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THE ROLE OF THE LECTIN BINDING PROTEIN, GALECTIN-3, ON
REGULATING THE TUMOR SPECIFIC CD8 T CELL RESPONSE AFTER GM-CSF
VACCINATION
By
Theodore S. Kouo
A dissertation submitted to Johns Hopkins University in conformity with the
requirements for the degree of Doctor of Philosophy
Baltimore, Maryland
March 2013
ABSTRACT
BACKGROUND: Galectin-3 is a 31 kD carbohydrate-binding lectin that is overexpressed by many human malignancies. It also modulates T cell responses through a
diverse array of mechanisms including induction of apoptosis, TCR cross linking in
CD8+ T cells, and T cell receptor (TCR) down regulation in CD4+ T cells. It is also seen
as a major regulator protein of the innate immune response. However, very few studies
exist that describe its overall role in regulating a tumor-specific immune response in a
tolerogenic host.
METHODOLOGY/PRINCIPAL FINDINGS: We found that patients responding to a
granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting allogeneic
pancreatic tumor vaccine developed post immunization antibody responses to galectin-3
on a proteomic screen. We used the HER-2/neu (neu-N) transgenic mouse model to
study galectin-3 binding on adoptively transferred high avidity neu-specific CD8+ T cells
derived from TCR transgenic mice. Here, we show that galectin-3 binds preferentially to
activated antigen-committed CD8+ T cells only in the tumor microenvironment (TME).
Galectin-3 deficient mice exhibit improved CD8+ T cell effector function and increased
expression of several inflammatory genes when compared with wild type (WT) mice.
We also show that galectin-3 complexes with LAG-3, and LAG-3 expression is necessary
for galectin-3 mediated suppression of CD8+ T cells in vitro. Lastly, galectin-3 deficient
mice have significantly elevated levels of circulating plasmacytoid dendritic cells
(pDCs), which are superior to conventional dendritic cells (cDCs) in activating CD8+ T
cells.
ii
CONCLUSION/SIGNIFICANCE: Binding of galectin-3 to cell surface glycoproteins
on immune cells suppresses the pro-inflammatory immune response. Thus, inhibiting
galectin-3 in conjunction with CD8+ T cell directed immunotherapies should enhance the
tumor specific immune response.
Advisor:
Dr. Elizabeth M. Jaffee (Reader)
Thesis Committee:
Dr. Jonathan Powell (Reader)
Dr. Jonathan Schneck
Dr. Ronald Schnaar
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ACKNOWLEDGEMENTS
I would like to thank my advisor, Dr. Elizabeth Jaffee, for her guidance and
continued support throughout my PhD work in her lab as well as after transitioning back
to medical school. I have no doubt that I will continue to seek her guidance throughout
my career. Dr. Jaffee was able to perfectly blend the freedom to explore with the right
amount of direction that I required. I would like to thank Dr. Todd Armstrong for his
advice and support, and the many extra hours he put in both in the lab and in the mouse
room to help me complete this project. I also appreciate the feedback and expertise of
my thesis committee: Drs. Jonathan Powell, Jonathan Schneck, and Ronald Schnaar.
I would like to thank my parents who survived many hours of yelling and arguing
to instill the importance of a good education and hard work in a stubborn young child
who just wanted to waste the day watching TV and playing video games. There is no
doubt that I would not have been able to accomplish what I have without the knowledge
that my parents would be there to catch me when I fail. Lastly, I would like to thank my
fiancé, Jennifer Lee, whose love and strength has pushed me to overcome the many lows
of graduate school and medical school.
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TABLE OF CONTENTS
ABSTRACT ........................................................................................................................ ii
ACKNOWLEDGEMENTS ............................................................................................... iv
TABLE OF CONTENTS .................................................................................................... v
LIST OF TABLES ............................................................................................................ vii
LIST OF FIGURES ......................................................................................................... viii
CHAPTER 1: INTRODUCTION ....................................................................................... 1
Seroproteomics identifies galectin-3 as a protein of interest .......................................... 1
Galectin-3 and its role in peripheral tolerance ................................................................ 2
HER-2/neu model and TCR transgenic mice.................................................................. 4
Overview of Thesis Work ............................................................................................... 5
CHAPTER 2: CHARACTERIZATION OF POST-TREATMENT SEROLOGIC
RESPONSES IN PDA PATIENTS WITH > 3 YEAR DFS SURVIVAL ......................... 7
Introduction ..................................................................................................................... 7
Materials and Methods .................................................................................................... 8
Results ............................................................................................................................. 9
Discussion ....................................................................................................................... 9
CHAPTER 3: CHARACTERIZATION OF TUMOR SPECIFIC CD8+ T CELLS WITH
SURFACE GALECTIN-3 ................................................................................................ 16
Introduction ................................................................................................................... 16
Materials and Methods .................................................................................................. 18
Results ........................................................................................................................... 24
Discussion ..................................................................................................................... 38
CHAPTER 4: DETERMINING THE ROLE OF GALECTIN-3 IN THE
INFLAMMATORY RESPONSE ..................................................................................... 63
Introduction ................................................................................................................... 63
Materials and Methods .................................................................................................. 64
Results ........................................................................................................................... 65
Discussion ..................................................................................................................... 68
CHAPTER 5: SUMMARY.............................................................................................. 77
APPENDICES .................................................................................................................. 84
v
Appendix A ................................................................................................................... 84
LIST OF REFERENCES .................................................................................................. 85
CURRICULUM VITAE ................................................................................................. 106
vi
LIST OF TABLES
Table 1. Antibody-reactive proteins identified by a functional seroproteomics approach ............ 11
vii
LIST OF FIGURES
Figure 1. Vaccine-induced galectin-3 antibody responses correlate with improved DFS
(1:400 Dilution). ............................................................................................................... 12
Figure 2. In vitro neutralization of galectin-3 by purified IgG from post-vaccination
patient serum. .................................................................................................................... 14
Figure 3. Increased surface galectin-3 on high avidity CD8+ T cells in the tumor
microenvironment. ............................................................................................................ 43
Figure 4. Activation of CD8+ T cells increases surface bound galectin-3. ...................... 44
Figure 5. LAG-3 but not PD-1 expression is required for galectinproduction by T cells. ....................................................................................................... 46
Figure 6. Ingenuity Pathway Analysis of gene expression differences between galectin3hi CD8+ TILS vs. galectin-3lo CD8+ TILS identified CD66 and ST6GAL1 as genes of
interest. .............................................................................................................................. 48
Figure 7. Co-staining of CD66 and Galectin-3 on high avidity CD8+ TILs. ................... 49
Figure 8. In vitro dephosphorylation of ZAP-70 after cross-linking of CD66 with
galectin-3........................................................................................................................... 51
Figure 9. Galectin-3 binding to the surface of CD8+ T cells is determined by α2,6sialylation. ......................................................................................................................... 52
Figure 10. Galectin-3 binding on adoptively transferred alloreactive CD8+ T cells in a
model of graft versus host disease. ................................................................................... 54
Figure 11. Pretreatment of mouse tumors with methyl ester sialidase inhibitor does not
increase surface galectin-3. ............................................................................................... 56
Figure 12. Removal of galectin-3 from the surface of high avidity CD8+ T cells requires
inhibition of galectin-3 expression in the tumor stroma and the T cell. ........................... 57
Figure 13. Removal of surface galectin-3 from high avidity CD8+ T cells leads to
improved effector function. .............................................................................................. 58
Figure 14. Galectin-3 knockout mice have improved tumor free clearance over wild type
mice in both adoptive transfer and endogenous tumor vaccine models. .......................... 59
Figure 15. Removal of galectin-3 increases expression of inflammatory molecules in high
avidity CD8+ T cells. ........................................................................................................ 61
viii
Figure 16. Western blot analysis of TIL lysates confirm S100A8 expression in Gal-3
KO/KO TIL but not Gal-3 WT/WT TIL........................................................................... 62
Figure 17. TNFα and IL-1 are increased in whole tissue lysates of galectin-3 KO VDNs
when compared with galectin-3 WT VDNs...................................................................... 71
Figure 18. CD11c+ cells are increased in galectin-3 KO mice in comparison with
galectin-3 WT mice........................................................................................................... 72
Figure 19. An increase in pDCs in galectin-3 KO neu-N mice is associated with lower
numbers of MDSCs........................................................................................................... 73
Figure 20. Plasmacytoid DCs from vaccinated galectin-3 KO mice are capable of
activating more antigen-specific naïve CD8+ T-cells than conventional dendritic cells
from vaccinated galectin-3 KO mice. ............................................................................... 75
Figure 21. N-glycosylation of co-regulatory molecules serves as the signal 4 required for
optimal T cell activation. .................................................................................................. 82
ix
CHAPTER 1: INTRODUCTION
Seroproteomics identifies galectin-3 as a protein of interest
Pancreatic cancer is the fourth leading cause of death due to cancer and accounts
for approximately 40,560 deaths per year with the incidence increasing on a yearly basis.
Current treatment options for pancreatic cancer can prolong survival but seldom lead to
complete remission or cure of the disease. The 5-year survival rate for pancreatic cancer
is still only 7% for all stages combined (1). Our lab has developed an immunotherapeutic
approach utilizing irradiated allogeneic pancreatic ductal adenocarcinoma (PDA) cell
lines genetically modified to secrete granulocyte macrophage colony stimulating factor
(GM-CSF) (2). In combination with chemotherapy, to deplete Treg cells, this approach
has been shown to be successful in activating the immune response in mouse models of
pancreatic cancer to eradicate the tumor.
Serum was collected from 60 patients in a Phase II clinical trial study at pre- and
post-vaccination time points to examine whether a vaccine-induced antibody response
occurs and, if so, what tumor associated antigens (TAA) are targeted. Patients were
divided into >3 year disease free survival (DFS) and < 3 year DFS groups for analysis.
The 3 year cut off was chosen because patients surviving disease free after 3 years are
likely to remain disease free. These experiments identified several protein bands that
patients in the > 3 year DFS group developed vaccine-induced antibody responses to
versus patients in the < 3 year DFS group. In total, 11 different proteins, including
galectin-3, were identified by this method.
1
Galectin-3 and its role in peripheral tolerance
One of the greatest hurdles in the development of a successful cancer vaccine is
peripheral tolerance. Because TAAs are often recognized as self- antigens by the immune
system, T cells that have strong reactivity against these self-antigens are often tolerized
through various mechanisms before they have the opportunity to kill tumor cells. A great
deal of research has focused on understanding the role of co-regulatory proteins on the
surface of T cells; however, despite the fact that oligosaccharide chains exist on nearly
every plasma membrane protein as well as occupying a large volume of surface area
known as the glycocalyx, the role that glycan-lectin interactions play in the activation
state of immune cells remains controversial.
Galectin-3 is a 31 kD lectin with a 1 mM affinity for lactose. It is a unique
member of the galectin family due to the fact that it is a chimeric protein with an Nterminal domain that has a Bcl-2 like motif conferring anti-apoptotic properties to
galectin-3 intracellularly. The N-terminal domain also allows galectin-3 to oligomerize
into pentamers in order to from high avidity interactions with its ligand and cross-link
glycoproteins (3). Galectin-3 has been shown to reduce the affinity of the TCR for its
cognate MHC I-peptide ligand by forming TCR-galectin-3 lattices that sequester the TCR
from its CD8+ co-receptor (4). Additionally, galectin-3 has also been shown to induce
apoptosis in CD8+ T cells via interactions with CD29 and CD7 (5). In CD4 cells,
galectin-3 has been demonstrated to induce internalization of the TCR leading to
disruption of the immunological synapse (6). In addition to its role in the adaptive
immune response, galectin-3 has been shown to be critical in the innate immune response
as well. It has been shown to be necessary for 2-integrin independent recruitment of
2
neutrophils (7). In dendritic cells (DCs), galectin-3 has been shown to influence the
strength of antigen activation (8). Galectin-3, therefore, is a ubiquitous protein that is
involved in every step of the immune response.
Galectin-3 and its role in the PDA tumor microenvironment
Several studies have demonstrated a critical role for stromal elements in the
development of cancer. Interactions between mesenchymal cells and epithelial cells
through direct cell-to-cell contact and soluble factors are necessary for maintenance of
regulated cell growth and tissue architecture. Perturbations of these mesenchymal cells
have been shown to lead to tumorigenesis. The main mesenchymal cell in pancreatic
cancer is the pancreatic stellate cell, which is believed to be central for the development
of the desmoplastic reaction that predominates in pancreatic cancer. Pancreatic stellate
cells can be activated by cancer cells, and secrete a variety of growth factors, proteases,
and cytokines that promote tumor growth, immune evasion, and metastasis. Signaling
through the TGF- pathway in particular is responsible for the development of fibrosis
and induction of T regulatory cells that suppress a tumor-specific immune response (9).
A role for galectin-3 in fibrosis has been demonstrated in mouse models of
hepatic fibrosis as well as in human studies of fibrosis in heart disease (10, 11). In hepatic
fibrosis, the activation of hepatic stellate cells (HSCs) to differentiate into extracellular
matrix (ECM) secreting myofibroblasts is influenced heavily by the profibrinogenic
cytokine TGF-, and is believed to be dependent on the presence of extracellular
galectin-3, which is rapidly internalized by HSCs. Galectin-3-/- HSCs are deficient in
growth, smooth muscle actin expression, and pro-collagen expression. The addition of
exogenous recombinant galectin-3 to galectin 3-/- primary HSCs restored a wild type
3
phenotype to galectin-3-/- HSCs. Because galectin-3 has been shown to be an important
factor in the development of fibrosis in various other organs, it is likely to be important in
establishment of the tumor microenvironment (TME) in PDA, which is characterized by
dense desmoplasia.
