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Characterization of invariant natural killer T cells in a novel humanized HBV-transgenic model

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Characterization of invariant natural killer T cells in a novel humanized HBV-transgenic model

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Content
i

CHARACTERIZATION
OF
INVARIANT
NATURAL
KILLER
T
CELLS
IN
A
NOVEL

HUMANIZED
HBV-‐
TRANSGENIC
MODEL

by

Agnieszka
Lawrenczyk

A
Thesis
Presented
to
the

FACULTY
OF
THE
USC
GRADUATE
SCHOOL

UNIVERSITY
OF
SOUTHERN
CALIFORNIA

In
Partial
Fulfillment
of
the

Requirements
for
the
Degree

MASTER
OF
SCIENCE

(MOLECULAR
MICROBIOLOGY
AND
IMMUNOLOGY)

August
2013

Copyright
2013

Agnieszka
Lawrenczyk

ii

Acknowledgements

First
I
would
like
to
thank
my
mentor,
Dr.
Weiming
Yuan,
for
giving
me
the

opportunity
to
join
his
lab
and
for
all
the
help
and
guidance
he
has
provided
during

this
process.

I
am
also
grateful
to
Xiangshu
Wen,
M.D.,
Ph.D.,
for
teaching
me
every
step
of
FACS

analysis,
including
experimental
design,
tissue
isolation,
fluorescent
antibody
cell

staining,
use
of
the
FACS
machine,
and
data
analysis.
Likewise,
I
greatly
appreciate

the
help
and
feedback
from
the
Ou
laboratory
and
Yongjun
Tian,
PhD.,
in
the

development
of
this
project.

I
am
also
thankful
to
our
lab
manager
Seil
Kim,
for

teaching
many
laboratory
skills
I
utilized
in
this
project,
and
all
of
my
lab
members

Ran
Xiong,
Zheng-‐
xi
Dai,
and
Pooja
Valkari.

I
would
like
to
express
my
great
gratitude
to
Dr.
Axel
Schonthal
and
Dr.
James
Ou,

for
being
part
of
my
Master’s
Thesis
Committee,
and
offering
me
their
insight
and

support.

This
whole
experience
also
would
not
have
been
possible
without
the
support
of
my

classmates,
family,
and
friends.
Thank
you!

iii

Table
of
Contents

Acknowledgements
ii

List
of
Figures

List
of
Tables

iv

v

List
of
Abbreviations

vi-‐vii

Abstract
ix-‐x

Introduction
1

1.
Hepatitis
B
and
its
pathogenesis
2

2.
Immune
response
to
HBV
2

3.
Chronic
Hepatitis
B
Infection
5

4.
Natural
Killer
T
Cells
8

5.
CD1d
Antigen
Presentation

6.
Lipid
Antigens

10

11

7.
Differences
between
human
and
mouse
CD1d/NKT
lipid

Presentation
systems

12

8.
NKT
cells
in
anti-‐viral
and
anti-‐HBV
immune
responses
14

9.
Experimental
Design
15

Materials
and
Methods
16

1.
Generation
of
HBVtg
hCD1d-‐KI
mice:
16

2.
Liver
Mononuclear
Cell
Isolation
16

3.
Multi-‐color
antibody
staining
for
Flow
Cytometry
16

4.
Statistical
Analysis
18

Results
19

1.
Characterization
of
immune
development
and
NKT
cell

populations
in
the
HBVtg
hCD1d-‐KI
model.

19

2.
Characterization
of
NKT,
NK,
and
Conventional
T
Cells
in
the
HBVtg

hCD1d-‐KI
model.

20

3.
Characterization
of
invariant
NKT
cells
in
the
HBVtg
hCD1d-‐KI

Model.

23

4.
Characterization
of
invariant
NKT
subpopulations
in
the
HBVtg

hCD1d-‐KI
model.

23

5.
Characterization
of
Conventional
CD4+
and
CD8+
expression
in
the

HBVtg
hCD1d-‐
KI
model.

28

6.
Characterization
of
PD-‐1
Expression
in
lymphocytes
of
the
HBVtg

hCD1d-‐KI
model.

30

Discussion
35

1.
Summary
of
Results
35

2.
Implications
and
Possible
Mechanisms
36

3.
Future
Directions
39

References
43

iv

List
of
Figures

Figure
1
Cellular
signaling
mechanisms
involved
in
chronic
Hepatitis
B

infections.

7

Figure
2
iNKT
–
Leukocyte
Interactions
and
Cytokine
Signaling
9

Figure
3
Antigen
Presentation
Comparison
Between
MHCI,
CD1d,
and

MHCII

10

Figure
4
Lipid
Chemical
Structures
12

Figure
5
Characterization
of
Immune
Development
in
HBVtg
hCD1d-‐KI

mice

20

Figure
6
Characterization
of
NKT,
NK,
and
Conventional
T
Cells
in
the

HBVtg
hCD1d-‐
KI
model.

21

Figure
7
Decline
of
NKT
Cells
in
HBVtg
mice
22

Figure
8
Characterization
of
invariant
NKT
cells
in
the
HBVtg
hCD1d-‐
KI

model.

24

Figure
9
Characterization
of
invariant
NKT
cell
subpopulations
in
the

HBVtg
hCD1d-‐
KI
model.

26

Figure
10
Characterization
of
Conventional
CD4+
and
CD8+
Expression

in
the
HBVtg
hCD1d-‐
KI
model.

29

Figure
11
Characterization
of
PD-‐1
Expression
in
CD8+
T
Cells
in
the

HBVtg
hCD1d-‐
KI
model.

33

v

List
of
Tables

Table
1

Fluorochrome
conjugated
mAbs
17

Table
2
Fluorochrome-‐antibody
excitation
and
emission
17

vi

List
of
Abbreviations

APC
(cell)

APC
(dye)

APC-‐
ef780

CCL3

Antigen
Presenting
Cell

Allophycocyanin

Allophycocyanin-‐
efluor780

Chemokine:
Macrophage
Inflammatory
Protein-‐1α

CCR4

CD28

CD4

CD8

Chemokine
Receptor
Type
4

CD28-‐
Antigen:
T
Cell
Specific
Surface
Glycoprotein

T
Helper
Cell

Cytotoxic
T
Cell

CHB
Chronic
Hepatitis
B

CTL
Cytotoxic
Lymphocyte

DC
Dendritic
Cell

DN

Ef450

FAS

FAS-‐L

FBS

FC

FCγRII/III

FITC

FSC

Double
Negative
T
Cells

Efluor450:
Replacement
dye
for
pacific
blue

Apoptosis
Antigen
I

Apoptosis
Antigen
I-‐
Ligand

Fetal
Bovine
Serum

Fragment
crystallizable
(region
of
antibody)

FC
gamma
receptor
2/3

Fluorescein
isothiocyanate

Forward
Scatter

HBV
Hepatitis
B
Virus

HBeAg
Hepatitis
B
e
Antigen

vii

HBsAg

HBx

Hepatitis
B
s
Antigen

Hepatitis
B
Virus
X
Protein

HCC
Hepatocellular
Carcinoma

hCD1d
Human
CD1d

HCV
Hepatitis
C
Virus

HIV
Human
Immunodeficiency
Virus

HPV
Human
Papilloma
Virus

HSV-‐2
Herpes
Simplex
Virus-‐2

HPV
Human
Papilloma
Virus

IL-‐10
Interleukin-‐10

IL-‐12
Interleukin-‐12

IFNα
Interferon
α

IFNγ

iTreg

Kb

Interferon
γ

Induced
T-‐
regulatory
cell

Kilo
base

KI
Knock-‐
In

LCMV

mAb

mCD1d

mCD8a

NIH

NK2GD

nm

Lymphocytic
Choriomeningitis

Mouse
Antibody

Mouse
CD1d

Mouse
CD8-‐alpha

National
Institute
of
Health

Natural
Killer
Type
II
Glycoprotein
Domain

Nanometer

viii

PBS
Phospho-‐buffered
Saline

PD-‐1

PD-‐L1

PE

PE
Cy7

Percp-‐Cy5.5

SSC

Programmed
Death-‐1

Programmed
Death-‐
Ligand-‐1

R-‐phycoerythrin

R-‐phycoerythrin-‐
Cyanine
7

Peridinin-‐chlorophyll-‐protein
complex-‐
Cyanine
5.5

Side
scatter

TCR
T
Cell
Receptor

Tg
Transgenic

TGF
β
Tumor
Growth
Factor
β

TH-‐1
T
Helper
Cell-‐1

TH-‐2
T
Helper
Cell-‐2

TH-‐17
T
Helper
Cell-‐17

TNFα
Tumor
Necrosis
Factor
α

Treg
T
regulatory
cell

ix

Abstract

Hepatitis
B
virus
gives
rise
to
chronic
infection
in
350
million
people
world
wide,

and
infection
can
result
in
the
development
of
liver
cirrhosis,
liver
failure
or

hepatocellular
carcinoma.
Current
therapies
merely
treat
the
infection
and
prevent

progression,
because
there
is
no
cure.
There
have
been
promising
studies
in
the

clearance
of
HBV
DNA
in
murine
models
with
the
use
of
iNKT
stimulating
lipid

αGalCer.
However,
iNKT
stimulation
of
αGalCer
has
not
been
an
effective
measure
in

anti-‐viral
or
anti-‐cancer
clinical
trials.
It
is
speculated
that
these
differences

between
human
and
mouse
reactivity
to
αGalCer
is
based
on
the
differences
of

CD1d/NKT
lipid
presentation
systems
between
the
two
species.
Previously,
our
lab

has
generated
an
hCD1d
KI
model
to
support
a
human
like
NKT
cell
environment.
In

order
to
investigate
the
role
of
invariant
NKT
cells
in
HBV
and
to
allow
us
to
test

potential
therapeutic
lipids
for
HBV
in
a
more
human
like
environment,
we

generated
a
novel
HBVtg
hCD1d-‐KI
model.

