cells for 4 hours. After incubation, the plates were
placed in centrifuge carriers and spun at 1200 rpm for 5-10 minutes. Then the Optiphase
Supermix scintillation fluid was added to each well of the Wallac 96 well sample plate.
100 µl of supernatant was then removed from each well, taking care not to disturb the
13
pellet. Supernatants was placed in the sample plate and mixed with the scintillation fluid.
The sample plate was then sealed with a plastic sealer and placed in a Wallac plate frame
for counting. The killing capacity was measured and calculated based on the level of
specific chromium release in the supernatant.
% specific cytotoxicity = experimental cpm – background release cpm x 100
total release cpm – background release cpm
Inhibition Assay
ALAKs were harvested and sorted on Day 6 or 7. The ALAKs were then
expanded and used in the assay day 3 after sorting. BALB/c splenocytes in a single cell
suspension were plated in a flat-bottom 96-well plate at 2.5x10 5, 5x10 5, or 7.5x10 5
cells/well with 1, 2.5, or 5 µg/ml Concanavalin A (ConA). Plates were incubated with a
titration of sorted subsets or whole ALAKs starting at a 1:1 ratio. On day 2 or 3, 1 µCi
Thymidine/well was added and incubated for 16-18 hours before harvest with a Tomtec
harvester. After drying for 24 hours, it was read on a Wallac counter.
Flow-based Killing Assay
The Effector B6 ALAK cells were prepared as above and harvested on day 7.
Resting BALB/c NK cells were purified using the NK Enrichment Negative Selection Kit
(STEMCELL, BC, Canada). BALB/c CD4+ T cells were purified using T Cell
Enrichment Kit (STEMCELL). The cells were washed in PBS and labeled with 5 µM/mL
Carboxyfluorescein succinimidyl ester (CFSE Invitrogen) at 5x10 6 cells/ mL at 37o C in
a waterbath for 10 minutes. They were then washed twice with 20% FBS (Gibco) in PBS
14
to neutralize. Effectors were serially diluted in triplicates in 96-well round bottom plates.
The labeled targets were added with 100 IU/mL of rh-IL2. The plates were incubated for
24, 48, or 72 hours. On the harvest day for each plate, the plates were gently vortexed to
disperse the pellets and 3 µl of 7-Amino-actinomycin D (7-AAD, BD Biosciences) was
added per well to stain the dead cells. The assay plate acquisition was performed on the
Fortessa Flow Cytometer 96-well plate reader.
Veto Mixed Leukocyte Reaction (MLR)
To investigate the effect of the allogeneic NK cells as suppressors of alloresponses responder BALB/c spleen cells, were made into single cell suspension. The
stimulator cells were prepared from C57BL/6 spleen cells in a single cell suspension and
irradiated at 850 cGy. B6 whole ALAKs or sorted subsets were titrated starting from a
10:1 ratio. BALB/c spleen cells and irradiated B6 spleen cells were plated in 96-well
round-bottomed plates in triplicates at 7x105, 5x105, 2.5x105, and 1.25x105 cells/well in
cRF-10 media. The plates were incubated for 2-5 days, and then pulsed with 3Hthymidine, to measure the proliferation of the responders (BALB/c), for 16 to 18 hours
prior to harvesting and counting.
IVIS Imaging
The mice were pre-treated with anti-ASGM-1 (Wako Chemicals, Richmond, VA)
to remove the host NK cells. The test groups were also either irradiated with 850 cGy
total body irradiation (TBI) or no irradiation. The luciferase-GFP transduced JM8A3.N1
15
mESCs were harvested, titrated at 1x106, 5x106 and 10x106 and injected intravenously
(i.v.). Before imaging on each day, the mice received an intraperitoneal (i.p.) injection of
Synthetic D-luciferin (Biosynth, Naperville, IL). The mice were then imaged on various
days using the IVIS 100 imaging system (Xenogen, Alameda, CA).
Teratoma Model
At two days prior, the appropriate SCID groups were given Poly I: C (BD
Biosciences), with/without 4D1 (BD Biosciences) to deplete the G2/A+ subset or 5E6
(BD Biosciences) to deplete the C/I+ subset or anti-ASGM-1 to deplete whole NK
population. On day 0, the mice were given a subcutaneous injection of 1x106 mESCs into
the right flank or an intravenous injection of 20x106 mESCs. Some groups were given
i.p. 50,000 IU/ mL of rhIL-2. On days 1 and 2 those groups received another 50,000
IU/mL i.p. injection of rhIL-2. On day 3, 6, 10, and 17 the subsets and ASGM-1
depletion was repeated with/without Poly I: C. The tumor growth was monitored every
other day by palpation and the size was recorded using a dial caliper. The tumor size was
calculated by measuring the length and width.