HER-2/neu model and TCR transgenic mice
HER-2/neu is a 185 kD protein, and a member of the erbB oncogene family. The
erbB family consists of type I tyrosine kinase receptors that are related to but distinct
from the epidermal growth factor receptor (EGFR) family. These proteins form homo and
heterodimers upon ligand binding with HER-2 being the most favorable binding partner.
HER-2/neu is overexpressed in 13-23% of breast cancers resulting in prolonged
activation of MAPK pathways (12). The HER-2/neu mouse model is on the FVB/N
mouse strain with the rat HER-2/neu proto-oncogene driven by the mouse mammary
tumor virus (MMTV) promoter. In this setting, female mice develop focal mammary
tumors by 4 months of age, and are tolerant to HER-2/neu containing orthotopic tumors
(NT2.5) and vaccination (13). However, supplementing vaccination with
cyclophosphamide (CY) to deplete T regulatory cells (Tregs) allows for the expansion of
neu-specific CD8+ T cells leading to tumor clearance in 10-20% of tumor challenged
mice.
Using overlapping peptide libraries, our lab identified the immunodominant
epitope of HER-2/neu, RNEU420-429,, against which the majority of tumor-specific CD8+
T cells were reactive after vaccination with 3T3NeuGM, a whole cell GM-CSF vaccine
that overexpresses HER-2/neu (14). Furthermore, dilutional cloning allowed us to further
differentiate this pool of tumor-specific CD8+ T cells into high avidity and low avidity
4
populations as measured by staining with MHC I tetramers containing the RNEU420-429
peptide (15). High avidity CD8+ T cells have T cell receptors (TCRs) characterized by
V4 and V1.1 side chains while low avidity CD8+ T cells have TCRs found to use the
V2 and V5 chains. For further analysis of these two populations in an adoptive transfer
setting, we generated high avidity and low avidity TCR transgenic mouse lines on an
FVB/N Thy1.2 background. The TCR transgenic mice contain pre-rearranged gene
segments for the V and the V TCR side chains so that all T cells in the high avidity
TCR transgenic mouse should be a clonal population expressing the high avidity TCR,
and likewise for the low avidity transgenic mouse. The Thy1.2 marker provides a
convenient way to label adoptively transferred cells after they have been injected into
recipient mice.
Overview of Thesis Work
The overall goal of this thesis is to understand the survival benefit of patients who
developed high titers of anti-galectin-3 antibodies in response to whole-cell GM-CSF
vaccination, and understand the effect of galectin-3 on modulating the anti-tumor
immune response. This has been accomplished by:
1. Demonstrating the ability of purified IgG from post-vaccination patient serum
to neutralize galectin-3’s inhibitory effect on IFNproduction by maximally
activated CD8+ T cells in vitro.
2. Characterizing phenotypic and functional differences associated with the
presence of surface galectin-3 on tumor-specific CD8+ T cells through surface
marker characterization, whole genome microarray studies, and tumor
survival experiments between galectin-3 WT and galectin-3 KO mice.
5
3. In vitro studies identifying putative binding partners through which galectin-3
may mediate CD8+ T cell suppression.
4. Demonstrating expansion of the plasmacytoid dendritic cell population after
deletion of galectin-3, and characterizing its influence on CD8+ T cell
activation.
6
CHAPTER 2: CHARACTERIZATION OF POST-TREATMENT SEROLOGIC
RESPONSES IN PDA PATIENTS WITH > 3 YEAR DFS SURVIVAL
Introduction
Our lab previously developed a functional proteomic approach to identify PDA
associated proteins that might serve as targets of the immune response (16). This
approach utilized pre- and post-treatment sera from patients who received two GM-CSFsecreting whole pancreatic tumor cell lines as vaccine and compared serologic reactive
proteins between treatment responders DFS > 3 years and non-responders (early
recurrence after a single vaccine or DFS < 3 years). Paired sera from 60 subjects treated
on a recently reported phase 2 study were evaluated by western blot. Proteins that
demonstrated an increase in post-treatment serologic responses were purified by 2-D gel
and mass spectrometry. Eleven proteins were identified (Table 1), two of which were
serologic targets recognized by multiple patient sera. The first protein, annexin A2,
induces an epithelial to mesenchymal transition (EMT) in pancreatic tumor cells and
promotes pancreatic tumor metastases (16). The second protein, galectin-3, is a
galactoside-binding protein and a known cancer-associated protein secreted by a number
of tumor types (17). Galectin-3, like annexinA2, induces post-treatment serologic
responses that correlate with improved DFS and overall survival (OS) (Fig. 1A-C).
ELISA results from all 60 patients in the Phase II trial showed that 67% of patients in the
> 3 year DFS group had a greater than 2-fold increase in anti-galectin-3 antibody titers,
and only 9.5% of patients in the < 3 year DFS showed a similar increase. In contrast,
vaccination does not alter immune responses against the positive control antigen,
influenza A.
7
The humoral arm of the immune response is critical for aiding T-cell mediated
tumor rejection (18-23). Antibodies are known to elicit their effects through a diverse
array of mechanisms such as antigen neutralization, complement fixation, ADCC, and
promotion of cross-presentation by dendritic cells (24). It was hypothesized that posttreatment serologic responses against galectin-3 improves DFS survival by enhancing
CD8+ T cell function through neutralization of galectin-3 binding to the cell surface. The
addition of exogenous galectin-3 to activated CD8+ T cells in vitro has previously been
shown to induce apoptosis, and suppress both IFNproduction and cellular proliferation.
Thus, the presence of anti-galectin-3 neutralizing antibodies was assessed by the ability
of purified IgG form patient sera to block galectin-3 mediated suppression of CD8+ T
cells in vitro.
Materials and Methods
In vitro activation and suppression of CD8+ T cells with exogenous galectin-3
CD8+ T cells were negatively isolated from total splenocytes using Dynal CD8+
negative isolation kits (Invitrogen), and stimulated for 3 days with anti-CD3/CD28
(Invitrogen) beads at a T cell to bead ratio of 1:1 according to the manufacturer’s
recommendations (25). For suppression studies, cells were incubated with the indicated
amount of recombinant human galectin-3. Cells were also incubated with a 5 fold molar
concentration of purified IgG from patient’s serum, as described in the figures. After 3
days of activation, CD8+ T cells were assessed for IFN production by ICS. Beads were
removed by magnetic separation and cells were plated with fresh anti-CD3/CD28 beads
in a 96-well assay plate at a T cell to bead ratio of 1:1 in the presence of monensin
8
(GolgiStop, Invitrogen) for 5 hours at 37C. Following the 5-hour incubation period, ICS
studies were conducted as detailed in Chapter 3.
Results
Patients develop neutralizing antibodies against galectin-3 after receiving whole cell
GM-CSF PDA vaccine
Galectin-3 is known to inhibit T cell activation by direct T cell binding (26). We
therefore tested our hypothesis that post-treatment serologic responses against galectin-3
improves DFS survival by enhancing CD8+ T cell function through inhibition of galectin3 binding to the T cell surface. First, we assessed the ability of recombinant human
galectin-3 to inhibit IFN production by CD8+ T cells maximally stimulated in vitro with
anti-CD3/CD28 beads. The minimal concentration necessary to observe a significant
reduction in IFN production was 25 µg/mL. Next, we tested whether purified IgG from
pre-vaccinated and post-vaccinated serum from patients with > 3 year DFS could prevent
this reduction. We chose to compare sera from patient 3.027, who exhibited the highest
post-vaccination anti-galectin-3 antibody titers in the DFS group, and patient 3.052
whose antibody titers were significantly lower. All antibodies were co-incubated with
galectin-3 at a 5:1 molar ratio. For both patients, we found that IgG from post-vaccine
serum but not pre-vaccine serum could attenuate the suppression observed at 25 g/mL
of galectin-3 (Figure 2A-B).
Discussion
These results provide a mechanism by which vaccine induced anti-galectin-3
antibodies may confer long-term DFS survival in patients with PDA by improving CD8+
9
T cell function; however, it remains to be elucidated whether these antibodies function in
the same manner in vivo. Galectin-3 targeted therapies using small molecule inhibitors
are already under investigation for the treatment of chronic organ failure (27-30) and
cancer (31-34). Similar to neutralizing antibodies, small molecule inhibitors also provide
a method to neutralize galectin-3 binding; however, antibodies possess the additional
ability to mediate clearance of antibody-antigen complexes by macrophages via the Fc
domain. Because extracellular galectin-3 exists in equilibrium with surface-bound
galectin-3, persistently elevated titers of anti-galectin-3 antibodies could promote
galectin-3 clearance and dissociation from the cell-surface (35-37). Multiple
conformations of galectin-3 exist depending on phosphorylation of the N terminus as well
as whether or not the CRD is engaged with a ligand. Evidence also demonstrates that the
N-terminal domain is also involved in carbohydrate recognition (38, 39); therefore,
immunization with recombinant galectin-3 alone may not be sufficient to develop
effective neutralizing antibodies. Rather, immunization strategies involving galectin-3 in
its native conformation with bound ligand, such as in whole cell GM-CSF vaccines,
would be preferred.
10
Table 1. Antibody-reactive proteins identified by a functional seroproteomics
approach
11
Figure 1. Vaccine-induced galectin-3 antibody responses correlate with improved
DFS (1:400 Dilution).
A) Pre-vaccine titers shown for patients who were either disease-free >3 years
(DFS>3yr), disease-free less than 3 years (DFS<3yr), patients who received only a single
vaccine before recurring (single Vac), or healthy donors (Donors). B) Post-vaccine titers
for patients divided by DFS >3 years (left panel) or < 3 years (right panel) shown before
the first vaccine (pre-Vac), 28 days after the first vaccine (Vac 1), after chemoradiation
which was given after the first vaccine (post-Rx), and after two additional vaccines given
after completing chemoradiation (Vac 3). C) Titers shown over time for 8 of the 12
patients demonstrated DFS>3 years. These patients received a total of 5 vaccinations
(Vac 1, Vac 2, Vac 3, Vac 4, and Vac 5), the first one before chemoradiation, the 2nd, 3rd,
and 4th each one month apart beginning one month after completing chemoradiation, and
the 5th, 6 months after completing the 4th vaccination.
12
A) Pre-vaccine titers
P = 0.0199
Anti-Galectin-3 Ab (OD4 5 0)
0.6
P = 0.0494
0.4
0.2
on
or
s
c
Va
e
D
gl
Si
n
D
D
FS
FS
>
<
3y
r
3y
r
0.0
B) Post-vaccine titers
P = 0.0015
P = 0.0025
Anti-Galectin-3 Ab (OD450)
0.6
P = 0.0117
P = 0.0337
P = 0.0007
0.4
0.2
C) Titers over time of DFS patients
0.8
9
12
Anti-Galectin-3 Ab (OD450)
27
29
0.6
31
47
52
53
0.4
0.2
0.0
Pre-Vac
Vac1
Post-Rx
Vac2
Vac3
Vac4
13
Vac5
c3
Va
x
t-R
Va
Po
s
c1
c
Pr
e-V
a
c3
Va
x
t-R
Po
s
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a
c
0.0
Figure 2. In vitro neutralization of galectin-3 by purified IgG from post-vaccination
patient serum.
Antibodies were purified from patient serum by Protein G isolation, and then incubated
with healthy donor T cells and recombinant human galectin-3 as described in materials
and methods. A) Gating of IFN+ CD8+ T cells on histogram plots and percentages of
IFN+ CD8+ T cells after a 3 day anti-CD3/CD28 activation in the presence of various
conditions as shown in the graph for patient 3.052. B) Percentages of IFN+ CD8+ T cells
after a 3 day anti-CD3/CD28 activation in the presence of various conditions as shown in
the graph for patient 3.027.
14
A) Patient 3.052
B) Patient 3.027
15
CHAPTER 3: CHARACTERIZATION OF TUMOR SPECIFIC CD8+ T CELLS
WITH SURFACE GALECTIN-3
Introduction
Galectin-3 has been demonstrated to possess a diverse array of functions in tumor
cells including intracellular signaling (40-44), apoptosis (45-48) trafficking and
metastasis (49-51), and angiogenesis (52-54). The diversity of galectin-3’s many
functions can be attributed to the ubiquity of oligosaccharide chains on nearly all plasma
membrane associated proteins. Unlike the high degree of specificity needed for proteinprotein interactions that govern the majority of receptor-ligand pairings, glycan-lectin
interactions allow for a single lectin to interact with multiple different receptors at once.
This phenomenon enables molecules such as galectin-3 to influence the function of a
broad spectrum of different cell types.
Due to the many pleiotropic effects of galectin-3, it is also an attractive target for
therapeutic intervention in a variety of human diseases. In addition to its involvement in
tumorigenesis, galectin-3 also has immunomodulatory functions. Galectin-3 can reduce
the affinity of the TCR for its cognate major histocompatibility complex (MHC) I-peptide
ligand by forming TCR-galectin-3 lattices that sequester the T cell receptor (TCR) from
its CD8+ co-receptor (4, 55). Furthermore, galectin-3 induces apoptosis in CD8+ T cells
via interactions with CD29 and CD7 (56) and internalization of the TCR leading to
disruption of the immunological synapse in CD4+ T cells (57). Galectin-3 also influences
the strength of antigen activation in dendritic cells (DCs) (58, 59). In addition, galectin-3
is necessary for β2-integrin independent recruitment of neutrophils (60).