Thus
in
this
study
we
seek
to

characterize
the
population
of
iNKT
cells
in
this
new
HBVtg
hCD1d-‐KI
model.
As

reported
in
human
chronic
HBV
patients,
we
found
that
the
iNKT
cells
are

significantly
lower
in
this
new
HBV-‐transgenic
model.
Among
the
decreased
levels

of
iNKT
cells,
there
was
a
significant
decrease
in
the
population
of
CD4+
iNKT
cells

accompanied
by
a
significant
increase
of
CD4-‐
iNKT
cells.
Similarly,
there
was
an

overall
significant
decrease
of
total
NKT
Cells.
There
was
an
overall
increase
in
CD8+

conventional
T
Cells,
and
a
significant
increase
of
total
conventional
T
cells.
Our

study
also
found
that
conventional
T
cells
had
and
significant
upregulation
of
PD-‐1,

which
was
restricted
to
the
CD8+
population.
In
our
study,
the
levels
of
PD-‐1
among

x

the
iNKT
cell
populations
were
not
affected;
suggesting
the
decrease
of
iNKT
cells

was
not
related
to
PD-‐1
expression.
Altogether,
our
results
support
that
our
new

HBVtg
can
a
useful
new
model
for
study
of
HBV
pathogenesis
as
well
as
exploration

and
validation
of
novel
anti-‐HBV
therapeutic
approaches.

1

Introduction

Among
the
2
billion
people
worldwide
infected
with
HBV
[3],
an
estimated
350

million
are
chronically
infected
[59].
Chronic
Hepatitis
B
has
been
named
a
“silent

killer”,
because
its
development
is
asymptomatic
and
lasts
up
to
30
years
[1].

Chronic
HBV
infection
can
lead
to
liver
cirrhosis,
fibrosis
and
hepatocellular

carcinoma
(HCC)
[3],
killing
approximately
1
million
people
each
year
[59].
Nearly

53%
of
HCC
cases
are
HBV
related
[52].
The
HBV
infections
considered
to
be
chronic

when
there
is
presence
of
HBsAg
particles
in
the
blood
for
more
than
6
months
[34].

The
adult
immune
system
is
usually
able
to
clear
the
initial
HBV
infection,
but
in

children
it
is
not
as
efficient.

In
fact,
90%
children
infected
with
HBV
become

chronically
infected
[48,
3],
compared
to
5-‐10%
of
adults
[52].
Therapy
for
CHB

includes
the
use
of
nucleoside/nucleotide
analogs
(NA)
and
type
I
interferon
(IFNα).

However,
these
treatments
are
not
always
available,
especially
in
developing

countries
where
there
is
an
increased
risk
for
HBV
infection
[1].
These
medications

are
only
effective
in
controlling
the
infection,
and
have
to
be
taken
for
long
periods

of
time,
making
them
very
expensive.
NA
treatment
is
less
susceptible
to
drug

resistance,
but
it
is
still
not
able
to
achieve
full
viral
control,
while
IFNα
has
many

adverse
side
effects,
and
is
vulnerable
to
drug
resistance
[42].
There
are
vaccines
for

HBV,
but
the
vaccine
is
preventive
and
is
not
helpful
to
those
that
have
already
been

infected.

2

1.
Hepatitis
B
and
its
pathogenesis:

Hepatitis
B
is
a
3.2
kb
double
stranded
DNA
enveloped
virus
that
infects

hepatocytes.
HBV
viral
proteins
can
alter
gene
expression
and
induce
oncogenesis

by
promoting
cell
proliferation
independent
of
growth
factors,
resistance
to
growth

inhibition
and
apoptosis,
tissue
invasion
and
metastases,
and
altering
cellular

energy
metabolism.
In
addition,
the
viral
gene
coding
for
the
HBx
protein
can

randomly
integrate
into
the
host
genome
during
regeneration
of
infected

hepatocytes,
directly
transforming
them
[3].
Chronic
hepatitis
B
infection
leads
to

persistent
inflammation,
liver
damage
and
cell
death
[72],
giving
rise
to
a
vast
array

of
chronic
liver
diseases.
The
greatest
danger
of
CHB
is
that
its
pathogenesis
is

typically
asymptomatic
and
these
progressive
liver
diseases
develop
in
silence.

Chronic
inflammation
can
develop
into
liver
fibrosis,
where
extracellular
matrix
is

deposited
around
the
liver
tissue,
forming
septa.

As
the
extracellular
matrix

continues
to
accumulate,
the
fibrotic
septa
eventually
surround
hepatocyte
islands,

creating
nodules
and
leading
to
liver
cirrhosis.
The
distressed
pathology
of
the

cirrhotic
liver
impedes
its
proper
function.

In
cirrhosis
there
is
a
progressive

decrease
of
vasculature
and
an
increased
hypoxic
environment,
which
aggravates

cell
damage
and
inflammation.
Consequently,
this
hypoxic
environment
inhibits

liver
regeneration,
leading
to
the
pathogenesis
of
HCC
[3].

2.
Immune
responses
to
HBV:

HBV
is
not
directly
cytopathic
[72]
and
once
it
has
infected
the
hepatocyte,
HBV

does
not
replicate
right
away
[9].
The
immune
response
to
HBV
can
be
separated

3

into
three
phases:
immune
tolerance,
immune
breakthrough
and
immune
clearance

[9].
The
liver
plays
a
key
role
in
many
physiological
processes
and
continuously

interacts
with
the
blood
circulation
where
it
is
exposed
to
toxins,
intestinal
flora
and

dietary
proteins
[44].
Thus
under
normal
conditions
LSEC
and
Kupffer
cells
secrete

anti-‐inflammatory
IL-‐10
and
TGFβ,
creating
a
tolerogenic
environment
and

suppressing
potential
inflammation
[12,
44].
The
liver
is
also
not
very
proficient
in

retaining
and
activating
CD8+
T
cells
[48].
During
immune
tolerance,
HBV
acts
to

suppress
this
already
tolerogenic
hepatic
microenvironment,
and
exploits
the
liver’s

inefficiency
in
CD8+
CTL
activation,
allowing
it
to
reach
an
immunotolerant
state

through
the
induction
of
T
cell
anergy
and
specific
T
cell
deletion
[48].
CTLs
are

critical
in
the
elimination
of
pathogens
and
secrete
cytolytic
mediators,
including

granzyme
B,
perforin,
IFNγ
and
TNFα
cytokines
[25].

Immune
tolerance
consists
of
attenuation
of
the
innate
and
adaptive
host
responses.

In
response
to
HBV
infection,
resident
hepatic
immune
cells
increase
their

production
of
IL-‐10
and
TGFβ
anti-‐inflammatory
cytokines
and
reduce
their

expression
of
Toll
Like
Receptors
(TLR),
leading
to
inactivation
of
innate
immunity

[9].
Specifically,
viral
protein
HBeAg
can
reduce
the
hepatocyte,
Kupffer
Cell,
and

monocyte
expression
of
TLR
2
[60],
promoting
liver
tolerance.
Most
importantly,

there
is
an
overall
lack
of
IFN-‐Type
I
induction,
which
is
critical
in
the
initiation
of

the
antiviral
immune
response.
In
patients
with
an
acute
HBV
infection,
levels
of

proinflammatory
cytokines
remain
at
undetectable
levels
for
about
30
days
post

infection
[53],
demonstrating
the
lack
of
immune
activation
in
the
early
stages
of
the

4

HBV
infection.
It
is
believed
that
HBV
can
evade
innate
immunity,
because
of
the

lack
of
IFNα/β
induction
[55].
However,
it
has
recently
been
shown
that
the
lack
of

innate
activation
is
rather
caused
by
the
active
suppression
of
immune
cells
by
HBV

viral
proteins
[7],
such
as
HBeAg
[61].
Hepatocyte
production
of
IFNα/β
recruits

APCs,
Kupffer
cells
and
DCs
into
the
liver
and
mediates
their
activities.
In
response

to
IFNα/β,
APCs
produce
IL-‐18
and
CCL3
chemokines,
activating
NK
and
NKT
cells

[9].
NK
cells
are
an
integral
part
of
innate
immunity
which
serve
to
recognize
and

lyse
virally
infected
cells,
specifically
those
that
have
lost
their
MHC
class
I
receptors

and
express
cell
stress
ligands
[7].
Further
immune
suppression
is
mediated

through
the
secretion
of
IL-‐10
by
hepatic
DCs.
IL-‐10
activates
CD4+CD25+FoxP3+

Tregs,
which
function
in
T
cell
inhibition,
further
suppressing
the
activation
of

adaptive
immunity.

Additionally,
upregulation
of
co-‐inhibitory
Programmed
Death

receptor-‐1
(PD-‐1)
leads
to
the
exhaustion
of
CD8+
CTLs
[72].
During
this
stage
of

HBV
infection,
there
is
no
liver
damage,
but
since
antiviral
mechanisms
have
been

halted,
HBV
actively
replicates
within
the
hepatocytes
[9,
72].

Factors
that
influence
the
transition
from
the
immune
tolerance
stage
into
the

immune
breakthrough
stage
are
still
undetermined.
As
the
infection
progresses
into

the
immune
breakthrough
phase,
there
is
a
strong
increase
in
the
production
IL-‐12,

IL-‐18
and
IFNα
cytokines,
and
decrease
IL-‐10
production,
leading
to
the
activation

of
innate
immunity.
Cells
of
innate
immunity
are
recruited
into
the
liver
and

increase
hepatic
cytotoxicity,
thereby
reducing
HBV
replication.
However,
PD-‐1

expression
on
CD8+
T
cells
makes
them
inefficient
in
viral
clearance
and
the

5

presence
of
Tregs
counteracts
the
production
of
antiviral
and
proinflammatory

cytokines.
This
counteracting
mechanism
reduces
the
effectiveness
of
the
innate

immune
system
and
the
cellular
exposure
to
proinflammatory
factors
results
in
liver

damage
[9,
72].