16
RESULTS
NK cell sorting and subset purification. B6 ALAKs were prepared and ready for
use on day seven. The adherent cells were harvested from the flasks and shaken
vigorously. The cells were stained with CD45, CD3, CD122, Ly49 G2, Ly49 A, and
Ly49 C/I. Using a sorter, the stained cells were then separated into the non-inhibitory
Ly49 C/I and inhibitory Ly49 G2/A subsets. Due to the low number of cells collected
from sorting, the cells were placed in a three day culture for enrichment. Our lab has
previously determined that NK cells in culture are viable for up to two weeks, therefore
our culture time post sort is limited. The Ly49 C/I subset has been demonstrated to
recognize H2b (B6) MHC-I and therefore to inhibit self-lysis in B6 mice, but mediate the
rejection of H2d (BALB/c) allografts. Ly49 G2 and Ly49 A subsets recognize H2d
(BALB/c) MHC-I. When looking at the whole NK population in Figure 1a prior to
sorting, there is approximately 15% of C/I subset and 40% of the G2/A subset present.
Directly after the sort, there was greater than 95% purity of each population present as
seen in Figure 1b. A purity check after a three day culture post sort shows each
population maintained its phenotype. The C/I subset showed purity greater than 95% and
G2/A showed purity greater than 93% as seen in Figure 1c.
The next step was to determine whether the ALAKs maintained their functionality
using a chromium release assay. B6 ALAKs were sorted according to inhibitory and
non-inhibitory Ly49 subsets (G2/A and C/I) and cultured for three more days. Since
0
0
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10
3
10
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anti-Ly49G2 FITC
20110915 APsortpuricheck A55.jo
Layout
A
17
anti-Ly49C/I PE
Whole ALAKs pre-sort
10
5
10
4
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10
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14.8
13.1
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32.6
20110915 APsortpuricheck A55.jo
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Ly49C/I+ sorted cells
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0.206
4
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13.1
10
2
93.4
10
3
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5
anti-Ly49G2 FITC
Figure 1. Whole ALAKs and sorted NK subsets purity check. The cells were stained
with CD45-PB, CD3-PC7, CD122-biotin + SA-APC, Ly49 G2, A (both FITC), C/I-PE
32.6 into C/I and G2/A39.4
antibodies and
sorted on a FACS Aria
subsets. (A) Whole(FlowJo
NKv9.4.3)
purity
3/29/12 4:16 PM
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check before the sort shows <15% Ly49 C/I subset and <40% Ly49 G2/A subset. (B)
Each subset had a purity >95% directly post-sort.
(C) Ly49 C/I subset maintained its
anti-Ly49G2 FITC
phenotype post three day culture maintaining purity >95%. Ly49 G2/A subset also
maintained its phenotype post culture with a purity >93%.
2
0
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ort
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C
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Ly49G2+ sorted cells
2
1.75
0
20110915 APsortpuricheck A55.jo
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Ly49C/I+ sorted
(FlowJo v9.4.3)
0.297
anti-Ly49C/I PE
B
.297
0
0
3.27
10
2
18
Figure 2. 51Cr release assay of YAC-1 killing by B6 sorted subsets vs. whole ALAKs
cultured for 3 days post-sort. YAC-1 target cells were labeled with 51Cr and incubated
for 4 hours with effectors either activated whole ALAKs, Ly49 C/I subset, or Ly49 G2/A
subset at 10:1 effector to target ratio (E:T). After 4 hours, supernatant was isolated and
counts were measured using a gamma counter. Results are displayed as percent specific
lysis calculated as follows:
[(experimental lysis – spontaneous lysis) / (maximum lysis – spontaneous lysis)] x 100.
Statistical analysis was performed using ANOVA analysis. No significant differences are
shown. Error bars represent standard error of the mean.
19
ALAKs viability is limited in culture, it was important to test the NK function post sort
before placing cells in any assay. The chromium release assay (Figure 2) shows they are
still functionally effective after 10 days of being in culture. There are no significant
differences between subsets and whole ALAK groups in their ability to kill YAC-1 cells,
a T cell lymphoma cell line which is a gold standard in determining NK cell cytotoxic
function.