16
While these studies have been critical for the elucidation of galectin-3’s impact on
immune cells, they were largely conducted in vitro by studying only a single population
of cells in isolation with the addition of exogenous galectin-3 into the culture medium.
However, because the immune response typically does not involve just one cell type
acting in isolation, but rather multiple cell types acting upon one another, these studies
still fail to present a complete picture of galectin-3’s role in the immune response. While
several in vivo studies have looked at the effect of removing galectin-3 on the innate
immune response to infectious diseases in galectin-3 knockout (KO) mice, very few have
been able to evaluate the effect of removing galectin-3 on the adaptive immune response
to tumors in a tolerogenic setting.
To elucidate the role of galectin-3 on CD8+ cancer specific T cells, we crossed
HER-2/neu transgenic (neu-N) mice which develop natural HER-2/neu (neu) expressing
mammary tumors and high avidity CD8+ TCR transgenic mice specific for the
immunodominant epitope recognized on the tumors of these mice, with galectin-3 KO
mice, and analyzed CD8+ T cell responses following treatment with a neu targeted
vaccine. Rather than focusing on galectin-3 signaling through individual cell surface
receptors as has been done in the past, the ability to compare tumor-specific CD8+ T cell
responses in galectin-3 wild type versus galectin-3 KO mice in response to a whole cell
GM-CSF vaccine enabled us to examine genome-wide changes in protein expression
caused by galectin-3. Here, we demonstrate in vivo that removal of galectin-3 increases
both the number of functional CD8+ T cells found in the tumor microenvironment and the
expression of inflammatory proteins by these T cells, leading to enhanced tumor rejection
in galectin-3 KO mice when compared with galectin-3 WT mice. Further, we show that
17
these changes occur independently of improved TCR signaling (4), demonstrating the
vast potential for lectin-glycan mediated regulation of CD8+ T cell responses.
Materials and Methods
Mice
HER-2/neu (neu-N) and LGALS3-/- (galectin-3 KO) mice were purchased from
Jackson Laboratories (Bar Harbor, ME), bred and housed in the Johns Hopkins animal
facility. High avidity T cell receptor (TCR) transgenic mice were generated as previously
described, and avidity was confirmed by tetramer staining (61). Galectin-3 KO mice were
backcrossed for 6 generations using a marker assisted selection (i.e. “speed congenic”)
approach. Mouse genomes were assessed at the DartMouse™ Speed Congenic Core
Facility at Dartmouth Medical School. Genetic background at the final backcross
generation was determined to be 99.75% for the desired FVB/N background.
Backcrossed galectin-3 KO mice were bred with neu-N and high avidity TCR transgenic
mice to generate galectin-3 KO neu-N and galectin-3 KO high avidity TCR transgenic
mouse lines. All experiments were conducted with female mice between 6-12 weeks of
age according to protocols approved by the Johns Hopkins Animal Care and Use
Committee.
Cell lines and media
The NT2.5 is a neu expressing tumor cell line generated from spontaneously
arising tumors in female neu-N mice as previously reported (62, 63). The 3T3neuGM
vaccine line is genetically modified from 3T3 fibroblast cells to secrete GM-CSF and
express rat neu (64). The T2-Dq cell line is a TAP-2 deficient cell line that expresses the
18
Dq MHC I allele, and was generated as previously reported (64). All cell lines are
cultured and maintained according as previously reported (65).
Tumor, vaccine, chemotherapy, adoptive transfer, and GCS-100 administration
procedures
For tumor clearance studies, mice were tumor challenged with the minimal
tumorigenic dose of 5x104 NT2.5 tumor cells injected subcutaneously (s.c.) in the right
upper mammary fat pad on day 0, given 100 mg/kg cyclophosphamide (CY)
intraperitoneally (i.p.) on day 2, vaccinated with 3 simultaneous s.c. injections of 1x106
3T3neuGM cells in the bottom and right limbs on day 3, and given 2x106 adoptively
transferred high avidity CD8 T cells on day 4. GCS-100 was administered 3 times per
week i.p at a dose of 20 mg/kg. For tumor infiltrating lymphocytes (TIL) and lymph node
experiments, mice were given 2 simultaneous s.c. injections of 2x106 NT2.5 tumor cells
in the right and left upper mammary fat pads on day 0, CY on day 8, vaccine on day 9,
and 6x106 adoptively transferred CD8+ T cells on day 10. High avidity Thy1.2+ CD8+ T
cells were negatively isolated from spleens of female TCR transgenic mice and
adoptively transferred as previously described (65)
Peptides and antibodies
RNEU420–429 (PDSLRDLSVF) and the negative control peptide LCMV NP118–126
(RPQASGVYM) peptides were produced in the Johns Hopkins Biosynthesis and
Sequence Facility at a purity >95%. Antibodies used for flow cytometry studies were:
anti-CD8-FITC (BD Biosciences), anti-CD8-PE (BD Biosciences), anti-CD8-PerCP (BD
19
Biosciences), anti-CD8-APC (BD Biosciences), anti-galectin-3-AF647 (Biolegend), antigalectin-PE (R&D), anti-PD1-PE (eBioscience), anti-LAG-3-PE (eBioscience), antiThy1.2-PerCP (Biolegend), anti-CD44-Pacific Blue (eBioscience), anti-CD11b-PE (BD
Biosciences), anti-CD11c-FITC (BD Biosciences), anti-B220-APC (BD Biosciences),
anti-Ly6C-PerCP-Cy5.5 (eBioscience), anti-IFN-PE (BD Biosciences), anti-IFNPacific Blue (eBioscience), and anti-Granzyme B-APC (BD Biosciences). Cellular
division was assessed by labeling of high avidity CD8+ T cells with 1.5 mM CellTrace
CFSE cell proliferation kit (Invitrogen) prior to adoptive transfer (65). Apoptosis was
determined using Live/Dead fixable aqua stain (Invitrogen) and Annexin V-Pacific Blue
labeling (Biolegend) for phosphatidylserine. All antibody staining was conducted at 4C
for 20 minutes in FACS buffer (PBS, 5%FBS, 0.02% NaAzide). Samples were read by
LSR-II and FACS Caliber (BD Biosciences) flow cytometers. Analysis was performed
using FACS Diva (BD Biosciences) and Flow Jo (Tree Star, Inc).
Flow cytometry, intracellular cytokine staining (ICS) and tetramer analyses
Lymph nodes were dissected 3 and 5 days after adoptive transfer, and tumors
were dissected 5 days after adoptive transfer, and homogenized by mashing through 40
mM nylon cell strainers. Tumors were further processed by enzymatic digestion using
collagenase (1mg/mL, Gibco) and hyaluronidase (25 mg/mL, Sigma). After digestion,
cells were washed with RPMI before being trypsinized for 2 minutes with 0.25%
Trypsin-EDTA (Gibco). Lymphocytes were incubated for 5 hours at 37C in CTL media
with RNEU420–429 or NP118–126 peptide-pulsed T2-Dq target cells in the presence of
monensin (GolgiStop, BD Biosciences) at a lymphocyte to target ratio of 4:1. Cells were
20
surface stained for Thy1.2 expression prior to fixation/permeabilization using a mouse
ICS kit (BD Biosciences) to stain for intracellular IFN-g and Granzyme B. The percent of
IFN-g and Granzyme B producing cells was determined by subtracting the percentage of
cytokine producing cells in NP118–126 -pulsed samples from the percentage of cytokine
producing cells in RNEU420–429 -pulsed samples. Absolute numbers were calculated from
total cell counts performed on each sample prior to the 5-hour incubation.
Avidity was determined by dilutional tetramer analyses with the RNEU420–429/H2Dq tetramer. Samples were stained at a starting concentration of 1.9 M and dilutions of
1:100 were performed to assess avidity by flow cytometry.
In vitro activation and suppression of CD8+ T cells with exogenous galectin-3
CD8+ T cells were negatively isolated from total splenocytes using Dynal CD8+
negative isolation kits (Invitrogen), and stimulated for 3 days with anti-CD3/CD28
(Invitrogen) beads at a T cell to bead ratio of 1:1 according to the manufacturer’s
recommendations (25)
For suppression studies, cells were also incubated with the indicated amount of
recombinant mouse galectin-3. After 3 days of activation, CD8+ T cells were assessed for
IFN-g production by ICS. Beads were removed by magnetic separation and cells were
plated with fresh anti-CD3/CD28 beads in a 96-well assay plate at a T cell to bead ratio
of 1:1 in the presence of monensin (GolgiStop, Invitrogen) for 5 hours at 37C. Culture
media was also supplemented with fresh galectin-3 at the indicated concentrations.
Following the 5-hour incubation period, ICS studies were conducted as above.
21
In vitro pZAP-70 dephosphorylation
CD8+ T cells were isolated and stimulated in culture as previously described.
Briefly, 2x106 cells were stimulated at a 1:1 ratio of cells:CD3/CD28 beads in 2 mL of
XVivo-15 media (Lonza) with 30 U/mL rhIL-2. On Day 2 of stimulation, 1 mL of media
was replaced with fresh XVivo-15 and rhIL-2. On Day 3 of stimulation, beads were
removed from culture, and cells were allowed to rest for 24 hours with 0, 2, 5, 10, 20, 30
g/mL rmGalectin-3. The next day, cells were collected, washed, and resuspended in
100 L cold PBS. Cells were then incubated for 25 minutes with 20 g/mL biotinylated
anti-CD3 and anti-CD28, and then washed one with cold PBS. CD3 and CD28 were then
cross-linked by the addition of 10 g/mL streptavidin for 20 minutes. After crosslinking, stimulation was allowed to proceed at 37 C for the indicated time points prior to
lysis in lysis buffer (RIPA, sodium orthovanadate, PMSF, NaF, Protease Inhibitor). Cell
lysate was then quantified by BCA assay and western blotting was performed for
pZAP70 and total ZAP70.
Cloning and purification of recombinant mouse galectin-3
Total RNA was isolated from in vitro activated high avidity CD8+ T cells using
the RNEasy Mini Kit (Qiagen). Galectin-3 cDNA was amplified with Superscript III First
Strand Synthesis System (Invitrogen) and galectin-3 specific primers containing BamHI
and NdeI restriction sites: 5’GGAATTCCATATGGCAGACAGCTTTTCGCTTAACGATG-3’ (Forward) and 5’CGGGATCCTTAGATCATGGCGTGGTTAGCGCTGGTGAGGG-3’ (Reverse). .The
galectin-3 open reading frame (ORF) was then cloned into the pET-22B bacterial
22
expression vector (Novagen), which was transfected into BL21(DE3) chemically
competent bacterial cells (Invitrogen) according to manufacturer’s recommendations.
BL21(DE3) cells were cultured in Luria Broth (LB) at 37C and 200 rpm to an OD600 of
approximately 0.700 at which point protein expression was induced with the addition of 1
mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). Cells were incubated for an
additional 4 hours before being pelleted at 4C. Galectin-3 was purified from bacterial
cell lysate material by binding to lactosyl-agarose beads (Sigma) and elution with 200
mM lactose. Purified material was dialyzed into PBS, and endotoxin was removed using
the ToxinEraser Endotoxin Removal Kit (GenScript). Endotoxin was quantified to be less
than 1.0 EU/mL by the LAL assay (Pierce).
Co-Immunoprecipitation of galectin-3 and LAG-3
10 mg LAG-3 (Clone 410C9) or galectin-3 (M3/38) specific antibody and
corresponding isotype controls were conjugated to Protein G Dynabeads (Invitrogen) in
PBS followed by cross-linking with 10mM BS3 (66). CD8+ T cells were isolated and
activated as previously described. Cell surface proteins were cross-linked with 10 mM
BS(PEG)9 prior to cell lysis with CelLytic M (Sigma) supplemented with 100 mM
lactose and protease inhibitor. Conjugated beads were incubated at 4°C overnight with
CD8+ T cell lysates. After washing beads with TBST (Tris-Buffered Saline + 0.1%
Tween-20) the following day, bound proteins were eluted by boiling in sample buffer
under reducing conditions. Standard western blotting procedures were followed and
protein interactions were shown after developing membranes for 1 hour on high
chemiluminescence film.
23
Gene Expression Analysis
RNA was extracted using the Stratagene Absolutely RNA Nanoprep Kit.
Microarray hybridization and analyses were performed by the Johns Hopkins Deep
Sequencing and Microarray Core Facility using the NuGen amplification system and an
Affymetrix Exon 1.0 ST array. The data discussed in this publication are accessible
through GEO Series accession number GSE59454.
Statistical Analysis
Student’s t tests were performed using GraphPad Prism software assuming equal
variances. Log-rank tests were used for Kaplan-Meier plots. P values of less than 0.05
were considered to be statistically significant.
Results
Galectin-3 binds specifically to tumor-specific CD8+ T cells found in the tumor.