In
90%
of
adults
HBV
infections
are
self-‐resolving.
Elimination
of
HBV
is
achieved
in

the
immune
clearance
stage
of
the
infection,
when
the
innate
immune
system
is

“hyper
activated”
[9].
Immune
clearance
is
the
active
stage
of
the
HBV
infection,

where
there
is
a
profound
upregulation
of
IL-‐12,
IL-‐18
and
IFNα,
followed
by
DC,

NK,
Th17
cells,
and
monocytes
liver
infiltration
[9].
IL-‐12
and
TNFα
stimulate
IFNγ

secretion
and
proliferation
of
CD8+
T
Cells
[7].
The
infection
is
resolved
after

successful
seroconversion
of
the
HBeAg
into
anti-‐HBe
status
[61].
Successful
viral

clearance
is
likely
due
to
the
efficient
differentiation
and
maturation
of
memory
T

cells
and
the
downregulation
of
PD-‐1
by
CD8+
T
cells,
reversing
their
exhaustion
[8].

3.
Chronic
Hepatitis
B
Infection:

The
failure
of
the
immune
system
to
clear
HBV
leads
to
chronic
liver
inflammation,

and
is
thought
to
be
established
by
the
unrelenting
presence
of
HBeAg
[61].
The

progression
of
chronic
hepatitis
B
is
asymptomatic
and
can
last
up
to
30
years
[61].

However
in
its
pathogenesis,
there
is
evidence
of
progressive
inflammatory
liver

damage
and
viral
persistence
[72].
Chronic
inflammation
is
accompanied
by

simultaneous
tissue
destruction
and
repair
[66].
The
continuous
recruitment
of

inflammatory
cells
results
in
their
incessant
activity
which
subsequently
causes
cell

6

death
[9]
and
liver
damage
[19].
Beginning
the
cycle
of
their
continuous
activity,

macrophages,
lymphocytes
and
other
MNCs
infiltrate
to
the
site
of
injury.
Once
in

the
liver,
lymphocytes
produce
chemokines
which
subsequently
stimulate

macrophages,
which
further
activate
lymphocytes,
leading
to
chronic
and
persistent

inflammation
[66],
exacerbating

the
liver
damage.

Since
HBV
is
a
non-‐cytopathic
virus,
the
progression
of
chronic
hepatitis
is

attributed
to
a
weakened
host
responses
against
the
viral
infection.
Primarily,
HBV

persistence
is
believed
to
be
a
direct
result
of
HBV-‐specific
CD8+
and
CD4+
T
cells

failing
to
eliminate
HBV
[11].
In
CHB
there
is
a
marked
exhaustion
of
CD8+
CTLs,

which
are
the
main
line
of
defense
against
HBV.
There
is
also
supporting
data
that
in

patients
with
chronic
HBV,
that
there
is
ineffective
CD4+
T
cell
priming
during
the

early
stages
of
infection.
This
results
in
decreased
CD8+
CTL
potential
to
mount
an

efficient
antiviral
response
[11].

Patients
who
develop
chronic
HBV
infection
also

have
fewer
T
cells
and
show
functional
impairment
of
T
helper
cells,
both
in
the

peripheral
blood
and
the
liver
[9].

Another
feature
common
to
CHB
is
T
cell
anergy.
T
cells
become
anergic
when
they

fail
to
respond
to
previously
encountered
antigenic
stimuli
by
functional
antigen

presenting
cells
(APCs)
[10].
The
loss
of
T
cell
function
is
a
gradual
process
in
which

T
cells
become
exhausted
through
different
stages
of
functional
impairment
[48].
In

CHB,
CD8+
CTLs
lack
the
ability
to
proliferate
and
produce
cytokines,
specifically
IL-‐
2
and
IFNγ,
as
well
as
displaying
an
overall
reduction
in
their
cytotoxic
abilities.
This

7

CTL
dysfunction
is
attributed
to
the
exceptionally
high
levels
of
viral
antigens,

HBsAg
and
HBeAg
[42].
Additionally,
exhausted
CTLs
have
altered
chemotaxis,

migration
and
adhesion
[21].

The
innate
immune
system
also
greatly
contributes
to
the
pathogenesis
of
CHB.
The

function
of
the
innate
NK
cells
is
determined
by
the
integration
of
stimulatory
and

inhibitory
signals
received
from
the
environment.
If
the
production
of
IL-‐10
by

hepatic
DCs
[7]
is
in
equilibrium
with
the
proinflammatory
cues,
NK
cell
function
is

in
a
deadlock,
resulting
inadequate
production
of
IFNγ
[72].

Moreover,
NK
cells
in

CHB
patients
induce
the
expression
TRAIL
(TNF
related
apoptosis
ligand)
on

hepatocytes
[72]
leading
to
hepatocyte
cell
death.
Therefore,
the
response
of
NK

cells
in
regards
to
the
HBV
infection
considerably
contributes
to
HBV
persistence

(Chang,

et
al,
2007)

Figure
1:
Cellular
signaling
mechanisms
involved
in
chronic
Hepatitis
B

infections.

8

and
cell
cytotoxicity.
The
major
receptors
involved
in
pathogen
recognition,
the

TLRs,
are
also
repressed
during
the
CHB
infection
[9],
leading
to
defective
cell

signaling
and
potential
to
eradicate
virus
(Fig.
1).
The
overall
production
of
IL-‐10

and
TGFβ
by
hepatic
DCs
also
disrupts
the
ability
of
virus
specific
T
cells
to

proliferate
[42],
through
the
induction
of
immunosuppressive
Tregs.
Peng
et
al

reported
that
a
specific
population
of
Tregs,
expressing
CD4+CD25+,
showed
a

higher
frequency
in
CHB
patients
and
was
associated
with
high
viral
titers
and

HBeAg
[46],
signifying
their
role
in
HBV
related
immune
suppression.

4.
Natural
Killer
T
Cells:

Natural
Killer
T
cells
are
an
unconventional
subset
of
T
cells
that
is
stimulated
by

lipid
antigens.
NKT
cells
bridge
the
gap
between
innate
and
adaptive
immunity
and

are
able
to
activate
cells
in
both
immune
divisions
[6].
NKT
cells
are
further
divided

into
Type
I
NKT
cells
and
Type
II
NKT
Cells,
based
on
their
tetramer
binding
and

lipid
preference.
Type
I,
or
invariant,
NKT
cells
are
stimulated
by
αGalCer
lipid,

whereas
Type
II
cells
are
stimulated
by
sulfatide
[18].
NKT
cells
are
CD1d
restricted

and
are
phenotypically
distinct
from
conventional
T
Cells
by
an
additional

expression
of
the
NK
cell
marker
NK1.1
(mouse)
or
CD161
(human)
[29].
They
are

further
characterized
by
the
presence
of
an
invariant
α
chain
and
a
restricted
TCR-‐β

repertoire,
which
in
humans
is
Vα24Jα18
paired
to
Vβ11.
Murine
TCRs
are

comprised
of
Vα14Jα18
paired
to
Vβ7,
Vβ8,
or
Vβ2,
with
Vβ8
being
most

homologous
to
the
human
Vβ11
chain
[6].
There
are
two
main
subpopulations
of

iNKT
cells,
CD4-‐CD8α-‐
or
double
negative,
and
CD4+CD8α-‐
(CD4+).
The
populations

9

of
iNKT
cells
are
functionally
distinct.
When
human
CD4+
iNKT
cells
are
stimulated

with
αGalCer,
they
secrete
both
TH-‐1
and
Th-‐2
Cytokines,
while
DN
secrete
Th-‐1

cytokines
[49].

Activation
of
iNKT
cells
leads
to
a
robust
release
of
cytokines
that
have
potent

anticancer
and
antimicrobial
function,
and
a
significant
role
in
allergy
and

autoimmune
disease
(Fig.
2)
[7].
In
the
case
of
potent
lipid
stimulation,
TCR

engagement
is
the
only
signal
needed
to
induce
the
activation
of
iNKT
cells.

However,
when
the
ligand
affinity
to
the
TCR
is
not
as
strong,
a
secondary
co-‐
stimulatory
signal
is
required.
During
bacterial
and
viral
infections,
dendritic
cell

secretion
of
IL-‐12
can
also
potently
activate
iNKT
cells,
because
of
the
high
baseline

Figure
2:
iNKT
Leukocyte
Interactions

iNKT
Treg
IL-2
Contact-dependent
inhibition of iNKT proliferation,
cytokine secretion, cytotoxicity
Macrophage
Activation
DC
CD40
CD1d
CD40L
IFNγ
IL-12
IL-12R
NK
IFNγ
IFNγ
Cytolysis B Cell
Antibody Production
Memory Responses
iNKT- Induced maturation,
Cross Presentaion
IFNγ IL-4
TCR
T Cell
(Adapted
from
Juno
et
al,
2012)

10

IL-‐12
receptor
expression
on
iNKT
cells
[7].
Upon
αGalCer
stimulation,
iNKT
Cells

and
DCs
engage
in
a
reciprocal
pattern
of
activation.
NKT
Cells
rapidly
upregulate

their
Th-‐1
and
Th-‐2
cytokine
production
and
their
chemokine
receptors.
iNKT
cells

produce
large
amounts
of
cytokines,
such
as
IFNγ,
and
are
able
to
reinforce
NK
cell

activation,
which
is
particularly
important
in
viral
infections.
Additionally,
following

iNKT
cell
activation,
there
is
a
profound
increase
in
granzyme
B,
perforin,
TRAIL,

and
FASL
expression,
consequently
leading
to
cell
cytolysis
[29].
Invariant
NKT
cells

can
also
stimulate
the
upregulation
of
MHCI
and
MHCII
presentation
molecules,

priming
the
adaptive
immune
system
[19].