The next step was to show differential functional capabilities of the subsets. This
would be seen by the suppression of allogeneic (BALBc) splenocytes that were activated
by Concavalin A (Con A). BALBc (H2d) splenocytes inhibit Ly49 G2/A, but not Ly49
C/I bearing NK cells. We hypothesized that the activated sorted Ly49 C/I subset should
have a stronger inhibitory effect than the Ly49 G2/A subset on Con A induced H2d T cell
proliferation. ALAKs were cultured and stained to be sorted on a FACS Aria into Ly49
C/I positive and Ly49 G2/A positive subsets. The two day and three day assay, seen in
Figure 3a and 3b, with 2.5x105 BALBc splenocytes + 2.5 µg/mL Con A show noninhibitory Ly49 C/I subset function as more effective suppressor cells compared to the
Ly49 G2/A and whole ALAK populations.
A flow based killing assay was used to determine if NK cells can target NK cells
in the setting of activated allogeneic NK cells versus resting NK. This has important
implications with respect to potential veto effects towards allogeneic NK cells as well as
for further subset studies. Activated NK cells should be able to be the most efficient
killer cell in an allogeneic setting with inactivated NK cells since they are activated to kill
20
A
B
Figure 3. B6 Ly49 subset inhibition of BALB/c splenocytes. BALB/c splenocytes were
placed in assay with 2.5 µg/mL ConA for T cell stimulation. (A) BALB/c splenocytes
were cultured with non-inhibitory Ly49 C/I subset or inhibitory Ly49 G2/A subset for
two days at a 1:1 NK: Responder (BALB/c) ratio. (B) BALB/c splentocytes were
cultured with non-inhibitory Ly49 C/I subset or inhibitory Ly49 G2/A subset for three
days at a 1:1 NK: Responder (BALB/c) ratio. On day two or three, 1 µCi 3H/well was
added and incubated for 16-18 hours before harvesting with a Tomtec harvester and
counts were measured using a gamma counter. Statistical analysis was performed using
ANOVA analysis. Error bars represent standard error of the mean. *p<0.05, ***p<0.001.
21
A
B
C
Figure 4. Flow-based killing assay showing B6 ALAK vs. resting BALBc
NK cells and CD4+T cells. BALB/c CD4+ T Cells or resting NK target cells were CFSE
stained and cultured for one day (A), two days (B), or three days (C) with highly
activated B6 NK cells beginning at a 10:1 effector to target ratio (E:T). Cells were
harvested and stained with the cell death marker 7-amino-actinomycin D (7-AAD) and
analyzed with a FACs flow cytometer. Statistical analysis was performed using ANOVA
analysis. Error bars represent standard error of the mean.
22
faster and more efficiently and a longer incubation time may allow death ligand-mediated
killing to occur. Allogeneic T cells should be an NK cell target as well. No killing of
either BALB/c resting NK or CD4+ T cells was seen in the 24 or 48 hour assay (Figure
4a and 4b). Looking at % CFSE+7-AAD- (live) cells at 72 hours, both NK and CD4+ T
cells showed susceptibility to allogeneic killing by activated NK cells with the highest
killing at the highest E:T (10:1).
The MLR assay was performed to determine if H2b veto ALAK can specifically
kill H2d splenocytes. Allogeneic ALAKs should suppress proliferation due to a veto
effect on attacking T cells. The BALB/c splenocytes were cultured with irradiated B6
splenocytes to initiate a response. This assay measures the suppression effects of the B6
ALAKs on the proliferation of BALB/c cells. Figure 5 shows the Ly49 C/I + subset exert
significant suppressive effects on the thymidine incorporation of responders (H2d
BALB/c) compared to both Ly49 G2/A+ subset and whole ALAKs. However, controls
show that the baseline MLR did not work.
In order to obtain in-vivo data, the mESCs were examined for the expression of
GFP. The Luciferase-GFP transduced JM8A3.N1 ESCs were analyzed by flow
cytometry to determine the GFP expression. As expected, in Figure 6 the normal there
was less than 1% of mESCs that were GFP+. More than 90% of live cells that were
transfected were shown to express GFP.
Once GFP expression was confirmed, the mESCs were used for imaging as
explained in Figure 7. The study was performed to determine if luciferase-transduced
JM8A3.N1 (B6) mouse ESCs could be visualized with the IVIS imaging system.
23
Figure 5. NK veto T cells in MLR assay. Activated B6 whole NK cells, Ly49 C/I, or
Ly49 G2/A subsets were cultured in a 4 day MLR assay with irradiated B6 splenocytes as
stimulators and BALB/c splenocytes as responders. On day 4, 1 µCi 3H/well was added
and incubated for 16-18 hours before harvesting with a Tomtec harvester and counts were
measured using a gamma counter. Statistical analysis was performed using ANOVA
analysis. Error bars represent standard error of the mean. *p< 0.05.