A few studies have already implicated galectin-3 in the regulation of CD8+ T cell
responses through several different mechanisms including induction of apoptosis and
TCR cross linking that results in preventing CD8+ T cell function (4). We therefore
employed the neu-N mouse model of mammary tumors for which we have developed
neu-specific TCR transgenic mice to further evaluate the role galectin-3 plays in
modulating neu-specific anti-mammary cancer T cell responses within the tumor
microenvironment. We previously reported that our whole cell GM-CSF vaccine cell
line, 3T3NeuGM, induces potent antitumor immune responses against the
24
immunodominant neu protein expressing CD8+ T cell epitope, RNEU420-429 in the
parental non-tolerant FVB/N mice but not in the tolerant neu-N mice (63). Low doses of
cyclophosphamide (CY) given with the vaccine results in the depletion of Tregs and
allows for improved high avidity RNEU420-429 -specific CD8+ T cell responses that are
associated with the cure of pre-existing tumors in 30% of neu-N mice. Adoptive transfer
of the high avidity T cells given together with CY and vaccine mediates long-term tumor
control and produce significantly more cytokines in the majority mice suggesting that
vaccine alone does not produce enough activated T cells to take on larger tumor burdens
(65). CD8+ T cells derived from these TCR transgenic mice express the Thy1.2 surface
marker, which allowed us to differentiate galectin-3 binding on adoptively transferred
high avidity CD8+ T cells from endogenous CD8+ T cells that express the Thy1.1 surface
marker, and are thought to be tolerized and nonfunctional against the neu antigen. Thus,
adoptive transfer of high avidity CD8+ T cells into tolerized neu-N mice provides the
opportunity to study a functionally well-defined population of T cells with uniform
avidity upon which the effects of galectin-3 can be evaluated in vivo.
We first evaluated adoptively transferred high avidity CD8+ T cells for expression
of surface galectin-3. To optimize recovery of tumor infiltrating lymphocytes (TIL) in
our adoptive transfer neu-N model, mice were first given a large tumor burden of two
simultaneous subcutaneous injections of 2x106 neu-expressing NT2.5 tumor cells in both
the right and left upper mammary fat pads on Day 0, followed by low dose CY (100
mg/kg) on Day 8, whole cell GM-CSF vaccine on Day 9, and adoptive transfer of 6X106
high avidity CD8+ T cells on Day 10. TIL were isolated from the mammary tumors 5
days following adoptive transfer which is the optimal time point of T cell trafficking into
25
the tumor microenvironment (65). For an accurate sampling of different immune
environments, we isolated CD8+ T cells from excised tumor tissue, lymph nodes, and
spleens. The largest number of T cells with expression of galectin-3 occurred on TILs,
with relatively negligible amounts by T cells isolated from the tumor draining node
(TDN) and spleen (Figure 3A). In addition, galectin-3 staining on TILs exhibited a
significant increase in mean fluorescence intensity (MFI) relative to what was observed
in the TDN and spleen suggesting that individual T cells bound more galectin-3 when in
the tumor microenvironment. To determine whether the increase in surface galectin-3
was due to an overall increase in extracellular galectin-3 from increased expression and
secretion by tumor cells, or due to increased expression of galectin-3 binding targets
specifically by high avidity CD8+ T cells in the tumor microenvironment, we further
compared galectin-3 staining on endogenous non-specific CD8+ T cells versus galectin-3
staining on high avidity CD8+ T cells in the TIL. Endogenous CD8+ T cells in TIL do not
appear to stain for surface galectin-3 to the same high levels as observed for adoptively
transferred high avidity CD8+ T cells (Figure 3B). These data suggest that galectin-3
selectively binds T cells that traffic into the tumor microenvironment relative to other T
cell residing sites, and T cells that are tumor antigen committed rather than all T cells
within the tumor microenvironment. Taken together, these data suggest galectin-3 does
not simply bind indiscriminately to every cell it encounters, but that there is a certain
degree of specificity governing galectin-3 binding.
Increased binding of galectin-3 occurs on activated CD8 T cells with an exhausted
phenotype
26
Due to the predominance of galectin-3 binding on CD8+ T cells only after they
have trafficked into the tumor microenvironment, and the increased binding observed on
high avidity CD8+ T cells over other CD8+ T cells in the tumor microenvironment which
were previously shown to be low avidity neu-specific, we next determined whether or not
galectin-3 binds preferentially to the most activated T cells. We found that CD8+ T cells
maximally activated in vitro with anti-CD3/CD28 beads show a significant increase in
galectin-3 MFI after 3 days in culture (Figure 4A). Because the majority of effector
CD8+ T cells found in the tumor have already been activated and undergone terminal
differentiation, we used lymphocytes isolated from TDNs, which contain populations of
T cells at several different stages of cellular division, to illustrate the differences in
galectin-3 binding during the various stages of T cell activation. Carboxyfluorescein
succinimidyl ester (CFSE) labeling of adoptively transferred T cells demonstrated that
the presence of surface galectin-3 was associated with only the most CFSE dilute cell
populations, further establishing a link between galectin-3 binding and cellular
differentiation. In addition, co-staining of galectin-3 with CD44, a cellular adhesion
molecule highly expressed only on effector/memory CD8+ T cells, revealed that galectin3 surface binding occurs only on cells that co-express CD44 indicating that galectin-3
binds preferentially to terminally differentiated effector T cells (Figure 4B).
Next, we evaluated TIL for the phenotypic differences of T cells associated with
increased galectin-3 binding. First, we separated high avidity CD8+ T cells in TIL into
galectin-3 high (galectin-3hi) versus galectin-3 low (galectin-3lo) T cells and analyzed
each population for the co-expression of other known regulatory molecules, specifically
programmed death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) because these
27
two markers are inhibitory receptors often involved in T cell tolerance (67). PD-1
expression often increases in a tolerogenic setting when CD8+ T cells fail to receive the
appropriate co-stimulatory signals. Several tumor cell types express the PD-1 ligand
B7H1, and signaling through PD-1 is one mechanism by which tumor cells can evade the
immune response (68). The role of LAG-3 is still controversial, but its expression has
been shown to increase during antigen stimulation and is thought to play a homeostatic
role in T cell expansion through the regulation of calcium flux (69). We observed an
increase in the expression of the inhibitory markers PD-1 and LAG-3 only on galectin-3hi
CD8+ T cells, with the majority of galectin-3hi T cells being PD-1 and LAG-3 double
positive when compared with galectin-3lo cells (Figure 4C). In addition, galectin-3hi cells
also have higher levels of Annexin V staining when compared with galectin-3lo cells
(Figure 4D). Despite the increase in expression of PD-1 and LAG-3, we did not observe
any differences in interferon gamma (IFN) expression associated with galectin-3 binding
in vivo (data not shown). Thus, galectin-3 binding on CD8+ T cells occurs almost
exclusively on differentiated effector T cells. Furthermore, increased binding is observed
on T cells displaying an exhausted or tolerized phenotype with elevated expression of
PD-1 and LAG-3, and higher Annexin V labeling.
LAG-3 is necessary for galectin-3 mediated suppression of IFN in vitro
These data so far provide a link between elevated galectin-3 binding and
increased PD-1 and LAG-3 expression on high avidity neu-specific CD8+ T cells. Next,
we evaluated whether PD-1 and LAG-3 surface proteins are directly involved in galectin3 associated downstream effects on T cell function. Both these molecules are known to
28
be heavily glycosylated and possess several potential binding sites for galectin-3 (70, 71).
We first confirmed previously reported findings that addition of exogenous galectin-3
inhibits IFN production in CD8+ T cells in vitro (26). Consistent with previously
published reports, we also demonstrated inhibition starting at 50 mg/mL of galectin-3
after a 3-day stimulation of CD8+ T cells with anti-CD3/CD28 beads (data not shown).
Next we determined whether galectin-3 can inhibit IFN production in PD-1
knockout and LAG-3 knockout CD8+ T cells when compared with wild type controls.
Although exogenous galectin-3 did not significantly alter IFN production of PD-1
knockout T cells, LAG-3 knockout T cells produced maximum IFN even at the highest
concentrations of exogenous galectin-3. In contrast, wild type CD8+ T cells had a 20%
reduction in IFN production at concentrations of 150 mg/mL or higher of exogenous
galectin-3 (Figure 5A and 5B). Furthermore, co-immunoprecipitation studies
demonstrated a physical interaction between LAG-3 and galectin-3. Using whole cell
lysates of activated CD8+ T cells, we were able to isolate LAG-3 with an anti-galectin-3
antibody, and vice versa (Figure 5C). Next, cross-linking of cell surface proteins prior to
cell lysis allowed us to immunoprecipitate a LAG-3/galectin-3 complex migrating
between 150 and 250 kD with a LAG-3 specific antibody (Figure 5D). Importantly, we
were not able to co-immunoprecipitate PD-1 with galectin-3. These data establish a link
between LAG-3 and galectin-3 on antigen-committed CD8+ T cells, and suggests a new
mechanism by which galectin-3 may regulate CD8+ T cell function.
These data establish for the first time a link between LAG-3 and galectin-3
expression on antigen-committed CD8+ T cells, and suggests a new mechanism by which
galectin-3 may regulate CD8+ T cell function. Conversely, this data also provides a
29
possible mechanism through which LAG-3 can still regulate CD8+ T cell function
independent of its known ligand MHC II.
Expression of CEACAM1 and ST6GAL1 are increased in galectin-3hi CD8+ T cells
We examined changes in gene expression of adoptively transferred high avidity
galectin-3hi CD8+ TILs versus high avidity galectin-3lo CD8+ TILs with Affymetrix
cDNA hybridization arrays (Figure 6). We chose to focus our attention on CD8+ TILs
due to the high percentage of these cells that are galectin-3hi relative to T cells seen in
peripheral lymphoid organs. The expression of CEACAM1 (CD66) was found to be twofold higher in galectin-3hi cells. CD66 is of particular interest because it has been
identified as the major receptor for galectin-3 in human neutrophils (72). Furthermore,
the cross-linking of CD66 has been demonstrated to result in recruitment of phosphatases
that dephosphorylate the TCR signaling molecule Zap-70 (73). CD66 expression was
verified by flow cytometry staining, and remains a potential binding partner of interest
due to a strong linear correlation with galectin-3 staining (Figure 7A-C). However,
western blot studies performed to demonstrate Zap-70 dephosphorylation with increasing
amounts of exogenous galectin-3 added to galectin-3 -/- CD8+ T cells were inconclusive,
and no significant differences were observed.
The addition of 5g/mL exogenous
galectin-3 may have lead to a decrease in Zap-70 phosphorylation observable at the 6 to 8
minute time points; however, these results were minimal and could not be repeated
(Figure 8).
The expression of an additional gene of interest, ST6GAL1, was found to be twofold lower in galectin-3hi cells. ST6GAL1 encodes the enzyme, beta-galactoside alpha-
30
2,6-sialyltransferase 1, which catalyzes the addition of sialic acid to galactose containing
residues. It has previously been shown that modification of galactose residues with sialic
acid might inhibit binding of galectin-3 (74). Indeed, labeling of T cells with Sambucus
nigra lectin to detect 2,6-linked sialic acid residues on terminal galactose residues
revealed higher staining of T cells isolated from peripheral lymphoid organs than from
TIL, which corroborates the observation that the percentage of galectin-3hi CD8+ T cells
in the TIL is significantly higher than observed in the tumor draining lymph node or the
spleen (Figure 9A). In addition, removal of sialic acid residues with sialidase increases
the amount of galectin-3 detected on the surface of CD8+ T cells after a 5 hour
stimulation with T2-Dq cells pulsed with RNEU420–429 (Figure 9B). Interestingly, antigen
exposure appears to increase surface galectin-3, which suggests a role for autocrine
secretion from the CD8+ T cell.
Determination of factors influencing sialylation of CD8+ T cells
Previous work has demonstrated the importance of cell surface glycan chains, and
their impact on T cell function by influencing receptor signaling on the T cell surface (75,
76). Further, activation of CD8+ and CD4+ T cells can lead to extensive remodeling of
cell surface glycoproteins by altering expression of glycan transferases. Notably, the
amount of sialylated glycans was dramatically reduced (77). Our observations thus far
that terminal sialylation of glycan chains appears to inhibit galectin-3 binding are
consistent with what has been described in the literature. However, this does not explain
why activated, terminally differentiated CD8+ T cells in the periphery remain sialylated
while those in the TME do not. To test whether or not desialylation of CD8+ T cells and
31
galectin-3 binding is specific to the TME or a result of antigen exposure, we used a
mouse model of acute graft versus host disease where adoptively transferred H-2b CD8+ T
cells develop an alloresponse against H-2d splenocytes in the host mouse (78). At 8
weeks post-adoptive transfer we observed marked splenomegaly reflecting the degree of
engraftment by adoptively transferred cells (Figure 10A). If galectin-3 binding were due
solely to antigen exposure and glycan remodeling, then one would expect to see increased
surface galectin-3 on engrafted H-2b CD8+ T cells; however, the amount of surface
galectin-3 detected by flow cytometry was similar to the level seen in our neu-N mouse
model (Figure 10B). Therefore, we reasoned that the TME must provide conditions that
promote galectin-3 binding that are not satisfied elsewhere. Because of low sialylation
observed in the TME, we wondered if a sialidase could be secreted either by tumor cells
or other cells in the TME. Using the neu-N mouse model, we tested to see if directly
injecting a sialidase inhibitor into mouse tumors could reduce the amount of galectin-3
binding observed on adoptively transferred high avidity CD8+ TILs (Figure 11).
However, we observed no difference between treated and untreated controls indicating
that either the drug treatment did not penetrate into the TME as we had hoped or that
other factors in the TME must be responsible for glycan remodeling and galectin-3
binding in and in vivo setting.