5.
CD1d
Antigen
Presentation:

CD1d
is
an
MHC-‐I
like
antigen
presenting
molecule,
which
presents
lipids
to
NKT

cells.
Figure
3
illustrates
the
comparisons
between
MHC-‐I,
CD1d,
and
MHCII

molecules,
by
APCS
(provided
by
Weiming
Yuan).

The
structure
of
CD1d
contains
2

hydrophobic
channels:
A’
pocket
which
binds
to
alkyl
chains
containing
up
to
26

carbons
and
sphingosine
binding
F’
pocket
[37].
Upon
αGalCer
binding,
hCD1d

Figure
3:
Antigen
Presentation
comparison
between
MHCI,
CD1d,
and
MHCI
I

molecules

11

undergoes
a
conformational
change
from
its
empty
to
filled
state,
marked
by
shifts

in
the
side
chain
tryptophan
residues.
The
structure
of
the
CD1d-‐α-‐GalCer
tetramer

encompasses
the
lipid
and
CD1d
α1
and
α2
helix
interactions
but
does
not
support

direct
interactions
between
CD1d
and
the
NKT
TCR.

The
interaction
with
the
TCR
is

accomplished
solely
through
the
galactose
polar
head
moiety
of
the
lipid
[32].

6.
Lipid
Antigens:

Invariant
NKT
cells
can
recognize
both
endogenous
and
exogenous
lipid
antigens.

Analogs
of
αGalCer
elicit
different
responses
in
iNKT
cells.
For
example,
lipids
with
a

truncated
sphingosine
chain,
as
in
the
structure
of
OCH,
have
a
low
affinity
to
CD1d

and
induce
a
Th-‐2
like
response
[56].
Since
the
polar
head
moiety
of
the
lipid
chain

facilitates
most
of
the
interactions
between
the
CD1d-‐lipid
tetramer
and
the
iNKT

TCR,
alterations
in
this
ring
structure
are
likely
to
influence
the
iNKT
response.
α-‐
Carba-‐GalCer
is
an
analog
of
α-‐GalCer,
where
the
5a’-‐
oxygen
atom
within
the

pyranose
ring
was
replaced
with
a
methylene
group
(Figure
4).
Stimulation
of
iNKT

cells
with
this
new
compound
resulted
in
a
Th-‐1
response
[56].
Tashiro
et
al

explained
that
this
lipid
had
a
much
more
stable
linkage,
compared
to
αGalCer,

allowing
it
to
be
resistant
to
galactosidases
activity
and
be
more
stable
in
vivo.

Additionally,
αGalCer
interacts
with
the
Vα24
iNKT
via
the
CDR3
TCR
region,

whereas
α-‐Carba-‐GalCer
had
additional
interactions
with
CDR1
[56].

Notice
that
a

more
a
stable
interaction
between
the
lipid
and
TCR
leads
to
a
more
sustained
TH1

polarity.

12

Lipid
antigens
can
also
be
used
as
vaccine
adjuvants.
The
TH-‐1
polarity
induced
by

α-‐Carba-‐GalCer
has
proved
to
be
a
successful
adjuvant
in
malaria
and
some

influenza
virus
vaccines
[65].
Additionally,
α-‐Carba-‐GalCer
also
has
superior

abilities
in
activating
NK
and
CD8
T
Cells.

7.
Differences
between
human
and
mouse
CD1d/NKT
lipid
presentation
systems:

The
mouse
and
human
CD1d
molecules
are
conserved
and
demonstrate
a
65.4%

amino
acid
sequence
homology
[37].
However,
the
examination
of
αGalCer
binding

to
hCD1d
and
mCD1d
revealed
different
orientation
of
the
polar
head
group
[15].
It

is
likely
that
the
resulting
difference
in
the
conformation
of
the
polar
head

significantly
alters
the
affinity
of
the
human
CD1d-‐
αGalCer
complex
to
the
TCR
[45].

A.
B.

α-‐GalCer

α-‐Carba-‐GalCer

(Tashiro
et
al,
2010)

Figure
4:
Lipid
chemical
structures
of
(A)
α-‐GalCer
and
(B)
α-‐Carba-‐GalCer.

13

αGalCer
has
had
powerful
tumor
therapeutic
potential
in
vivo
but
was
translated

into
many
unsuccessful
clinical
trials.
In
these
trails
it
was
found
that
the
αGalCer

induced
IFNγ/IL4
ratio
in
patients
was
much
lower
than
in
mouse
models.

This

could
be
a
result
of
an
overall
much
weaker
response
in
human
patients,
or
because

of
a
different
immune
polarization
between
the
Th-‐1
and
Th-‐2
phenotype.
These

differences
can
be
attributed
to
the
diversity
between
the
hCD1d
and
mCD1d

structure
and
the
different
affinities
they
have
for
lipid
antigens,
resulting
in
altered

antigen
presentation
between
the
hCD1d
and
mCD1d
APCs
[45].

The
population
and
distribution
of
iNKT
cells
in
humans
and
mice
are
also

substantially
different.
In
humans,
iNKT
cells
make
up
0.1-‐0.2%
of
T
cells,
and

0.001-‐
3%
or
total
leukocytes
in
the
blood,
bone
marrow
and
spleen
[54].
iNKT
cells

are
not
highly
enriched
in
the
liver.
Yet
their
highest
concentration
of
0.5%
resides

in
this
organ
[54].
In
contrast,
mouse
livers
contain
up
to
30%
of
iNKT
cells
[7],

indicating
just
how
fewer
of
these
cells
are
found
within
the
human
immune
system.

Nevertheless,
these
cells
have
extraordinary
potential
and
can
stimulate
many

effector
mechanisms
that
can
potentially
rid
the
body
of
the
persistent
HBV

infection.
iNKT
cells
can
stimulate
the
induction
and
differentiation
of
CD8+
T
cell,

that
are
lacking
in
CHB,
and
can
prime
the
adaptive
immune
system
[19],
yielding
a

stronger
antiviral
response.
By
increasing
granzyme
B
and
perforin,
they
can
also

directly
participate
in
the
lysis
of
HBV
infected
cells
[29].

14

8.
NKT
cells
in
anti-‐viral
and
anti-‐HBV
immune
responses:

NKT
and
hCD1d
cell
function
is
altered
in
many
viral
infections.
HIV,
HSV-‐2
and
HPV

are
known
to
directly
interfere
with
the
antigen
presenting
function
of
hCD1d,

leading
to
impaired
iNKT
activation.
There
is
a
significant
drop
in
the
populations
of

NKT
cells
in
HIV-‐1
and
LCMV
infections
[14].
HBV
specific
NK,
NKT
and

polymorphonuclear
leukocytes
are
found
in
the
inflammatory
lesions
of
the
liver

but
these
cells
are
not
effective
in
clearing
the
virus,
and
cause
hepatocellular

damage
[17].
Furthermore,
the
NKG2D
receptor
expressed
on
NK
and
NKT
cells,

recognizes
stress
ligands,
has
been
directly
implicated
in
HBV
pathogenesis
16.

Human
CD4-‐
iNKT
cells
show
a
high
expression
of
NKG2D
and
blocking
of
the

NKG2D
receptor
has
been
shown
to
decrease
the
pathogenic
effects
caused
by
HBV

[49].

Activation
of
iNKT
cells
promotes
the
loss
of
tolerance
to
HBV
specific
CD8+T
cell

antigens
in
mouse
models
[24].
Additionally,
αGalCer-‐activated
iNKT
cells
have
been

shown
to
promote
CD8+T
cell
differentiation
during
mouse
MCMV
viral
infections

[49].
More
evidence
for
NKT/HBV
interactions
are
presented
by
HBV
transgenic

mouse
models,
where
upon
αGalCer
stimulation,
activated
NKT
cells
block
HBV

replication,
and
produce
large
amount
of
IFNγ
and
are
able
to
eradicate
HBV
DNA

[72].

However,
in
clinical
trials
αGalCer
therapy
showed
no
effect
in
patients
being

treated
for
chronic
hepatitis
B
[64].

15

9.
Experimental
Design:

The
host
range
for
HBV
is
limited
to
humans
and
chimpanzees
[59].
For
these

reasons,
transgenic
mouse
models
have
been
generated
to
facilitate
the

investigation
of
HBV
pathogenesis.
In
order
to
better
study
the
human
CD1d
lipid

presentation
and
the
properties
of
human
CD1d-‐restricted
NKT
cells,
our
lab

previously
generated
a
novel
human
CD1d-‐knock-‐in
mouse
as
an
in
vivo
model
[63].

In
this
model,
human
CD1d
supports
the
development
of
an
NKT
cell
population

closely
resembling
that
in
human.
In
this
CD1d-‐humanized
background,
we
intend

to
study
how
HBV
interacts
with
the
human-‐like
NKT
cell
population
in
the

repertoire,
phenotype
and
abundance
of
NKT
cells.
HBV
transgenic
models
have

been
generated
and
widely
used
in
HBV
pathogenesis
[67].
This
particular
model

contains
DNA
that
is
1.3
times
larger
than
the
HBV
genome,
allowing
it
to
produce

high
titers
of
HBV
DNA.
The
HBV
transgene
is
found
on
Chromosome
11,
and
the

location
of
the
hCD1d
is
on
Chromosome
3,
thus
the
two
transgenes
can
be
readily

incorporated
into
the
same
mouse
strain.
Our
lab
crossed
the
previously
generated

hCD1d
KI
strain
with
HBV
(wtTg05)
provided
by
the
Ou
laboratory,
to
yield
a
mouse

model
containing
the
HBV
transgene
in
an
hCD1d
knock
in
background.