20110820 IVISpreinjectionA48.jo
Layou
24
SSC-A
Normal mESC Control
GFP-luc mESC
10
5
105
104
104
0.886
103
2
102
0
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2
3
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91.4
103
4
10
5
0
10
2
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10
5
FITC-A
Figure 6. GFP expression check of Luc-GFP vector in mESCs. Luciferase-GFP
transduced JM8A3.N1 ESCs were analyzed by flow cytometry to determine GFP
expression. In the control, <1% of mESC show GFP expression. More than 90% of live
cells were shown to express GFP.
3/29/12 5:14 PM
Page 1 of 1
(FlowJo v9.4.3
25
Deplete groups 2-4
with anti-ASGM-1
B6 or
BALBc
Mice
-2
0
Transfer animals;
inject 3mg/mouse
in 200ul i.p.
luciferin and image
using IVIS
1
2
3
4-10
Days
Irradiate with 950 cGY TBI
and inject 1x106, 5x106, or
10x106 mESCs i.v.
Figure 7. IVIS imaging timeline. The mice were pre-treated with anti-ASGM-1 on day 2. The test groups were also either irradiated with 950 cGy total body irradiation (TBI)
or no irradiation. The luciferase-GFP transduced JM8A3.N1 mESCs were harvested,
titrated at 1x106, 5x106 and 10x106 and injected intravenously (i.v.). Before imaging on
each day, the mice received an intraperitoneal (i.p.) injection of Synthetic D-luciferin.
The mice were then imaged on days 1-4 using the IVIS 100 imaging system.
26
A
27
B
Figure 8. IVIS imaging of mESCs in B6 mice. B6 mice were NK depleted on day -2,
followed by 950 cGY TBI on day 0. All groups were transferred to the imaging center on
day 1 and imaged using 3 mg/mouse in 200 µl i.p. D- luciferin. (A) Shows a dorsal view
of mice that received 5x106 million mESCs. (B) Shows a ventral view of mice that
received 10x106 million mESCs.
28
Syngeneic, irradiated, NK-depleted mice were expected to mount minimal
rejection to B6-derived ESCs. This environment provides the best chance to visualize the
mESCs if these cells survived the injection process and remain in the circulation. There
were four groups of two mice that all received treatments shown in Figure 7. No
luciferin signal was detectable in the mice.
This study was repeated in BALB/c mice. Both irradiated and non-irradiated
animals showed a stronger decrease in mESC signal in the NK-replete group compared to
the NK-depleted group on day 1. While the difference was visible on both day 0 and day
1 in the irradiated animals, it was only apparent on day 0 in the non-irradiated animals
(Figure 9a). Figure 9b shows 1x106 mESCs given to BALBc mice with TBI.
Subcutaneous injection of 1x106 gave a clearly visible signal. Although there was a low
number of mESCs injected, cell survival may have been stronger due to the localized
injection site. However, there was no clear trend in NK-depleted in comparison to undepleted animals. One mouse from the NK depleted group showed no signal for mESCs
immediately after being given the injection. mESCs were seen on day one, but the no
mESCs were visible again until day 10.
Embryonic stem cells and teratomas express high levels of NKG2D ligands,
relatively low level of MHC I, and therefore function as good targets for NK cells. The
mouse in-vivo study will determined the best route and dose of NK and cytokine
administration to prevent or treat allogeneic teratoma. The treatment schedule was as
seen in Figure 10. The teratoma growth varied between treatment groups. Teratoma
29
TBI
No TBI
AA
3 hrs
+ anti-ASGM-1
-
+ anti-ASGM-1
-
Day 1
+ anti-ASGM-1
-
+ anti-ASGM-1
-
B
3 hrs
+ anti-ASGM-1
-
Day 1
Day 7
Day 10
+ anti-ASGM-1
-
+ anti-ASGM-1
-
+ anti-ASGM-1
-
Figure 9. IVIS imaging of mESCs in BALBc mice. (A) Luciferase-transduced
JM8A3.N1 (B6) mESCs in-vivo with the IVIS imaging system, comparing NK-depleted
versus non-depleted and TBI versus non-irradiated BALBc mice using the model
described in Figure 7. Mice received 10x106 mESCs i.v. (B) shows mice receiving 1x106
mESC s.c with 950 cGy TBI imaged at 3 hours, day 1, day 7, and day 10 post injection.