Depletion of extracellular galectin-3 leads to improved T cell function
Despite our findings that galectin-3 suppresses IFN production by high avidity
antigen-specific CD8+ T cells in vitro, we were not able to observe a similar effect on
TILs in vivo when we sorted on galectin-3hi and galectin-3lo T cells. We reasoned that this
32
discrepancy may be due in part to the observation that galectin-3 binds to the majority of
CD8+ T cells that traffic into the TIL, and that a truly galectin-3 negative T cell
population does not exist in the TIL. While categorizing cells into galectin-3hi or galectin3lo may be sufficient to study MFI shifts in surface protein expression, it may not be
sufficient for examining functional differences. Further, galectin-3 can be expressed and
secreted by a variety of cell types, making it difficult to study the impact of its absence
using the current model (79). Thus, in order to accurately determine a functional role for
galectin-3 in vivo, we bred the neu-N and high avidity TCR transgenic mice onto a
galectin-3 genetic knockout background.
We first evaluated the genetic knockout of galectin-3 in only the neu-N mouse
and found that it was not sufficient to completely abolish galectin-3 binding on the
surface of high avidity CD8+ T cells, although it did result in an overall decrease in
galectin-3 expression (Figure 12A). However, galectin-3 knock out in both the high
avidity CD8+ T cell and the neu-N recipient mouse completely abolished galectin-3
binding on high avidity CD8+ T cells (Figure 12B). This model allows the removal of
galectin-3 from TILs without disrupting galectin-3 expression in the transplantable tumor
cells themselves, thus demonstrating that the major source for galectin-3 are tumorreactive CD8+ T cells and cells belonging to the tumor stroma rather than the tumor itself.
Based on prior reports, galectin-3 can mediate CD8+ T cell anergy by sequestering
the TCR from the CD8 co-receptor, raising the threshold of activation (62). We therefore
evaluated the outcome of reduced galectin-3 on TCR avidity by tetramer staining. As
expected, removal of galectin-3 from the surface of high avidity CD8+ T cells did not
33
result in a significant change in tetramer binding, confirming that any observed changes
in T cell function is not a result of increased TCR signaling (Figure 12C).
Next, we evaluated the effect on CD8+ T cell function in TIL populations after
genetic removal of galectin-3 from the neu-N recipient alone, in the T cell alone, or in
combination. We observed an increase in the total number of IFN and Granzyme B
producing T cells only when galectin-3 is knocked out in both the T cell and the recipient
mouse. Depletion of galectin-3 in CD8+ T cells alone or in the recipient mouse alone was
insufficient to cause an improvement in CD8+ T cell effector function as measured by
cytokine production (Figure 13A-B). This observation is consistent with our earlier
finding that elimination of galectin-3 expression is required in both the recipient mouse
and the adoptively transferred T cell to completely remove galectin-3 from the cell
surface.
After demonstrating improved effector function after removal of galectin-3, we
next tested the ability of high avidity CD8+ T cells to promote long-term tumor control in
a galectin-3 null environment. We previously determined that a minimum of 4x106 high
avidity CD8+ T cells was required to achieve long-term tumor control in >75% of neu-N
mice (ref). Thus, to illustrate the increase in efficacy of CD8+ T cells in galectin-3 null
mice, we wanted to see if galectin-3 knockout (KO) high avidity CD8+ T cells could
mediate a similar level of clearance using half the amount of T cells required for clearing
tumor in galectin-3 wild type (WT) mice. We compared long-term tumor control in WT
neu-N mice receiving 2x106 WT high avidity CD8+ T cells, WT neu-N mice receiving
2x106 WT high avidity CD8+ T cells in addition to treatment 3 times a week with the
galectin-3 inhibitor GCS-100, galectin-3 KO neu-N mice receiving 2x106 galectin-3 KO
34
high avidity CD8+ T cells, and galectin-3 KO neu-N mice without adoptive transfer
(Figure 14A). In addition, we also compared long-term tumor control in WT neu-N mice
receiving adoptive transfer of either 2x106 galectin-3 WT or KO high avidity CD8+ T
cells (Figure 14B). All mice were given the minimal tumorigenic dose of 5x104 NT2.5
cells on Day 0 followed by CY on Day 1, whole cell GM-CSF vaccine on Day 2, and
adoptive transfer on Day 3. Consistent with our functional data, we only observed
improved tumor-free survival in the galectin-3 KO mice that had received galectin-3 KO
CD8+ T cells with 90% of mice tumor free at the end of 60 days versus 50% of the
control mice. Galectin-3 WT mice that received galectin-3 KO CD8+ T cells did not show
any survival advantage over control mice. Interestingly, treatment with the galectin-3
inhibitor, GCS-100, resulted in an initial delay in tumor growth. Furthermore, because
100% of galectin-3 KO mice that did not receive adoptive transfer of high avidity CD8+ T
cells rapidly developed tumors by Day 20, we show that tumor clearance in these mice is
mediated by tumor-specific high avidity CD8+ T cells, which may not be present at high
enough numbers in the endogenous host without adoptive transfer.
To confirm the more general role of galectin-3 in regulating antigen-specific T
cell responses, we used the transplantable Panc02 tumor model to compare endogenous
anti-tumor T cell responses in galectin-3 KO mice versus WT mice. The Panc02 tumor
cell line has many similarities to human PDA, and has been used previously to study
PDA specific vaccine induced in vivo T cell responses and the role of regulatory T cells
(Tregs) in preventing antitumor immunity (80). Mice were given a tumor burden of
2.5x105 Panc02 tumor cells injected subcutaneously in the right flank on Day 0 followed
by CY (100 mg/kg) and an anti-CD25 antibody, PC61 (50 mg) on Day 2 (to deplete
35
Tregs), and a whole cell GM-CSF vaccine on Day 3. Mice were then followed for 60
days for tumor growth. We observed that at the end of the 60 day period, a significantly
higher percentage of galectin-3 KO mice were tumor free versus control WT mice
(Figure 14C). Thus, these data provide for the first time evidence that galectin-3
expression by tumor-specific high avidity CD8+ T cells inhibits their function in vivo, and
that the source of galectin-3 is provided by both the T cells and the tumor stromal cells.
Galectin-3 removal leads to increased expression of pro-inflammatory genes in CD8+ T
cells
Due to the increase in the numbers of IFN and Granzyme B producing high
avidity CD8+ T cells observed when galectin-3 is knocked out, we were interested in
identifying other molecules whose expression may also be affected by the presence of
galectin-3 on the cell surface. We used whole genome microarray analyses of galectin-3
WT CD8+ TILs isolated from galectin-3 WT neu-N mice (WT/WT), galectin-3 WT CD8+
TILs isolated from galectin-3 KO neu-N mice (WT/KO), and galectin-3 KO CD8+ TILs
isolated from galectin-3 KO neu-N mice (KO/KO). Adoptively transferred high avidity
CD8+ T cells in TILs were purified from the endogenous T cell population by FACS
sorting on Thy1.2. and global gene expression patterns were compared between groups.
We found that in both KO/KO and WT/KO CD8+ T cells, molecules associated with
inflammatory response processes were increased relative to WT/WT cells. Specifically,
CCL8, CXCL16, Gp49a/Lilrb4, Lyz1/Lyz2, RNASE3, CCL13, Chi3l3/Chi3l4, CYBB,
SHC1, TYROBP, CD86 were upregulated at least 2-fold or higher in in WT/KO CD8+ T
cells. Some similar and some additional genes were upregulated 2-fold or higher in
36
KO/KO CD8+ T cells including CCL8, CXCL16, Gp49a/Lilrb4, Lyz1/Lyz2, RNASE3,
C3AR1, C5AR1, CXCL9, S100A8, S100A9, SPI1, IRF1, Tlr13, GAB2, Sirpb1a (Figure
15). Analyses of upstream regulator pathways predicted activation of the TNFα, IFN,
and IL-6 signaling pathways in the KO/KO CD8+ T cells but not in WT/KO CD8+ T cells,
suggesting a possible mechanism behind why increased effector function is observed
only in a completely galectin-3 null mouse (see Appendix A for Table 2).
Increased expression of the genes CCL8, CXCL16, Gp49a/Lilrb4, Lyz1/Lyz2, and
RNASE3 are observed in both WT/KO and KO/KO groups in comparison with WT/WT
mice, suggesting the expression of these genes are influenced by the presence of galectin3 specifically in the recipient mouse but are independent of whether or not galectin-3 is
expressed in CD8+ T cells. In contrast, C3AR1, C5AR1, CXCL9, S100A8, S100A9, SPI1,
IRF1, Tlr13, GAB2, and Sirpb1a are only increased in expression when galectin-3
expression is completely eliminated. A comparison between WT/KO and KO/KO CD8+
T cells reveal very few changes with respect to inflammatory response genes with the
exception of S100A9, which is often found to be co-expressed with S100A8, and has also
been shown to stabilize S100A8 expression via a posttranscriptional mechanism (81).
S100A8 and S100A9 are EF-hand Ca2+ binding proteins that are heavily involved in
several inflammatory processes and possess tumoricidal properties in vitro. These
inflammatory response genes have been shown to be expressed by neutrophils, but not
yet by T cells (82). We show for the first time that S100A8 expression is increased
almost 7-fold in galectin-3 KO/KO CD8+ T cells when compared with WT/WT cells
(Figure 16).
37
Discussion
Our data describe three novel findings supporting galectin-3 as a regulator of
antigen-specific T cell activation within the TME. First, galectin-3 selectively targets T
cells only after they have been activated and traffic into the TME. In addition, the T cells
themselves and host derived cells within the TME, but not the tumor cells, are the major
sources of galectin-3 that mediate galectin-3 dependent T cell suppression. Second,
galectin-3 regulation of activated T cells leads to genome wide changes in inflammatory
gene expression. Third, galectin-3 co-expresses with LAG-3 on activated and terminally
differentiated T cells, and functional LAG-3 is required for galectin-3 mediated T cell
suppression.
We show for the first time that galectin-3 regulation of T cells occurs primarily in
the TME, and selectively targets only tumor-specific T cells. In addition, this is the first
example that galectin-3 mediates T cell suppression as a result of autocrine secretion of
galectin-3 by the T cells themselves, as well as by non-tumor cells likely residing in the
tumor stroma. Other groups have also observed that activated CD8+ T cells readily bind
more galectin-3 than naïve T cells (83). One hypothesis proposed by Demotte et.al. and
others is that activation of CD8+ T cells leads to changes in glycosylation machinery that
increases the number and accessibility of LacNAc motifs available for galectin-3 binding
(84). However, glycan remodeling resulting from activation still fails to fully explain why
only a small percentage of activated, effector CD8+ T cells in peripheral lymphoid tissue
stain positive for galectin-3 binding while the majority of activated CD8+ T cells in the
tumor do bind galectin-3. Thus, the TME must provide other conditions that are not
satisfied elsewhere to promote galectin-3 binding in order to promote an
38
immunosuppressive environment. Preliminary experiments with sialidase inhibitors were
not successful in either reducing surface galectin-3 or increasing glycan sialylation. These
findings are consistent with microarray results demonstrating 2-fold downregulation of
ST6GAL1 in galectin-3hi staining CD8+ T cells. Future work should focus on clarifying
regulation of glycan remodeling in CD8+ T cells that reside in peripheral lymphoid
tissues versus in the TME.
The leading assumption has always been that tumor cells are a major source for
galectin-3 (85). While this may be the case for certain tumor types, our data demonstrate
for the first time that tumor cells are not always the source of extracellular galectin-3.
Rather, galectin-3 expression by tumor-specific CD8+ T cells and stromal cells within the
TME, result in cancer-specific CD8+ T cell suppression. A likely candidate in the stromal
compartment could be fibroblasts as they have been shown in models of fibrosis to
secrete large amounts of galectin-3, and are also known to be important for establishing
the TME (86). Our findings highlight the importance of targeting not just tumor cells, but
also stromal cells for effective cancer immunotherapy. These data also imply that T cells
may regulate their own activity via the autocrine secretion of galectin-3, but interestingly,
this only appears to occur in the TME. Future studies should focus on identifying the
specific tumor stromal cell types that secrete galectin-3 as well as what other additional
factors provided by the TME promote galectin-3 binding to T cells.
This is also the first study to conduct a genome wide analysis evaluating changes
that occur in tumor-specific CD8+ T cells resulting from binding of extracellular galectin3 in an in vivo setting. The data demonstrates that removal of galectin-3 leads to
increased expression of proinflammatory genes within CD8+ T cells providing additional
39
evidence that galectin-3 is a member of a suppressive network of signals. Recent data
from several groups demonstrate the capacity for galectin-3 to suppress T cell function by
inducing T cell anergy via TCR clustering, and that these T cells can be rescued by
removing surface galectin-3 (55). Our gene array findings demonstrate that the
mechanisms of T cell regulation by galectin-3 extends beyond TCR signaling, and
includes altering T cell fate at the gene expression level. These findings are not surprising
given the extent of glycosylation occurring on cell surface proteins, and the importance of
T cell co-receptors for overall T cell function. Furthermore, while these previous studies
were able to reduce the amount of galectin-3 bound to the surface of T cells using
antibodies or inhibitors, they were not able to completely eliminate galectin-3 binding
from the surface of the T cell. We show that after optimizing for TCR avidity, further
improvements in T cell function were still attainable after complete elimination of
galectin-3 from the cell surface. Taken together, these observations suggest multiple
layers of T cell inhibition by galectin-3, and that TCR avidity is just one mechanism in
the broader scheme of galectin-3 regulation. Future research should focus on improving
galectin-3 targeted therapies to increase elimination of bound galectin-3 in order to
maximize T cell function. Additional studies should further elucidate the role of the
S100A8/9 signalizing pathway as a potential mediator of T cell lytic activity.