Goal
of
this

study
is
to
characterize
an
HBV
mouse
model
in
containing
a
population
of
NKT
cells

closely
resemble
that
of
humans.
This
will
allow
us
to
test
new
anti-‐HBV
therapeutic

lipids
that
will
be
more
compatible
with
human
immune
conditions.
Furthermore,
a

valid
animal
model
will
allow
us
to
study
the
human-‐specific
anti-‐viral
immune

responses
and
the
role
of
iNKT
cells
in
these
responses.

16

Materials
and
Methods

1.
Generation
of
HBVtg
hCD1d-‐KI
mice:

C57BL/6
hCD1d-‐KI
mice
from
our
laboratory
[63]
were
crossed
with
HBVtg
wt05

mice
from
Dr.
James
Ou
[67].
The
HBV
gene
368
bp
(1601-‐1233)
was
confirmed
by

PCR
with
primers:
3’-‐HBV-‐PCR:
tgc
aga
ggt
gaa
gcg
aag
tgc
aca
cgg
acc
/
5’-‐HBV-‐PCR:

cat
gcg
tgg
aac
ctt
tgt
ggc
tcc
tct
gcc,
in
a
standard
PCR
cycle.
The
mice
used
for
this

experiment
were
3
month
old
females.

2.
Liver
mononuclear
cell
isolation

A
30mL
perfusion
was
done
into
the
hepatic
portal
vein
with
1X
PBS
at
room

temperature.
The
liver
was
excised
and
cells
were
homogenized
through
a
40
μm

nylon
cell
strainer
(BD
Falcon
#352340)
with
4°C
FACS
buffer
(2%FBS,
0.02%
NaN3

,1X
PBS)
and
centrifuged
at
4°C
at
500g
for
7
minutes.
Liver
monocytes
were
further

isolated
with
25mL
of
25°C
sterile
Percoll
(Percoll
mixture:
33.75%
Percoll
in
1X

PBS),
and
centrifuged
at
25°C
at
700g
for
12
minutes.
Cells
were
incubated
with
1X

Red
Blood
Cell
Lysis
Buffer
(Biolegend)
on
ice
for
2
minutes.
Cells
were
washed
3

times
with
4°C
FACS
buffer
and
centrifuged
for
5
minutes
at
500g.
After
the
first

wash,
the
cells
were
again
filtered
through
a
40
μm
nylon
cell
strainer.
After
the
last

wash,
the
cells
were
resuspend
with
1mL
of
4°C
FACS
buffer,
and
counted.

2.
Multi-‐color
antibody
staining
for
flow
cytometry:

Liver
cells
were
counted
by
hemacytometers.
For
Flow
Cytometry
antibody
staining,

2
million
cells
were
used
per
sample.
The
cells
were
incubated
with
1:100
FC
Block

17

to
block
the
FCγRII/III,
for
15
minutes
in
4°C
dark
conditions,
followed
by
cell

staining
with
fluorochrome
conjugated
mouse
antibodies
for
30
minutes
also
in
4C°

dark
conditions.
The
descriptions
of
the
fluorescent
mAbs
used
in
this
experiment

are
shown
in
Table
1
and
their
excitation
and
emission
range
is
shown
in
Table
2.

Flow
cytometry
was
performed
with
FACS
CantoII
(BD)
and
the
data
was
analyzed

by
FlowJo
(Tree
Star
Inc.)

Table
1.
Fluorochrome
conjugated
mAbs:

Antibody
FACS
Channel
Company
Catalog
No.
Concentratio
n

CD1d-‐PBS57
PE
NIH
#15957
1.2mg/mL

mTCRβ
Percp-‐Cy5.5
Ebioscience
#
45-‐5961-‐82
0.2mg/mL

NK1.1
Ef450
Ebioscience
#
48-‐5941-‐82
0.2mg/mL

mCD8a
APC-‐eF780
Ebioscience
#
47-‐008182
0.2mg/mL

CD4
APC
Ebioscience
#17-‐0041-‐83
0.2mg/ml

PD-‐1

FITC
Ebioscience
#11-‐9981-‐81
0.5mg/mL

CD28
PeCy7
Ebioscience
#25-‐0281-‐81
0.2mg/ml

Table
2:
Fluorochrome-‐
Antibody
Conjugate
Excitation
and
Emission

Fluorochrome
Fluorescence
Excitation
Emission

APC
Red
650
nm
660
nm

APC-‐ef780
(APC
Cy7)
Infrared
650
nm

785
nm

Ef450
(Pacific
Blue)
Blue
401
nm
452
nm

FITC
Green
494
nm

520
nm

PE
Yellow
496
nm
578
nm

PeCy7
Infrared
496
nm
785
nm

Perp-‐Cy5.5
Far
Red
482
nm
695
nm

18

4.
Statistical
Analysis:

Statistics
are
displayed
as
mean
±
standard
deviation.
Statistics
were
calculated
by

2-‐tailed
T
test
analysis
in
Microsoft
Excel
software.

19

Results

1.
Characterization
of
immune
development
and
NKT
cell
populations
in
the
HBVtg

hCD1d-‐KI
model:

Immune
development
and
the
major
lymphocyte
populations
within
the
hCD1d-‐KI

model
have
already
been
characterized
[63].
The
total
NKT
cell,
and
iNKT
cell

populations
have
an
abundance
and
phenotype
closely
resembling
that
of
humans.

The
hepatic
mononuclear
cell
(MNC)
population
consisted
of
6.13%
NKT
cells
and

1.25%
iNKT
cells.
In
addition,
the
iNKT
cell
population
displayed
had
higher
Vβ8

usage
(82.4%).
This
demonstrated
that
the
hCD1d
knock-‐in
gene
influenced
the

murine
iNKT
cells
to
take
a
more
human
like
course
in
development
[63].

The
HBV

transgene
has
been
previously
mapped
to
Chromosome
11,
although
the
exact

insertion
site
has
not
been
defined
(Dr.
James
Ou,
unpublished
results).
This
allowed

us
to
breed
the
HBV
transgene
into
the
hCD1d-‐KI
mice
(CD1d
localized
on

Chromosome
3
in
mice).
To
investigate
whether
the
HBV
transgene
has
an
impact

on
immune
development,
particularly
that
of
iNKT
cells,
we
also
characterized
the

major
lymphocyte
populations
in
the
central
and
peripheral
immune
organs.
We
did

not
detect
substantial
difference
of
thymic
development
in
HBVtg
hCD1d-‐KI
mice

compared
to
control
hCD1d-‐KI
mice
(Xiangshu
Wen,
unpublished
results),

validating
that
the
presence
of
the
transgene
does
not
impact
the
iNKT
cell

development.

20

2.
Characterization
of
NKT,
NK,
and
Conventional
T
Cells
in
the
HBVtg
hCD1d-‐KI

Model:

A
recent
study
performed
by
Wang
et
al
exposed
a
significant
decrease
in
the
total

MNC
NKT
cells
within
their
HBVtg
mouse
model
[62].
This
model,
was
however

bread
in
WT
mice,
expressing
mCD1d.
We
intended
to
investigate
the
impact
of
HBV

on
the
total
population
of
NKT
cells
in
our
HBVtg
hCD1d-‐KI
model.
We
also

investigated
the
populations
of
NK
and
conventional
CD4
and
CD8
T
cells.
It
has

been
reported
that
the
numbers
of
NK
cells
are
also
altered
during
HBV
infection
in

humans
[2],
and
CD8+
conventional
T
cells
are
known
to
play
a
critical
role
in
HBV

clearance
[11].

(Xiangshu
Wen,
unpublished
data)

Figure
5:
Characterization
of
Immune
Development
in
HBVtg
mice.
Hepatic

MNCs
in
WT
and
HBV
wt05
tg
mice
were
isolated
and
their
iNKT
cell

populations
were
characterized
based
on
their
CD1d-‐tetramer+
and
TCRβ+

expression.
The
percentage
of
CD1d-‐tetramer
+TCRβ
iNKT+
cells
is
reported

at
the
population
gate.

21

To
characterize
the
NKT,
NK,
and
conventional
T
cell
populations,
we
isolated
liver

mononuclear
cells
(MNCs)
from
the
HBVtg
hCD1d-‐KI
mice
and
control
hCD1d-‐KI.

The
MNCs
were
stained
with
a
panel
of
fluorescent
antibodies
to
CD8,
CD4,
NK1.1,

TCRβ
and
mCD1d-‐tetramer
(Table
2)
that
would
define
cell
populations
that
are

critical
in
chronic
hepatitis
B
infection.

Figure
6:
Characterization
of
NKT,
NK
and
Conventional
T
cells
in
the
HBV

hCD1d-‐KI
model.
(A)
Hepatic
MNCs
from
HBVtg
hCD1d-‐KI
and
hCD1d-‐KI
mice

were
electronically
gated
on
TCRβ
and
NK1.1
to
identify
the
populations
of

NK1.1+TCRβ+
NKT
cells,
NK1.1+
TCRβ-‐
NK
cells
and
NK1.1-‐TCRβ+
conventional

T
cells.
The
percentage
of
NKT,
NK,
and
conventional
T
cells
are
reported
in
their

appropriate
quadrant.
(B)
The
average
population
of
NKT
cells,
conventional
T

cells
and
NK
cells
compiled
from
total
HBVtg
hCD1d-‐KI
and
hCD1d
mice
in
this

experiment.

A.

B.