30
Group 5: ASGM-1.
Groups 6,9,10: Poly I:C
Groups 7,9: anti-5E6
Groups 8,10: anti-4D11
SCID
Mice
-2
0
Groups 3,4:
50,000
IU/mL rhIL-2 i.p.
1
Groups 2,5-10: 1x106 mESC s.c.
Group 3: 1x106 mESC + 1x106 B6
ALAK s.c.
Group 4: 2x107 mESC i.v.
Groups 3, 4: 50,000 IU/mL rhIL-2
i.p
2
3
6
10
17
Days
Repeat subset,
ASGM-1
depletion,
and Poly I:C
Figure 10. Teratoma prevention model. At day -2 the appropriate SCID groups were
given Poly I: C, with/without anti-4D11 to deplete the G2/A+ subset or anti-5E6 to
deplete the C/I+ subset or anti-ASGM-1 to deplete whole NK population. On day 0, the
mice were given a subcutaneous injection of 1x106 mESCs into the right flank or an
intravenous injection of 20x106 mESCs. Some groups were given i.p. 50,000 IU/ mL of
rhIL-2. On days 1 and 2 those groups received another 50,000 IU/mL i.p. injection of
rhIL-2. On day 3, 6, 10, and 17 the subsets and ASGM-1 depletion was repeated
with/without Poly I: C. The tumor growth was monitored every other day by palpation
and the size was recorded using a dial caliper. The tumor size was calculated by
measuring the length and width.
31
Figure 11. Teratoma prevention. Teratoma growth shown using 1x106 mESC s.c. in the
right flank or 20x106 mESCs i.v. Growth was examined given the various treatment
groups described in figure 10. Tumor size was calculated by measuring the length and
width of the tumor. No significant differences are seen between groups.
32
growth was seen in all but one of the treatment groups. The group that received a
subcutaneous injection of NK cells and rhIL-2 with the mESCs showed no teratoma
growth (Figure 11). As expected, a higher teratoma growth was seen in the C/I depleted
treatment group compared to the G2/A depleted treatment group.
In order to examine the ALAKs differential killing capabilities, JM8A3.N1 teratoma
cells, JM8A3.N1 ESCs, and YAC-1 cells were labeled with chromium and measured for
percent lysis after incubation with ALAKs. Poor labeling of the teratoma cells is seen
because there was no evident lysis of the teratoma cells by the B6 ALAKs. The teratoma
spontaneous release wells had an average of 38%. This was higher than what was seen in
some of the test wells with the effector ALAKs, resulting in negative percent lysis. The
percent lysis for the YAC-1 and ESCs was between 25-30% (Figure 12).
33
Figure 12. mESCs, teratoma, and YAC-1 killing by B6 ALAKs. YAC-1, mESC, and
teratoma target cells were labeled with 51Cr and incubated for 4 hours with B6 ALAKs as
effectors at a 10:1 effector to target ratio (E:T). After 4 hours, supernatant was isolated
and counts were measured using a gamma counter. Results are displayed as percent
specific lysis calculated as follows:
[(experimental lysis – spontaneous lysis)/(maximum lysis – spontaneous lysis)] x 100.
Statistical analysis was performed using ANOVA analysis. No significant differences are
shown. Error bars represent standard error of the mean.
34
DISCUSSION
This project involved ex vivo activation and killing potential of NK cells and
CTLs as a basis for future use of these cells to veto stem cell rejection. In this study it
was shown that NK cells can be easily isolated from the spleens and bone marrow of
mice. These cells were also activated using cytokine recombinant human Interleukin-2
(rh IL-2) so that they can be used as potent effectors. It was determined through these
studies that NK cells maintain their effector functions. ALAKs were also successfully
sorted using a FACS sorter into the inhibitory and non-inhibitory subsets. It was seen
that these cells maintained their phenotype and functionality after a three day culture post
sorting, which was essential for use in both in vitro and in vivo experiments.
NK cell sorting yields over 90% purity for each subset post sort. High purity
yields for each subset can allow us to distinguish the functional difference between the
subsets. Although the purity was maintained, there were still a low number of cells that
collected. Even with enrichment for three days, there were less than 10x106 Ly49 G2/A
and 5x106 Ly49 C/I subsets on average. Another technique that can be utilized to collect
subsets is to in vivo subset deplete B6 mice to collect each subset. This can allow for
further testing in the in vivo models with NK subsets.