PD-1 and LAG-3 are two major co-receptors that have been shown to modulate T
cell function, and their co-expression has been shown to be associated with regulating
terminal T cell activation/exhaustion (67). Our finding that LAG-3 expression is
associated with galectin-3 binding, and further, is required for galectin-3 suppression in
vitro provides a new mechanism through which LAG-3 can regulate CD8+ T cells. LAG-
40
3 is capable of negatively regulating the function of CD8+ T cells despite the fact that
CD8+ T cells do not interact with LAG-3’s known ligand, MHC II. Anti-LAG-3 antibody
therapy was shown to reverse this effect in CD4+ depleted mice, which indicates a direct
role for LAG-3 on CD8+ T cells (87). The mechanism by which LAG-3 mediates CD8+ T
cell activity is unknown. LAG-3 can be extensively glycosylated, and as a result, would
be a likely target for galectin-3 binding. Our data provide the first evidence that LAG-3
signaling can be induced by galectin-3 cross-linking, and this may be one of the primary
means by which galectin-3 regulates T cell function. Furthermore, our coimmunoprecipitation studies of LAG-3 and galectin-3 support a direct physical
interaction. Lastly, it is interesting to point out that while increased PD-1 expression was
also associated with galectin-3 binding, we did not observe any functional relationship
between the two. Thus, it is possible that either galectin-3 does not bind PD-1 at all or
galectin-3/PD-1 interactions are irrelevant. The latter highlights the fact that not all
lectin-glycan interactions are functionally consequential. Further studies are required to
connect galectin-3 surface binding with signaling downstream of LAG-3.
Our work indicates that CEACAM1 (CD66) should remain a molecule of interest
given the strong correlation between galectin-3 surface binding and CD66 expression.
Work done in Jurkat T cells has demonstrated that cross-linking of CEACAM1 with
antibody was sufficient to lead to phosphorylation of ITIM motifs, which then allow for
the recruitment of phosphatases that dephosphorylate ZAP-70 and result in
downregulation of TCR signaling (73). The fact that we were not able to successfully
repeat these experiments with primary mouse T cells stimulated in vitro, and using
galectin-3 as a cross-linker for CEACAM1 rather than antibody could be due to several
41
reasons. First, mouse T cells express a variety of different glycoforms of CEACAM1 that
are not expressed by human T cells. Second, CEACAM1 exists in both long
(CEACAM1-L) and short (CEACAM1-S) isoforms in mouse cells, whereas it exists
almost exclusively in the long isoform in human cells (88). The cytoplasmic tail of the
long isoform is required for ZAP-70 dephosphorylation. As a result, ZAP-70
dephosphorylation may not be detectable in mouse cells due to the presence of
CEACAM1-S, and the variety of different glycoforms that exist which may or may not
be amenable to galectin-3 cross-linking. Therefore, future studies examining the
relationship between CEACAM1 and galectin-3 should utilize human cells rather than
mouse cells.
42
Figure 3. Increased surface galectin-3 on high avidity CD8+ T cells in the tumor
microenvironment.
A) Tissues were processed according to the procedure outlined in the materials and
methods section 5 days after adoptive transfer into neu-N mice. Galectin-3 surface
staining is shown for CD8+ T cells gated on Thy1.2 expression. B) Histogram overlays
show comparisons of galectin-3 MFI on adoptively transferred high avidity CD8+ T cells
(Thy1.2+) versus on endogenous CD8+ T cells (Thy1.2-) found in the tumor
microenvironment.
43
Figure 4. Activation of CD8+ T cells increases surface bound galectin-3.
A) CD8+ T cells were negatively isolated from splenocytes and stimulated in the presence
of 30 U/mL rhIL-2 with anti-CD3/CD28 beads. Galectin-3 surface staining is shown after
3 days of stimulation in comparison with naïve cells. B) High avidity CD8+ T cells were
labeled with 1.5 μM CFSE prior to adoptive transfer into neu-N mice. The tumor draining
node (TDN) was dissected 3 days after adoptive transfer and processed according to the
materials and methods section. Galectin-3 surface staining is shown with CFSE dilution
and CD44 expression for cells gated on Thy1.2. C+D) Tumor infiltrating lymphocytes
(TIL) were extracted from tumors 5 days after adoptive transfer according to the
materials and methods section. Cells were gated on Thy1.2 and divided into galectin-3hi
and galectin-3lo staining populations based on isotype staining controls. A comparison of
PD-1 and LAG-3 co-expression is shown for galectin-3hi and galectin-3lo populations in
C) and Annexin V staining in D).
44
45
Figure 5. LAG-3 but not PD-1 expression is required for galectin-3 suppression of
The effect of increasing concentrations of extracellular galectin-3 on the percentage of
IFN producing CD8+ T cells is shown for A) PD-1 knockout versus wild type T cells in
and B) LAG-3 knockout versus wild type T cells. Experiments performed at least 2
independent times with 3 replicates per sample with errors bars representing SD. C) Coimmunoprecipitation of LAG-3 and galectin-3 from activated CD8+ T cell lysates with
either a galectin-3 specific antibody (M3/38) or a LAG-3 specific antibody (410C9). D)
Co-immunoprecipitation of a LAG-3/galectin-3 complex with LAG-3 specific antibody
after cross-linking of cell surface proteins on activated CD8+ T cells prior to cell lysis as
described in materials and methods.
46
47
Figure 6. Ingenuity Pathway Analysis of gene expression differences between
galectin-3hi CD8+ TILS vs. galectin-3lo CD8+ TILS identified CD66 and ST6GAL1
as genes of interest.
Adoptively transferred high avidity CD8+ T cells were isolated from tumor tissue as
described in materials and methods and then sorted based on staining for Thy1.2 and
galectin-3. Galectin-3hi versus galectin-3lo cells were separated based on the top 5% and
bottom 5% of galectin-3 MFI. Gene expression changes were filtered based on fold
change > 2 and p > .05.
48
Figure 7. Co-staining of CD66 and Galectin-3 on high avidity CD8+ TILs.
A) Adoptively transferred high avidity CD8+ T cells were isolated from tumor, and
stained for surface galectin-3 as detailed in materials and methods. Cells were partitioned
into separate populations based on galectin-3 MFI. B) Histogram overlays comparing
CD66 MFI among cell populations with increasing galectin-3 MFI. C) Logarithmic
transformation of galectin-3 MFI vs. CD66 MFI demonstrate a linear correlation between
galectin-3 surface staining and CD66 expression.
49
50
Figure 8. In vitro dephosphorylation of ZAP-70 after cross-linking of CD66 with
galectin-3.
Galectin-3 KO CD8+ T cells were isolated and treated according to procedures outlined in
materials and methods. Cells were incubated with 5 g/mL galectin-3 for cross-linking.
After cell lysis, 7.5 g of total protein was loaded for each time point. Chemiluminescent
film was exposed for 3 minutes prior to development.
51
Figure 9. Galectin-3 binding to the surface of CD8+ T cells is determined by α2,6sialylation.
A) Bar graph demonstrating percent of adoptively transferred high avidity CD8+ T cells
with galectin-3 MFI greater than isotype control reveals majority of galectin-3 binding
occurs in the TME. Inset demonstrates inverse relationship between galectin-3 binding
and sialic acid as reflected by staining with FITC-Sambucus nigra lectin. B)
Pretreatment of high avidity CD8+ T cells isolated from TDN 3 days post adoptive
transfer with sialidase prior to 5 hour stimulation with either T2-Dq pulsed with NP or
specific peptide, RNEU420–429 increases binding capacity for galectin-3 on the T cell
surface.
52
53
Figure 10. Galectin-3 binding on adoptively transferred alloreactive CD8+ T cells in
a model of graft versus host disease.
6 x 107 H2-Kb splenocytes isolated from female C57Bl/6 mice were adoptively
transferred into female B6D2F1 mice, which possess the H2-Kb/d allotype. Spleens were
harvested at 8 weeks post-adoptive transfer. A) Percent of CD8+ T cell engraftment in
spleens demonstrated by gating on CD8+ H2-Kb+/d- T cells. Inset – size of spleens
reflecting degree of engraftment. B) Histogram overlays demonstrate surface galectin-3
MFI on alloreactive CD8+ T cells isolated from B6D2F1 spleens, high avidity CD8+ T
cells from neu-N spleens, and high avidity CD8+ T cells from neu-N TIL.
54
55
Figure 11. Pretreatment of mouse tumors with methyl ester sialidase inhibitor does
not increase surface galectin-3.
A) Mouse tumors were directly injected with 100 M of a 10 mM solution of methylester conjugated sialidase inhibitor in DMSO on days 3 and days 4 post adoptive transfer
prior to TILs being extracted on day 5 and analyzed for surface galectin-3 by flow
cytometry. Histogram overlays comparing untreated vs. treated mice are shown. B) SNAFITC staining demonstrating no increase in sialylation as a result of sialidase inhibitor
treatment.
56
Figure 12. Removal of galectin-3 from the surface of high avidity CD8+ T cells
requires inhibition of galectin-3 expression in the tumor stroma and the T cell.
Tumor infiltrating lymphocytes (TIL) were isolated 5 days post adoptive transfer
according to the materials and methods section. Cells were gated for Thy1.2 expression.
A) Galectin-3 wildtype (WT) high avidity CD8+ T cells were adoptively transferred into
either galectin-3 WT (red) or galectin-3 KO neu-N (blue) recipient mice. Histogram
overlays show differences in galectin-3 MFI between the two groups with respect to
isotype control staining (orange). B) Galectin-3 staining is now shown on galectin-3 KO
high avidity CD8+ T cells adoptively transferred into a galectin-3 KO neu-N recipient
(blue) with respect to an isotype control (red). C) TCR avidity is compared between
galectin-3 WT high avidity CD8+ T cells from a galectin-3 WT neu-N recipient and
galectin-3 KO high avidity CD8+ T cells from a galectin-3 KO neu-N recipient. TCR
avidity was assessed by staining with MHC I Dq tetramer loaded with the RNEU420-429
(P50) peptide.
57
Figure 13. Removal of surface galectin-3 from high avidity CD8+ T cells leads to
improved effector function.
Effector function of TILs was assayed after elimination of galectin-3 in the CD8 T cell,
the tumor stroma, or both. Tumors were removed 5 days after adoptive transfer of high
avidity CD8+ T cells, and TILs were isolated according to the protocol outlined in
materials and methods. Cytokine expression was assessed by intracellular cytokine
staining (ICS) after a 5 hour incubation with a TAP-2 deficient cell line (T2-Dq)
expressing MHC I Dq loaded with either RNEU420-429 (P50) or LCMV NP (NP). The
absolute number is shown for Thy1.2+ CD8+ T cells / mg tumor expressing either A) IFNγ or B) Granzyme B.
58
Figure 14. Galectin-3 knockout mice have improved tumor free clearance over wild
type mice in both adoptive transfer and endogenous tumor vaccine models.
A) All neu-N recipient mice received the minimal tumorigenic dose of 5x104 NT2.5 cells
on day 0, followed by 100 mg/kg CY on day 2, and whole cell GM-CSF vaccine on day
3. Adoptive transfer groups (n=10) received 2x106 galectin-3 WT or KO high avidity
CD8+ T cells as indicated above on day 4. A control group of galectin-3 KO mice (n=7)
did not receive adoptive transfer in order to assess the endogenous anti-tumor response.
Mice were followed every 5 days for development of palpable tumor formation at the
injection site. One tailed p value is shown for KO/KO vs. WT/WT groups B) Mice were
treated as in A), and galectin-3 WT neu-N mice received adoptive transfer of either 2x106
galectin-3 WT high avidity CD8+ T cells (n=10) or 2x106 galectin-3 KO high avidity
CD8+ T cells (n=10). C) WT C57Bl/6 mice (n=15) or galectin-3 KO C57Bl/6 mice
(n=15) received 2.5x105 Panc02 tumor cells on day 0, 100 mg/kg CY and 50 μg PC61 on
day 2, and whole-cell GM-CSF vaccine on day 3. Mice were followed every 5 days for
development of palpable tumor formation at the injection site.
59
60
Figure 15. Removal of galectin-3 increases expression of inflammatory molecules in
high avidity CD8+ T cells.
This figure summarizes microarray findings comparing high avidity galectin-3 WT CD8+
TILs from galectin-3 WT neu-N mice (WT/WT) (n=4), high avidity galectin-3 WT CD8+
TILs from galectin-3 KO neu-N mice (WT/KO) (n=4), and high avidity galectin-3 KO
CD8+ TILs from galectin-3 KO neu-N mice (KO/KO) (n=4). Analysis was performed
using Ingenuity Pathway Analysis (IPA) software using a filter for linear fold changes ≥
2, and p ≤ .05. Molecules listed above the arrows between each group are immuneassociated genes found to be upregulated between groups. Molecules listed below each
group are immune-associated genes found to be upregulated in comparison to the
WT/WT control. CCL8, CXCL16, Gp49a/Lilrb4, Lyz1/Lyz2, and RNASE3 were found to
be upregulated in both WT/KO and KO/KO groups in comparison with WT/WT. (*)
denotes transcription factors.