22

Our
results
demonstrated
a
statistically
significant
decrease
of
NKT
Cells
(p=0.009)

in
the
HBVtg
hCD1d-‐KI
model
(Figure
6).
The
average
population
of
NKT
cells
in
the

HBVtg
hCD1d-‐KI
model
and
hCD1d-‐KI
model
3.97±0.37
and
8.59±1.40,
respectively.

This
result
is
consistent
with
the
earlier
study
completed
by
Wang
et
al,
verifying

the
HBV
dependent
decline
of
hepatic
NKT
cells
(Figure
7).
Conversely,
the
NKT
cell

population
in
the
Wang
et
a
l
study
decreased
from
19.3%
in
control
mice
to
8.3%
in

the
HBVtg
model
(Figure
7)
[62].
This
difference
can
be
explained
by
our
generation

of
the
HBVtg
model
and
using
an
hCD1d-‐KI
background.
Within
hCD1d-‐KI
mice,

characterized
by
Wen
et
al,
there
is
a
much
lower
population
of
NK1.1+TCRβ+
NKT

cells
[63].
Additionally,
there
is
an
increase
of
the
total
amount
of
conventional
T

cells
(p=0.11).
Surprisingly,
there
was
no
significant
difference
in
the
NK
cell

populations
between
the
two
groups.

NK
cells
are
believed
to
play
a
role
in
liver

damage
during
CHB
infections
[2].

Figure
7:
Decline
of
NKT
Cells
in
HBVtg
mice.

(Wang
et
al,
2012)

23

3.
Characterization
of
invariant
NKT
cells
in
the
HBVtg
hCD1d-‐KI
model:

We
also
intended
to
investigate
whether
the
decrease
of
NKT
cells
extends
into
the

iNKT
cell
populations.
Clinically,
there
are
reports
that
patients
with
chronic

hepatitis
B
have
a
significantly
lower
frequencies
of
circulating
iNKT
cells
compared

to
inactive
carriers
of
HBV
and
healthy
uninfected
individuals
[27].
To
characterize

the
iNKT
cell
frequencies
in
the
HBVtg
hCD1d-‐KI
model,
we
gated
liver
monocytes

on
TCRβ
and
mCD1d-‐tetramer
expression,
to
identify
the
TCRβ+

CD1d-‐tetramer+

population
of
iNKT
cells.

We
indeed
found
a
statistically
significant
decrease
of
iNKT
cells
in
the
HBVtg

hCD1d-‐KI
model
(p=
0.00117)
(Figure
8).
The
average
population
of
iNKT
cells
in

this
study
was
0.658%±0.09
in
the
HBVtg
hCD1d
KI
model
and
2.315%±0.219
in
the

uninfected
hCD1d
model.

The
population
of
hCD1d
iNKT
cells
is
consistent
with

previous
work
from
our
lab
[63],
and
from
previously
characterized
iNKT
cells
in

CHB
patients
[27],
validating
the
HBV
related
decrease
of
iNKT
cells.
Additionally,

other
viruses
also
have
demonstrated
an
impact
on
iNKT
cell
frequency.

HIV
has

been
shown
to
infect
CD4+
Vα24
NKT
cells,
and
cause
their
depletion
[51].

4.
Characterization
of
invariant
NKT
subpopulations
in
the
HBVtg
hCD1d-‐KI
model:

It
has
been
reported
that
the
balance
between
CD4+
and
CD4-‐
iNKT
cell
populations

is
altered
in
chronic
viral
infections
[27].
We
determined
if
the
ratio
of
CD4+
and

CD4-‐
iNKT
cells
is
different
than
the
previously
characterized
hCD1d
model
[63].

24

In
order
to
investigate
the
expression
of
CD4+
and
CD4-‐
in
the
iNKT
cells,

TCRβ+CD1d-‐tetramer+
cells
were
electronically
gated
for
their
CD4
and
CD8

expression.
Figure
9
demonstrates
the
distribution
of
CD4+
and
CD4-‐
iNKT
cells.

Figures
9A-‐9C
reveal
the
significant
decrease
in
CD4+
iNKT
cells
in
the
HBVtg

hCD1d-‐KI
model.
The
average
CD4+
iNKT
frequency
in
the
HBVtg
hCD1d-‐KI
and

hCD1d-‐KI
model
was
25.93±3.59
and
41.25±0.64,
respectively.

There
is
also
a

Figure
8:
Characterization
of
invariant

NKT
cells
in
the
HBVtg
hCD1d-‐KI

model.
(A)
Hepatic
MNCs
from
HBVtg

hCD1d-‐KI
and
hCD1d-‐KI
mice
were

isolated
and
their
iNKT
cell

populations
were
characterized
based

on
the
expression
of
TCRβ
and

reactiveness
to
the
CD1d-‐tetramer.

The
shown
percentage
of
CD1d-‐
tetramer+TCRβ+
cells
is
a

representative
value
of
these

populations.
(B)
The
average

population
of
iNKT
cells
was
compiled

from
all
HBVtg
hCD1d-‐KI
and
hCD1d-‐
KI
mice.

A.

B.

25

significant
increase
in
the
population
of
CD4-‐
iNKT
cells
in
the
HBV
hCD1d
model

(Figures
9A-‐9C).

The
average
frequency
of
CD4-‐
iNKT
cells
in
the
HBVtg
hCD1d-‐KI

and
hCD1d-‐KI
models
was
73.46±3.45
and
58.4±.07,
respectively.
We
noted
that
the

standard
deviation
for
the
HBV
infected
mice
is
slightly
larger,
most
likely
because

of
the
different
response
each
mouse
had
to
HBV,
such
as
the
viral
titer.

In
spite
of

this,
the
significant
alternations
in
CD4+
and
CD4-‐
iNKT
cell
populations
make
it

evident
that
regardless
of
viral
titer
and
HBV
pathogenesis;
there
is
a
great
impact

on
iNKT
cells.

The
ratio
of
CD4-‐
to
CD4+
iNKT
cells
was
also
greatly
impacted.
Figure
8D

demonstrates
that
there
was
also
significant
increase
in
the
ratio
of
DN
to
CD4+
cells

(p=0.03).
This
implies
that
HBV
may
cause
iNKT
cells
to
become
more
Th-‐1

polarized.
This
is
consistent
with
findings
from
a
clinical
study
in
which,
CHB

patients
with
significantly
decreased
frequencies
of
iNKT
cells,
also
had
significantly

lower
amounts
of
CD4+
iNKT
cells
than
in
those
of
inactive
carriers
[28].
However,

these
patients
did
not
have
an
increase
of
CD4-‐
iNKT
cells.
This
discrepancy
can
be

explained
by
other
variations
between
human
and
mouse
immune
systems,
because

in
mice
there
are
no
CD8+
iNKT
cells.
Thus
in
our
model
a
decrease
in
CD4+
cells

would
ultimately
result
in
an
increase
of
CD4-‐
iNKT
cells,
however,
in
humans
this

could
mean
an
increase
in
CD8+
iNKT
cells,
even
though
CD8+
populations
of
iNKT

cells
in
humans
are
miniscule.

26

The
importance
of
iNKT
cell
subpopulations
is
due
to
their
different
functions.
It
is

also
interesting
to
note
that
iNKT
cell
activation
by
a
potent
lipid
induces
a

production,
of
both
TH-‐1
and
TH-‐2
cytokines,
but
activation
with
IL-‐12
or
TLR

agonists
lead
strictly
to
a
TH-‐1
response
[65].
When
discussing
HBV,
in
the

Figure
9:
Characterization
of
invariant
NKT
cell
subpopulations
in
the

HBVtg
hCD1d-‐KI
model.
(A)
The
iNKT
cell
populations
from
HBVtg
hCD1d-‐
KI
and
hCD1d-‐KI
mice
were
electronically
gated
for
their
CD4
and
CD8

expression.
The
CD4+
iNKT
population
is
defined
by
CD4+CD8-‐
expression

and
the
CD4-‐
iNKT
population
is
defined
by
the
CD4-‐CD8-‐
expression.
The

population
percentage
for
each
category
is
shown
in
their
appropriate

quadrant.
(B)
The
average
population
of
CD4+
iNKT
cells
and
CD4-‐
iNKT

cells
was
compiled
from
all
HBVtg
hCD1d-‐KI
and
hCD1d-‐KI
mice.
(C)
The

level
of
CD4
expression
in
HBVtg
hCD1d-‐KI
and
hCD1d-‐KI
mice.
(D)
Ratio
of

CD4-‐
iNKT
cells
to
CD4+
iNKT
Cells
for
HBVtg
hCD1d-‐KI
and
hCD1d-‐KI
mice,

based
on
average
percentage
of
each
population.

*

27

clearance
phase
of
HBV
there
is
an
upregulation
of
IL-‐12
[72],
which
is
likely
to

enhance
iNKT
signaling.
However,
these
mice
are
trans-‐genetically
modified
and

their
HBV
gene
is
constitutively
expressed.
Consequently,
their
viral
titer,
which

could
be
different
between
individual
animals,
stays
consistent
through
out
the
life

span.

Because
of
this,
one
cannot
determine
the
stage
of
HBV
pathogenesis,
based

solely
on
cell
populations.

Sandberg
et
al
found
that
CD4+
and
CD4-‐
NKT
cells
differ
in
their
expression
of

homing
receptors.
CD4-‐
NKT
cells
expressed
high
levels
if
CD11a,
homing
receptor

to
peripheral
tissues,
and
low
levels
of
CD62L,
homing
receptor
for
secondary

lymphoid
tissues,
while
CD4+
NKT
cells
expressed
low
levels
of
CD11a
and
high

levels
of
CD62L.
This
suggests
that
the
CD4-‐
NKT
are
migrating
and
infiltrating
liver

tissue,
and
the
CD4+
NKT
cells
are
exiting
the
liver
and
moving
to
the
lymph
nodes

[51].
This
finding
can
provide
insight
as
to
whether
the
balance
between
CD4+
and

CD4-‐
iNKT
cells
is
altered
because
of
homing
of
CD4-‐
iNKT
cells
into
the
liver
or
the

migration
of
CD4+
iNKT
cells
out
of
the
liver
and
into
the
lymph
nodes,
or
if
it
is
due

to
the
specific
apoptosis
of
CD4+iNKT
cells.
Additionally,
CD4+
iNKT
cells
express

high
levels
of
CCR4
suggesting
they
can
migrate
to
additional
sites
[31],
suggesting

the
decline
of
CD4+
iNKT
cells
in
HBV
livers,
can
be
a
result
of
migration.