NK subset differential suppression effects were examined with an inhibition
assay. In the initial two day assay, the sorted subsets were cultured at1.5x106 cells/mL
for three days for cell enrichment before being placed in the assay. The Ly49 C/I subset
in the assay was deteriorating as evident by phenotype. It is possible that the cell density
during the post sort enrichment culture was too concentrated. The sorted cells in the
35
three day inhibition assay were cultured for enrichment at 0.5x106 cells/mL. As expected,
the non-inhibitory Ly49 C/I subset is able to suppress H2d proliferation more efficiently.
Since media was depleted, conditions were not ideal to show maximal proliferation
differences that subsets may have caused. Repeating at a lower ConA concentration or
for shorter amount of time may yield better results.
Although the primary mechanism examined to be responsible for NK cytotoxic
activities are through perforin/granzyme, there are other long term killing mechanisms
that can be examined using a flow based killing assay. A flow based assay can determine
if NK cells can target NK cells in the setting of activated allogeneic NK cells versus
resting NK. This assay has important implications with respect to potential veto effects
towards allogeneic NK cells as well as for further subset studies. BALB/c resting NK
and CD4+ T cells susceptibility to killing by activated B6 ALAKs were not seen until 72
hours. Also, there was a high cell death seen in the control wells with no effector cells.
This may have been due to high numbers of cells within the wells or to a low amount of
rhIL-2 in the assay. Higher dose of rhIL-2 needs to be examined. 100 IU/mL rhIL-2 is a
low dose; it’s possible that T cells will outcompete NK cells for the cytokine due to
CD25 affinity for IL-2. CD25 is the alpha chain of the IL-2 receptor, which is present on
a number of immune cells including T cells and can act as a high affinity receptor for IL2. Therefore, this assay needs to be repeated for a longer time period and with a higher
dose of rhIL-2.
A veto effect was determined using a MLR assay. Although this assay shows a
significant difference between the subsets, the MLR baseline control which contained
36
responders and stimulators only did not work. With only irradiated B6 stimulators in the
wells, we expected to see a higher proliferation of the BALB/c responders. The ALAK
population more closely behaves like the inhibitory Ly49 G2/A subset which is to be
expected since unsorted ALAKs consists of over 40% Ly49 G2/A and only 15% Ly49
C/I. The veto MLR assay should be repeated to see if there is a consistent pattern of
suppression with a greater effect by the Ly49 C/I+ subset.
NK cells play an essential role in the control of virus-infected cells and cancer
cells. This making NK cells an ideal candidate for teratoma prevention. It has been
previously determined that ES cells and teratoma express high amounts of NK activating
ligands and a low amount of MHC I. The experiment showed a higher teratoma growth
in the treatment group that received the intravenous injection of NK cells compared to the
treatment group that received the subcutaneous injection of NK cells. This may have
been due to the subcutaneous injection being localized to the injection site of mESCs and
the systemic more difficult to reach to the site of mESCs. However, it is possible that
there were issues with the administration process of the intravenous injection where the
injected cells either did not enter the bloodstream or had undergone high stress resulting
in increased cell death, leading to a higher teratoma growth. The in-vivo study has
provided us with information on utilizing NK cells to prevent or treat allogeneic
teratoma.
Overall, time and resources were the single biggest factor during this project. If
allowed to continue, all the studies initiated would be repeated, phenotype and function
would be assessed, NK cell subsets would continue to be evaluated, and tested in the
37
appropriate animal model. The data presented in this project is an early step that sets up
the parameters showing the ultimate goal of using donor NK cells as veto cells. Future
studies would include the investigation of mESCs that have been further differentiated.
This would allow us to determine the effects of the differential MHC expression on
recipient NK and CTLs at the various stages of development. Veto effects of donor NK
cells would be further investigated at the various stages of differentiation.
This study can not only be applied to stem cell therapies but also to whole organ
transplants. In addition, the study can help to expand our knowledge of NK cells and
tolerance induction. Ly49 have a functional human homolog in the killer cell
immunoglobulin-like receptor family (KIR). Therefore, our findings may have great
relevance for future clinical applications of NK therapies as the studies of human NK
subsets advance. Different subsets could be used as clinical tools and may offer
additional advantages when used as potential veto cells when compared to whole NK cell
populations. Donor NK cells will play the role of attacking the attackers from the
recipient’s immune system to allow the proper grafting of the donor stem cells. It is still
not clearly understood what prevents the proper grafting of stem cells. Thus, it is
necessary to investigate the role of the immune cells and how they view the incoming
donor stem cells.
38
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