61
Figure 16. Western blot analysis of TIL lysates confirm S100A8 expression in Gal-3
KO/KO TIL but not Gal-3 WT/WT TIL.
Adoptively transferred galectin-3 WT high avidity CD8+ T cells and galectin-3 KO high
avidity CD8+ T cells were purified by FACS sorting on cells expressing the Thy1.2
surface marker. Cells were then lysed and total protein concentration quantified by
measuring absorbance at 280 nm. 6 μg of total protein was loaded per sample.
62
CHAPTER 4: DETERMINING THE ROLE OF GALECTIN-3 IN THE
INFLAMMATORY RESPONSE
Introduction
The major aim of cancer immunotherapy is to induce an immune response against
tumor cells akin to the immune response observed against foreign antigens such as
viruses and bacteria, which are rapidly cleared by the immune system. In non-tolerized
FVB/N mice, GM-CSF vaccination against NT2.5 tumor cells can induce potent tumorspecific high avidity CD8+ T cells that produce IFN, TNF, and IL-2 (15, 65). Unlike
foreign antigens, tumor cells are recognized by the immune response as “self” antigens,
and thus several tolerogenic mechanisms exist in the periphery and the TME to prevent
the induction of a potent anti-tumor immune response. These mechanisms include Tregulatory cells (Tregs), tolerogenic antigen-presenting cells, myeloid-derived suppressor
cells (MDSCs), immunoregulatory molecules, and immunosuppressive cytokines (89-92).
As previously mentioned in Chapter 1, galectin-3 has been shown to be involved
in the regulation of both the innate and the adaptive immune response, and functions as a
regulatory molecule during every step of the continuum from acute inflammation to
chronic inflammation and fibrosis (93). Several published reports have suggested a proinflammatory role for galectin-3. It has been shown to be critical for neutrophil
recruitment and migration in mouse models of S. pneumoniae infection (94, 95), and
triggering the oxidative burst (96, 97). However, its impact on monocytes and
macrophages, which lie at the intersection between the innate and adaptive immune
response appears to be less clear. While some papers have shown galectin-3 to be
important for activation and recruitment of monocytes (98, 99), more recent papers have
63
shown the exact opposite, and that it inhibits monocyte to dendritic cell differentiation
(100, 101). Our microarray findings from galectin-3 KO mice discussed in Chapter 3
revealed upregulation of inflammatory pathways involving TNF, IFN, and IL-6.
Because many of the aforementioned studies involving galectin-3 and inflammation were
done in infectious disease models, we were interested in elucidating the impact of
galectin-3 deficiency on inflammation in the neu-N mouse tumor model where the
immune response is overwhelmingly tolerogenic.
Materials and Methods
Cytokine expression arrays
Mice were treated as in tumor challenge experiments, but did not receive
cyclophosphamide or adoptive transfer. Four days after vaccination, mice were
euthanized and cytokine expression in whole tissue lysates from VDNs was evaluated
using cytokine arrays. Briefly, VDNs from each experimental group were pooled and
homogenized as previously described in Chapter 3. Cells were lysed according to
manufacturer’s instructions and lysate was blotted with the Mouse Cytokine Antibody
Array, Panel A (R&D Systems, Minneapolis, MN). The dot blot was then scanned and
analyzed using VisionWorks LS (UVP, LLC, Upland, CA) to determine density per pixel
and fold-change in expression in galectin-3 KO cells in comparison with galectin-3 WT
cells.
Direct Ex Vivo Antigen Detection Assay
Mice were treated as in tumor challenge experiments, but did not receive
cyclophosphamide or adoptive transfer. Four days after vaccination, CD8+ dendritic cells
64
and plasmacytoid dendritic cells were isolated from spleen tissue using CD8+ dendritic
cell and plasmacytoid dendritic cell isolation kits (Miltenyi). CD8+ T cells were
negatively isolated from high avidity TCR transgenic mice and labeled with CFSE as
mentioned above. All cells were co-cultured at a 1:1 ratio in CTL media for 3 days before
evaluating CFSE dilution and cytokine production by FACS.
Results
Galectin-3 KO mice have increased expression of inflammatory cytokines in vaccine
draining nodes (VDNs) 4 days after receiving a GM-CSF vaccine
The microarray results discussed in Chapter 3 comparing high avidity galectin-3
KO/KO CD8+ TILs with high avidity galectin-3 WT/WT CD8+ TILs revealed
upregulation of inflammatory pathway genes associated with TNFIFNand IL-6.
Furthermore, increased expression of several inflammatory-associated molecules in
galectin-3 WT CD8+ TILs adoptively transferred into galectin-3 deficient hosts indicates
involvement of the host’s innate immune response in shaping the adaptive immune
response. Previous work has demonstrated the importance of the cytokine milieu
generated by the innate immune response in establishing an inflammatory or suppressive
environment for CD8+ T cells (102).
To confirm these findings and to begin to identify any differences in the innate
immune response to GM-CSF vaccination between galectin-3 KO and galectin-3 WT
mice, we analyzed tissue lysates of VDNs for differences in cytokine expression 4 days
after mice had received the GM-CSF vaccine. Four days post vaccination was chosen
because this is the time point that has been shown to coincide with trafficking of dendritic
cells into the draining lymph nodes (24). We found increases in TNFand IL-1 in the
65
lysates of galectin-3 KO VDNs when compared with galectin-3 WT VDNs (Figure 17).
IL-6 was not included in the array; however, IL-1 is also a pro-inflammatory cytokine,
and is capable of increasing IL-6 production (103, 104).
Inhibition of galectin-3 expression leads to an increase in plasmacytoid dendritic cells
(pDC) in galectin-3 KO mice
To begin to examine the impact of galectin-3 on cells of the innate immune
response, we first looked broadly for differences in CD11b+ and CD11c+ cell populations
in the lymph nodes of naïve galectin-3 KO and galectin-3 WT neu-N mice. Although not
specific to any single cell population, these markers help to differentiate dendritic cells
from macrophages and neutrophils. While we did not see significant differences in cells
expressing CD11b+, we did note a large increase in CD11c+ cell types. Because CD11c+
is predominantly expressed on dendritic cells, we further characterized this population of
cells for expression of B220 and Ly6C to distinguish between plasmacytoid dendritic
cells (pDC), which express CD11c+ B220+ Ly6C+, and conventional dendritic cells
(cDC), which express CD11c+ B220- Ly6C- (Figure 18). We observed a substantial
increase in numbers of CD11c+ B220+ Ly6C+ cells in the galectin-3 KO versus the
galectin-3 WT neu-N mice. Furthermore, the overall number of pDCs found in the lymph
node did not seem to vary significantly from the naïve mouse in response to tumor
challenge alone, or tumor challenge in addition to treatment with CY and whole cell GMCSF vaccine (Figure 19A). This finding indicates that galectin-3 may instead play an
intrinsic role in pDC homeostasis rather than solely during immune stimulation, as was
the case for CD8+ T cells. To test this possibility, we compared pDC populations in
66
untreated female galectin-3 KO neu-N mice, untreated female galectin-3 WT FVB/N
mice and untreated female galectin-3 WT neu-N mice. We found that pDCs were also
present in galectin-3 WT FVB/N mice, which would suggest, not surprisingly, that other
factors must also be involved in pDC regulation (Figure 19C).
A reciprocal relationship has been shown to exist between pDCs and myeloid
derived suppressor cells (MDSCs). Ablation of pDCs with the depletion antibody 120G8
leads to increased myelopoiesis and mobilization of MDSCs into peripheral lymphoid
organs, resulting in inhibition of autoimmunity (105). Because pDCs appear to be
significantly elevated in the absence of galectin-3, we looked to see if there was a
corresponding decrease in the number of MDSCs. In contrast to what was observed with
pDCs in galectin-3 KO mice, the number of MDSCs found in the lymph nodes is minimal
until immune activation with the whole cell GM-CSF vaccine. As expected, however, the
number of MDSCs found in galectin-3 KO mice is significantly lower than what was
observed in galectin-3 WT mice (Figure 19B).
Next, we determined whether or not pDCs directly activate T cells. Due to the
known plasticity of pDCs, their role in antitumor immunity remains controversial. While
pDCs are known to be involved in the promotion of inflammatory processes important
for reversing microbial infections (106), they are also known to be immunosuppressive in
the tumor microenvironment when galectin-3 is present (natural environment) (107-109).
To clarify the function of pDCs following a GM-CSF vaccination in the absence of
galectin-3, we evaluated their capacity to induce proliferation of naïve high avidity CD8+
T cells in vitro using a direct ex-vivo antigen detection (DEAD) assay. Our data indicate
that naïve T cells proliferate to a greater extent and produce more IFN when co-cultured
67
with both pDCs and conventional DCs than when co-cultured with either DC subtype
alone. Futher, co-culture of naïve T cells with just pDCs alone activated a significantly
greater number of T cells than with cDCs alone (Figure 20A-C). These findings support
a new role for pDCs in the promotion of an anticancer inflammatory response resulting in
enhanced CD8+ T cell stimulation in vaccinated galectin-3 KO mice.
Discussion
In addition to galectin-3’s direct effect on T cells, several reports have also
demonstrated galectin-3’s regulation of conventional dendritic cells, which would
indirectly impact T cells by influencing the strength of their activation as well as the
cytokine milieu they are activated in (59, 101, 110). We are the first to show a direct
correlation between inhibition of galectin-3 expression, and the expansion of pDCs.
Interestingly, LAG-3 has been reported to negatively regulate pDC homeostasis. Thus,
steady state levels of extracellular galectin-3 may be necessary to regulate pDC
expansion via LAG-3, without which, pDCs are allowed to proliferate unchecked.
Galectin-3 has also been shown to be a negative regulator of monocyte differentiation
into cDCs (101); therefore, it may be possible that a similar mechanism exists for pDC
differentiation from its precursor. Further, because pDCs are either directly or indirectly
involved with the homeostasis of both MDSC and Treg, dysregulation of pDCs may have
downstream consequences on these cell types (105). Indeed, our findings that MDSCs
proliferate significantly less in response to GM-CSF vaccination in galectin-3 KO mice
are consistent with the notion of reciprocal regulation between pDCs and MDSCs.
The observation that pDCs are present in untreated galectin-3 WT FVB/N mice
but are absent in untreated galectin-3 WT neu-N mice is puzzling.
68
While these
observations would make sense if the neu-N mice had initially received vaccine or a
tumor challenge to induce a state of tolerance that might suppress pDC expansion, these
mice were untouched. It may be possible that a low level of inflammation may already
exist due to the presence of microtumors in the neu-N mice, which could over time lead
to a state of tolerance even before the mice are tumor challenged or receive a neu-specific
vaccine. Expression of the MMTV promoter is increased during pregnancy and lactation,
but they are also expressed during embryogenesis and development of the virgin
mammary gland. Further, expression of the promoter is selective, but not specific, and is
expressed in lungs, kidneys, salivary glands, seminal vesicles, T-cells, testes, and prostate
(111).
The role of pDCs in tumor immunity remains unclear. While they are important
for antiviral immunity, they have also largely been associated with tolerance induction,
and poor prognosis in cancer. It may be possible that pDCs take on a stimulatory role in
the absence of extracellular galectin-3. Our microarray finding that the expression of
several inflammatory genes in T cells is increased as a result of galectin-3 deficiency in
only host-derived cells may help to support this hypothesis. Although these results may
be due simply to a reduction of surface galectin-3 on the T cell, a more compelling
argument is that the expansion of host-derived pDCs in the absence of galectin-3 helps to
skew the immune response toward inflammation. Galectin-3 binds preferentially to “self”
glycans, and thus, has been theorized to provide a dampening mechanism that keeps the
immune system at bay by raising the threshold of activation for immune cells (112).
Further studies will be necessary to characterize differences in cytokine production and
functionality of pDCs in the presence and absence of galectin-3.
69
Because pDCs are largely considered to be critical components of the innate
immune response rather than the adaptive response, and furthermore, have demonstrated
immunostimulatory capacities in the context of bacterial and viral infections, our data
illustrates the importance of properly evoking both arms of the immune system in the
appropriate context to combat cancerous cells. Indeed, cells associated with innate
immunity such as neutrophils and macrophages have increasingly been shown to be protumorigenic by causing rampant inflammation when allowed to accumulate unchecked
(113-115). This chronic inflammation in the TME then leads to development of an
immunosuppressive environment that is unfavorable for immunotherapy. However, with
further research, it may be possible to one day harness the inflammatory abilities of these
cells in a more controlled manner.
70
Figure 17. TNFα and IL-1 are increased in whole tissue lysates of galectin-3 KO
VDNs when compared with galectin-3 WT VDNs.
VDNs were isolated from mice 4 days after receiving GM-CSF vaccination, and
processed according to materials and methods.
71
Figure 18. CD11c+ cells are increased in galectin-3 KO mice in comparison with
galectin-3 WT mice.
Inguinal and axillary lymph nodes were dissected from naïve galectin-3 KO and galectin3 WT neu-N mice. Lymph nodes were processed according to the materials and methods
section. Cells were co-stained for CD11b and CD11c expression, and analyzed by
FACS.
72
Figure 19. An increase in pDCs in galectin-3 KO neu-N mice is associated with
lower numbers of MDSCs.