28

5.
Characterization
of
conventional
CD4+
and
CD8+
T
cells
in
the
HBVtg
hCD1d-‐
KI

model:

HBV-‐specific
CD8
+

CTLs
cells
play
a
major
role
in
the
clearance
of
Hepatitis
B
[11].
It

has
been
reported
that
when
CD8
+

T
Cells
are
depleted
at
the
peak
of
HBV
induced

viremia,
the
virus
will
not
be
cleared
efficiently,
and
will
be
followed
by
the
onset
of

viral
hepatitis
[11].
In
addition,
because
of
the
persisting
viral
presence,
the
virus

specific
T
cells
of
CHB
patients
lack
the
ability
to
proliferate
and
produce
cytokines

[48,
11].
Furthermore,
CD8+
T
cells
in
CHB
patients
become
exhausted
and
have

reduced
cytotoxic
abilities
[48].
Therefore,
we
investigated
the
CD4
and
CD8

expression
by
liver
MNCs.
Cells
were
gated
by
FACS
analysis
on
APC-‐CD4
and
APC-‐
Cy7-‐CD8.

Figures
10A
and
10B
show
the
frequency
of
liver
MNCs
expressing
CD4
and
CD8

markers.
There
is
a
slight
increase
in
the
number
of
CD8+
T
cells
in
the
HBVtg

hCD1d-‐KI
model,
compared
to
the
hCD1d-‐KI
(p=0.114),
28.23%±1.77
vs.

19.95%±48,
respectively.
Since
T
cell
dysfunction
is
related
to
viral
persistence
[22],

and
CD8+
T
cells
are
vital
to
viral
clearance
[30]
their
presence
is
valuable
indicator

of
the
progression
of
HBV.
However,
while
the
increased
numbers
of
CD8+
T
cells

are
most
likely
a
reflection
of
previous
stimulation
and
proliferation,
it
cannot
be

assumed
that
these
cells
are
functionally
active.

In
a
state
of
“split
anergy”
activated

29

CD8+T
cells
lose
their
ability
to
proliferate
and
become
non-‐responsive.
However,

these
cell
numbers
are
prevented
from
T
cell
deletion
through
“anergy

maintenance”
[10].

Thus
the
resulting
increase
of
CD8+
cells
in
these
HBV
mice
can

be
a
result
of
an
HBV
induced
anergic
state.

Figure
10:
Characterization
of
conventional
CD4+
and
CD8+
expression
in

the
HBVtg
hCD1d-‐KI
model.
(A)
Hepatic
MNCs
from
HBVtg
hCD1d-‐KI
and

hCD1d-‐KI
mice
were
electronically
gated
based
on
their
CD4+
and
CD8+

distribution.
The
percentage
of
each
population
is
indicated
in
its

appropriate
quadrant.
(B)
The
average
population
of
CD4+
and
CD8+
cells

was
compiled
from
all
HBVtg
hCD1d-‐KI
and
hCD1d-‐KI
mice.

30

There
was
some
variation
between
CD4+
populations,
but
there
was
no
meaningful

change
in
its
expression,
shown
in
Fig.
10B
(left
panel).

Since
there
was
a
dramatic

decrease
of
CD4+
iNKT
cells,
it
is
interesting
that
there
was
no
change
in
the
CD4+

population.
This
indicates
that
the
mechanism
behind
the
decrease
of
CD4+iNKT

cells
could
be
iNKT
cell-‐specific.
Data
from
HIV
studies
revealed
that
CD4+
NKT
cells

are
more
prone
to
infection
than
regular
CD4+
cells,
because
CD4+
iNKT
cells
have

higher
expression
of
HIV
co-‐receptor
CCR5,
making
them
much
more
appealing
to

HIV
[51].
This
suggests
that
the
CD4+
iNKT
cell
decrease
could
be
a
reflection
of
a

particular
receptor
confined
to
that
population,
ultimately
causing
their
depletion.

6.
Characterization
of
PD-‐1
expression
in
the
HBVtg
hCD1d-‐KI
model:

Finally,
we
intended
to
characterize
the
populations
of
cells
in
HBVtg
hCD1d-‐KI
mice

based
on
the
expression
of
co-‐modulatory
markers,
in
order
to
investigate
potential

mechanism
behind
the
expansion
or
depletion
of
certain
cell
populations.

PD-‐1
co-‐
inhibitory
marker
has
been
implicated
in
virus
specific
inhibition
of
CD8+
T
cells
in

HIV[13],
HCV
[16]
and
HBV
[35].
Most
importantly,
Chikuma
et
al
discovered
that

PD-‐1
defective
CD8+
T
cells
are
unable
to
undergo
anergy
[10].
PD-‐1
inhibition
plays

a
greater
role
in
CD8+
T
cells
than
CD4+
T
cells,
and
there
are
many
reports
that
the

blockage
of
the
PD-‐1
pathway
is
able
to
reverse
T
cell
exhaustion
[5].
We
thus

intended
to
further
investigate
the
role
PD-‐1
has
in
HBV
CD8+
T
cells.

Figure
11A
shows
that
there
is
a
significant
upregulation
of
PD-‐1
expression
in
the

total
number
of
conventional
T
cells
in
HBVtg
hCD1d
mice
(p=.037).
The
average

31

expression
frequencies
for
conventional
T
cells
in
HBVtg
hCD1d-‐KI
and
hCD1d-‐KI

mice
were
32.87±9.46
and
7.655±0.346,
respectively.
The
large
standard
deviation

in
the
HBVtg
hCD1d-‐KI
mice
is
likely
due
to
the
high
variation
of
PD-‐1
expression

levels
among
the
populations
of
CD4
and
CD8
T
cells
(see
below).
This
variation
may

also
be
affected
by
the
disease
status
of
the
mice,
based
on
factors
such
as
viral
titer.

Viral
titer
can
influence
cytokine
production,
thereby
causing
variations
in
the
anti-‐
HBV
response
between
individual
mice.
For
example,
IFNα/β
activity
in
mice
with

low
viral
titer
can
enhance
HBV
replication
[57].
This
implies
that
mice
with
lower

HBV
titer
may
have
larger
amount
of
immune
activation,
possibly
leading
to
cell

exhaustion.

Additionally,
we
also
determined
that
there
was
no
significant
change
in
PD-‐1

expression
in
NKT
and
NK
cells.
However,
NKT
cells
did
have
fluctuating
levels
of

PD-‐1
expression
between
HBVtg
hCD1d-‐KI
mice.
Fluctuating
levels
of
PD-‐1

expressed
by
NKT
cells,
have
been
seen
in
Moll
et
al
where
it
was
shown
in
a
clinical

study
that
the
NKT
cells
of
HIV
infected
patients
had
an
elevated,
but
varied
level
of

PD-‐1
expression,
in
compared
to
healthy
controls.

This
elevation
was
also
confined

only
to
the
CD4-‐
NKT
cell
population.
They
attributed
the
heterogeneity
to
be

correlated
to
the
proliferative,
but
not
IFNγ
producing
capacity
of
the
NKT
cells,
in

that
the
NKT
proliferation
decreased
with
PD-‐1
expression
[39].

32

Figure
11:
Characterization
of
PD-‐1
expression
in

CD8+
T
Cells
in
the
HBVtg
hCD1d-‐KI
model.
Liver

MNCs
were
first
electronically
gated
for
the
NK1.1-‐
TCRβ+
conventional
T
cell
population.
(A)
The

NK1.1-‐
TCRβ+
conventional
T
cells
were
gated
for

PD-‐1
against
SSC
to
determine
PD-‐1
expression.
The

graph
is
showing
average
percentage
of
PD-‐1

expression
in
HBVtg
hCD1d-‐KI
and
hCD1d-‐KI
mice.

(B)
NK1.1-‐
TCRβ+
cells
were
gated
on
CD4
and
CD8

to
isolate
the
CD8+
T
cell
population.
The
CD8+
T

cells
were
further
gated
on
PD-‐1
and
SSC
to

determine
the
expression
of
PD-‐1.
Each
gating
is

noted
with
the
percentage
of
positive
PD1

expression.

(C)
Levels
of
PD-‐1
expression
in
CD8+
T

Cells.
(D)
Levels
of
PD-‐1
expression
in
CD4+
T
Cells.

(E).
PD-‐1
expression
on
NKT
cells
compiled
from
all

HBVtg
hCD1d-‐KI
mice,
(F)
PD-‐1
expression
on
iNKT

cells
compiled
from
all
HBVtg
hCD1d-‐KI
and
hCD1d-‐
KI
mice.