Galectin-3 KO and WT neu-N mice were left untreated (n=3), challenged with 5x104
NT2.5 tumor cells (n=3), or challenged with 5x104 NT2.5 tumor cells and treated with
CY on day 2 and whole cell GM-CSF vaccine on day 3 (n=4). All mice were sacrificed
on day 6, and axillary and inguinal lymph nodes were dissected and pooled for each
mouse. Tissues were processed according to the Materials and Methods section, and
cells stained for expression of CD11b, CD11c, B220, and Ly6C. The total number of
cells was averaged for each lymph node, and is shown for A) pDCs (CD11c+ B220+
Ly6C+) and for B) MDSCs (CD11b+ Ly6C+). C) pDC populations in untreated, naïve 810 week old feamle galectin-3 KO neu-N, galectin-3 WT FVB/N, and galectin-3 WT
neu-N. Cells are gated on B220+.
73
74
Figure 20. Plasmacytoid DCs from vaccinated galectin-3 KO mice are capable of
activating more antigen-specific naïve CD8+ T-cells than conventional dendritic
cells from vaccinated galectin-3 KO mice.
pDCs, cDCs, and high avidity Thy1.2+ CD8+ T cells were isolated according to the
procedures listed in the Materials and Methods section. Cells were co-cultured at a 1:1
ratio as specified in the figure prior to being assessed for either A) CFSE dilution or B)
IFN production by flow cytometry. Cytokine expression was assessed by intracellular
cytokine staining (ICS) after a 5 hour incubation with a TAP-2 deficient cell line (T2-Dq)
expressing MHC I Dq loaded with RNEU420-429 (P50). C) Statistical comparison of cell
division in T cells + pDC vs. T cells + pDC + cDC.
75
76
CHAPTER 5: SUMMARY
Development of an anti-tumor immune response is a constant balance between
pro-inflammatory and anti-inflammatory signals. Activation of tumor-specific CD8+ T
cells involves a complex interplay amongst not only stimulatory and inhibitory receptors
expressed by the CD8+ T cell during priming, but also dendritic cells, CD4+ T cells, and
the cytokine milieu that surrounds them. The complex signals that are involved in T cell
activation are commonly simplified into 3 signals. Signal 1 refers to the TCR-MHCIpeptide interaction. Signal 2 refers to the interaction between stimulatory and inhibitory
co-receptors, such as CD28, PD-1, and LAG-3, with their ligands. Signal 3 refers to the
cytokine milieu in which T cell priming occurs. The cytokines IL-2, TNFand IFNare
considered “inflammatory cytokines” often associated with T cell activation while IL-4,
IL-10, and TGF are “anti-inflammatory cytokines” associated with suppression. All
three of these signals are in some way or another influenced by the degree and structure
of sugar chains, or glycosylation, attached to the cell surface receptors that mediate these
“signals”. Glycosylation of cell surface proteins occurs to such an extent that it forms a
thick coat known as the glycocalyx, which is responsible for many of the cell’s “social”
functions. In this thesis, we have explored the intricate role that galectin-3 plays in
modulating the immune response from its impact on CD8+ T cell function to its global
role in establishing a pro-inflammatory environment.
In Chapter 2, we described elevated anti-galectin-3 antibody titers in response to
GM-CSF vaccination in PDA patients with > 3 year DFS when compared with PDA
patients with < 3 year DFS. Using serum collected from the > 3 year DFS patients, we
found that purified IgG from post-vaccination serum but not pre-vaccination serum could
77
neutralize galectin-3 inhibition of CD8+ T cells in vitro. Future work should now focus
on developing effective vaccination strategies targeting galectin-3. Galectin-3 can exist
in multiple conformations, and neutralizing antibodies in vitro may not necessarily have
the same effect in vivo. Furthermore, considering the role galectin-3 has been
demonstrated to play in regulating the innate arm of the immune response, it would be
worth evaluating the effectiveness of galectin-3 targeted therapy even prior to delivery of
the GM-CSF vaccine. Last, it would be interesting to elucidate what allowed these
patients to break tolerance against a secreted, and ubiquitously expressed protein like
galectin-3. Given its involvement in wound healing and inflammation, it is highly
unlikely that the whole-cell GM-CSF vaccine was the first time that the patient’s immune
system was introduced to galectin-3 in the context of a “danger” signal.
In Chapter 3, we evaluated the effect of galectin-3 on tumor-specific high avidity
CD8+ T cells in the tolerogenic neu-N mouse model of breast cancer. We identified
several new findings. First, galectin-3 appears to bind selectively to terminally
differentiated, activated CD8+ T cells, and only in the TME. Second, galectin-3 can be
secreted from antigen-experienced CD8+ T cells in an autocrine fashion, and it is also
secreted by stromal cells in the TME. Third, lack of surface galetin-3 is associated with
improved CD8+ T cell effector function, tumor free survival, and upregulation of
inflammatory pathway molecules. Fourth, sialylation blocks galectin-3 binding, and
trafficking of CD8+ T cells into the TME results in glycan remodeling and removal of 26-sialic acid. Lastly, in vitro inhibition assays with galectin-3 identified LAG-3 as a
potential binding partner for galectin-3, which was verified by co-immunoprecipitation
studies of a LAG-3/galectin-3 complex. Microarray comparisons of galectin-3hi and
78
galectin-3lo also identified CEACAM1(CD66) as a potential binding partner of galectin-3.
Flow cytometry also confirmed these findings, and demonstrated a strong correlation
between CD66 expression and galectin-3 surface binding. While functional studies
assessing ZAP-70 dephosphorylation as a potential mechanism were unrevealing,
experiments were incorrectly performed using mouse cells, and should have been done
using human cells due to reasons that were discussed in Chapter 3. From our data, it is
clear that galectin-3 has an extensive impact on CD8+ T cell function. Whether or not it
exerts its effects directly by cross-linking cell surface glycoproteins or indirectly through
its effect on other cell populations remains controversial. Future studies should now
focus on demonstrating inhibition of CD8+ T cells by galectin-3 via cross-linking of
glycoproteins in vivo, and verifying the LAG-3/galectin-3 interaction in vivo as well.
In Chapter 4, we examined the effect of galectin-3 deficiency on tipping the
balance between pro-inflammatory signals and anti-inflammatory signals. As previously
described, galectin-3 is thought to raise the threshold of immune cell activation by
sequestering surface receptors from interacting with their ligands. Microarray data in
Chapter 3 revealed upregulation of inflammatory pathways involving TNFIFN, and
IL-6 in galectin-3 KO/KO CD8+ TILs versus galectin-3 WT/WT CD8+ TILs. We
confirmed this using western blotting of cytokine arrays using whole tissue lysates of
VDNs 4 days after GM-CSF vaccination, and found increased expression of TNF and
IL-1. Because cells of the innate immune response are often the first to establish the
cytokine milieu, we compared the neutrophil and monocyte populations in lymph nodes
of untreated galectin-3 WT neu-N and galectin-3 KO neu-N mice by assessing
differences in CD11b+ and CD11c+ populations by flow cytometry. We found that pDCs
79
were significantly increased in galectin-3 KO mice, and that these cells were superior to
cDCs at activating CD8+ T cells. Because pDCs are also present in galectin-3 WT
FVB/N mice at the same level but are completely absent in neu-N mice, expression of the
neu-N oncogene must be exerting an effect on pDCs even in virgin mice. Low level
expression of the neu-N oncogene in virgin mice, and development of low grade
inflammation resulting from immunosurveillance of dysplastic cells may initiate the
development of tolerance even at a very young age. Thus, future work should now focus
on inflammation as it relates to tumorigenesis, and how galectin-3 mediates this process.
The Pdx-1-Cre, LSL-Trp53R172H/-, LSL-KrasG12D/- (KPC) mouse model of PDA
development is ideal for these experiments. These mice contain a pancreatic tissuespecific knock-in allele of activated KRasG12D mutant as well as a knock-in allele of
p53R172H mutant. These mice recapitulate the progression of PanIN lesions to PDA
observed in human patients, but at an accelerated rate. We have already crossed these
mice onto a galectin-3 KO background.
In summary, these findings develop for the first time a comprehensive profile of
the extensive impact that galectin-3 plays at every stage of an anti-tumor immune
response in a tolerogenic mouse model. To our knowledge, we are the first to develop
such a model in a galectin-3 deficient background, which provided us with the unique
ability to evaluate in vivo the role of galectin-3 after immune priming with a whole-cell
GM-CSF secreting cancer vaccine in a mouse model that accurately recapitulates the
tolerance mechanisms of human patients. We propose that patients who develop antigalectin-3 antibody titers in response to vaccination are able to neutralize galectin-3’s
immunosuppressive effects either directly or by disruption of galectin-3/receptor lattices,
80
and as a result, mount a more effective cytotoxic CD8+ T cell response against tumor
cells. TCR engagement, co-receptor activation, and the cytokine milieu have long been
considered the three signals that determine T cell fate. However, consideration of protein
glycosylation as a “Signal 4” may now help to uncover a new class of molecules for
immunomodulation (Figure 21).
81
Figure 21. N-glycosylation of co-regulatory molecules serves as the signal 4 required
for optimal T cell activation.
In the vaccine draining node (VDN) where naïve T cell priming occurs, activating signals
are delivered via TCR recognition of peptide-MHC I complexes presented by dendritic
cells (Signal 1) and interactions between co-stimulatory molecules and their ligands
(Signal 2). Furthermore, cytokines secreted by regulatory cell populations also impact
cellular differentiation (Signal 3). Plasmacytoid dendritic cells (pDCs) are increased in
number in the absence of extracellular galectin-3, and the cytokines they secrete may
skew T cell differentiation in the direction of a more pro-inflammatory phenotype.
Activation of CD8 T cells leads to increased expression of the co-regulatory molecules
PD-1 and LAG-3 by terminally differentiated effector CD8 T cells. In the tumor
microenvironment, expression of PD-L1 by tumor cells can suppress T cell function via
PD-1. Changes in glycosylation of LAG-3 (Signal 4) make it a more favorable target for
galectin-3 binding, which in turn leads to cross-linking and activation of the LAG-3
signaling complex. Cross-linking of LAG-3 results in suppression of effector CD8 T cell
function. Lack of galectin-3 increases S100A8/9 expression, which may also lead to
optimization of lytic function.
82
83
APPENDICES
Appendix A
See file Table 2.xls in supplemental files.
84
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CURRICULUM VITAE
The Johns Hopkins University School of Medicine
Theodore Kouo
March 4, 2015
Educational History:
M.D., Ph.D. expected 2015
Program in Immunology Johns Hopkins University School
of Medicine
Mentors: Elizabeth Jaffee, M.D.
B.A.
2004
Molecular Biology and Genetics Northwestern University
Other Professional Experience:
Research rotation
2007
ORISE Fellow
Research elective
2004-2006
2002-2004
Lab of Dr. Elizabeth Jaffee, Johns Hopkins
University School of Medicine
Centers for Disease Control Influenza Division
Lab of Dr. Karla Satchell, Northwestern School of
Medicine
Scholarships and Fellowships:
2004
ORISE Fellowship
2003
ASM Undergraduate Research Fellowship
Academic Honors:
2004
Summa Cum Laude
2004
Phi Beta Kappa
2003
Oliver Marcy Scholars Award
2000-2004
Weinberg College of Arts and Sciences
Dean’s List
Northwestern University
Northwestern University
Northwestern University
Northwestern University
Publications:
Kouo T.S., Huang L.Q., Pucsek A.B., Cao M., Solt S., Armstrong T.A., Jaffee E.M.
(2015) Galectin-3 shapes anti-tumor immune responses by suppressing CD8+ T cells via
LAG-3 and inhibiting expansion of plasmacytoid dendritic cells. Cancer Immunology
Research. 2015 Feb 17. pii: canimm.0150.2014.
Weiss V.L., Lee T.H., Song H., Kouo T.S., Black C.M., Sgouros G., Jaffee E.M., and
Armstrong T.D. Trafficking of high avidity HER-2/neu-specific T cells into HER-2/neuexpressing tumors after depletion of effector/memory-like regulatory T cells. PLoS One.
2012. 7(2):e31962.
Payungporn S, Crawford P, Kouo T, Chen L, Pompey J, Castleman W, Dubovi E, Katz J,
Donis R. Influenza A virus (H3N8) in dogs with respiratory disease in Florida. Emerg
Infect Dis 2008 Jun;14(6):902-8.
Posters and Abstracts:
106
Kouo T.S., Bowman M, Huang L.Q., Armstrong T.D., and Jaffee E.M. (2013)
Regulation of tumor specific CD8 T cells via interactions between galectin-3 and cell
surface glycoproteins. AACR Tumor Immunology Conference. Miami, FL. Poster
Presentation.
Kouo T.S., Bowman M, Huang L.Q., Armstrong T.D., and Jaffee E.M. (2013)
Regulation of tumor specific CD8 T cells via interactions between galectin-3 and cell
surface glycoproteins. AACR Annual meeting 2013. Washington, DC. Poster
Presentation.
Kouo T.S., Chen L.M, Donis, R.O (2005). High Growth Reassortants of Canine
Influenza for Diagnostic, Vaccine, and Research Applications. American Society of
Virology 35th General Meeting. State College, Pennsylvania. Poster Presentation.
Kouo T.S., Satchell, K.F. (2004). Mapping the Active Domains of V. cholerae RtxA with
mAbs. American Society of Microbiology 104th General Meeting. New Orleans, LA.
Poster Presentation.
Service and Leadership:
2014
Transition to the Wards Patient Coordinator and Preceptor
2010-2012
TA for Medical Student Genes to Society Cell Physiology Course
107