33

We
then
focused
in
on
the
CD8+
T
cell
expression
of
PD-‐1.
Figures
11B
and
11C

illustrate
the
significant
upregulation
of
PD-‐1
expression
between
HBVtg
hCD1d-‐KI

and
hCD1d
CD8+
T
cells,
(p=0.008).
The
average
PD-‐1
expression
for
the
HBVtg

hCD1d-‐KI
model
and
hCD1d
KI
model
was
47.73±8.26
and
9.665±0.247,

respectively.
To
validate
this
CD8+
specific
PD-‐1
upregulation,
we
analyzed
the
PD-‐1

expression
of
CD4+
T
cells
(Figure
11D).
There
was
no
change
of
PD-‐1
expression

between
the
HBVtg
hCD1d-‐KI
and
hCD1d
CD4+
T
cells.
In
a
clinical
study
of
CHB

patients,
the
CD8+
T
cell
PD-‐1
expression
was
significantly
elevated
in
the
immune

clearance
stage
of
the
infection.
This
elevation
related
to
serum
ALT
levels
but
not

with
viral
DNA
nor
presence
of
HBeAg
[35].
Since
ALT
levels
indicate
liver
damage,

these
findings
demonstrate
that
CD8+
T
Cell
PD-‐1
expression
increase
with
liver

damage,
but
not
with
viral
factors.

We
can
infer
that
the
level
of
PD-‐1
expression

among
the
HBVtg
hCD1d-‐KI
mice
is
reflective
of
their
liver
damage.

We
speculated
that
the
decrease
of
iNKT
cells
in
the
HBVtg
hCD1d-‐KI
model
could

be
due
to
their
exhaustion.
Lee
at
al
found
that
in
patients
with
HCC,
the
level
of

their
iNKT
cells
correlated
with
PD-‐1
CD8+
T
Cells
[33].

It
has
also
been
shown
that

activated
iNKT
cells
upregulate
the
expression
of
PD-‐1
at
their
cell
surface,
and
Lee

at
el
showed
that
iNKT
cells
can
be
counter
balanced
by
PD-‐1
expressing
CD8+
T

cells
in
HCC
patients
[33].
HCC
is
one
possible
outcome
of
CHB,
leading
us
to
ponder

the
specific
features
in
CHB
that
may
facilitate
the
development
of
HCC.
We
however

did
not
see
any
change
in
iNKT
cell
expression
of
PD-‐1
(Figure
11F).
The
PD-‐1

expression
for
HBVtg
hCD1d-‐KI
and
hCD1-‐KI
was
2.067±1.49
and
1.94±1.73,

34

respectively.
This
finding
suggests
that
the
iNKT
cell
depletion
is
not
due
to

activation-‐induced
anergy.
However,
this
finding
is
from
one
particular
time
point
of

the
HBV
pathogenesis,
and
the
anergy
induction
and
subsequent
apoptosis
could

have
already
taken
place.

35

Discussion

1.
Summary
of
Results:

The
HBVtg
hCD1d
serves
to
provide
a
model
for
HBV
pathogenesis
in
vivo.

Mice
are

not
natural
hosts
of
HBV.
In
order
to
ensure
proper
infection,
an
HBVtg
model
was

created
and
subsequently
crossed
with
an
hCD1d-‐KI,
allowing
us
to
relate
the
HBV

pathogenesis
to
a
more
human
like
context.

This
model
is
a
useful
tool
in
studying

chronic
HBV
pathogenesis,
because
the
transgenic
mice
constitutively
express
the

viral
DNA,
preventing
them
from
clearing
the
infection.
This
can
provide
a
model

that
is
similar
to
humans,
where
the
immune
fails
in
controlling
HBV,
leading
to

chronic
infection.
With
this
model
we
can
identify
the
cell
populations
most

impacted
during
HBV
pathogenesis
and
providing
some
indications
into
the

important
mechanisms
involved.

In
this
experiment
we
characterized
the
invariant
NKT
cell
population
within
the

novel
HBVtg
hCD1d-‐KI
model.

We
have
demonstrated
that
in
this
model
there
is
a

significant
decrease
of
iNKT
cells,
accompanied
by
an
alternation
in
their

CD4+/CD4-‐
subpopulations.
Additionally,
there
is
a
decrease
in
the
overall
NKT
cell

population.
These
observations
have
been
consistent
with
a
previous
HBV-‐
transgenic
model
[62].
Our
data
is
also
consistent
with
clinical
studies
of
CHB

patients,
in
regards
to
declined
iNKT,
CD4+iNKT,
and
NKT
levels
[27].
Additionally,

we
showed
that
there
is
an
HBV-‐dependent
depletion
of
iNKT
cells,
demonstrating

their
critical
role
in
viral
infections
and
potential
as
a
target
for
viral
therapies.

36

We
further
validated
this
model
by
demonstrating
the
significant
upregulation
of

PD-‐1
expression
in
CD8+
T
cells,
which
has
been
well
documented.
The
PD-‐1

upregulation
was
largely
confined
to
CD8+
T
cells.

Additionally,
from
previous

studies
we
can
infer
that
the
level
of
PD-‐1
expression
among
the
HBVtg
hCD1d-‐KI

mice
is
could
be
indicative
of
their
liver
damage
[35],
it
would
also
be
useful
to

measure
the
levels
of
ALT
in
these
mice
and
compare
them
to
the
frequency
of
CD8+

T
Cells
and
PD-‐1
expression.

We
reasoned
that
the
decrease
of
iNKT
cells
could
be
also
caused
by
PD-‐1

expression,
potentially
resulting
in
apoptosis,
but
the
iNKT
cell
population
did
not

show
any
change
in
its
PD-‐1
expression.
Since
there
was
no
change
in
PD-‐1

expression
on
iNKT
cells,
we
speculated
that
their
depletion
is
unlikely
related
to

exhaustion.

2.
Implications
and
possible
mechanisms:

The
presence
of
iNKT
cells
in
HBV
infection
has
not
been
well
defined.
However,

there
are
viruses
that
have
been
shown
to
impact
the
iNKT
cell
population.
In
cases

of
HIV
and
LCMV
infections,
the
decrease
of
iNKT
cells
results
from
apoptosis,
but

through
two
very
distinct
pathways.
Van
der
Vliet,
et
al
showed
that
iNKT
cells

depleted
in
HIV-‐1
infection
undergo
Fas
mediated
apoptosis
[58].
In
contrast,
during

human
LCMV
infection,
the
iNKT
depletion
is
independent
of
Fas/FasL
expression

and
is
rather
a
result
of
direct
apoptosis
[20].
HBV
and
HIV
are
distinct
viruses
that

infect
different
cell
populations;
HBV
infects
hepatocytes
and
HIV
directly
infects

37

CD4+
T
cells.
In
spite
of
this
difference,
both
viral
infections
ultimately
alter
immune

cell
function
and
abundance,
which
leaves
the
possibility
that
they
may
have
some

similarities
in
the
depletion
of
certain
cellular
populations.

It
is
therefore
possible

that
iNKT
cell
population
could
be
depleted
by
direct
apoptosis
or
FAS
induced

apoptosis,
as
seen
in
HIV.

The
reduction
of
the
iNKT
cell
population
can
also
be
caused
by
the
down
regulation

of
the
hCD1d
receptor.
The
HBV
infection
could
cause
a
down
regulation
of
hCD1d

within
the
infected
hepatocyte
resulting
in
the
subsequent
depletion
of
iNKT
cells.

Zeissig
et
al
found
that
during
an
acute
HBV
infection
there
is
an
activation
of
non-‐
variant
type
II
NKT
cells,
which
indirectly
activate
Type
I
iNKT
cells
through
an

hCD1d
dependent
process.
In
this
study,
the
antigen
presentation
of
hCD1d

accomplished
by
a
process
mediated
by
the
presence
of
HBV
induced
lipids
[70].

Activated
Type
II
cells
are
known
to
recruit
type
I
NKT
cells
into
the
liver
and
via
DC

secretion
of
IL-‐12
and
MIP-‐2,
resulting
in
rapid
anergy
induction
of
the
Type
I
NKT

cells.
It
is
therefore
possible
that
stimulation
of
Type
II
NKT
cells
results
in
the

anergy
of
these
iNKT
cells
[18].
Additionally,
viruses
such
as
HSV
[69],
HIV
[50]
and

HPV
[38]
have
been
shown
to
interfere
with
CD1d
antigen
presentation,

independently
of
Type
II
NKT
cells.

Additionally,
because
the
production
of
IL-‐10
is
so
great,
it
is
possible
that
resident

hepatic
Tregs
can
inhibit
iNKT
expansion.
Induced
Tregs
(iTregs)
are
generated
in

an
environment
with
high
TGFβ
and
IL-‐2.
TGFβ
synergized
with
rapamycin
can

38

stimulate
human
iNKT
cells
to
induce
their
expression
of
Foxp3
[41].

However,
the

stimulation
of
the
iNKT
cells
with
TGFβ
was
done
ex-‐vivo,
so
it
does
not
take
into

account
other
physiological
factors
that
could
be
taking
place.
While
TGFβ
could

stimulate
the
iNKT
cells
to
convert
into
a
Foxp3+
regulatory
cell
population,
it
does

not
explain
the
decrease
of
CD4+
iNKT
cells.
Furthermore,
the
population
of
these

Foxp3+
iNKT
cells
suppressed
the
activity
of
total
CD4+
T
cells.
The
populations
of

CD4+
T
cells
in
our
study
were
not
altered,
further
rejecting
the
likelihood
of
iNKT

cell
conversion
to
Foxp3+
cells,
and
their
role
in
this
disease
model.

This
however

does
not
rule
out
the
possibility
iNKT
could
be
suppressed
by
Tregs.
Since
it
has

been
reported
that
iNKT
cells
are
able
to
inhibit
iTregs
in
vivo
via
IFNγ
[43],
it
is

possible
that
under
the
tolerogenic
environment
in
hepatitis,
Tregs
inhibit
iNKT

cells
and
limit
their
function.

There
is
evidence
that
Tregs
interact
with
NKT
cells

and
that
NKT
IL-‐2
and
IL-‐4
secretion
can
stimulate
the
proliferation
of
Tregs,
and

their
homing
to
the
liver.
Specifically,
the
IL-‐2
production
is
restricted
to
CD4+
CD1d

restricted
NKT
cells
[26].
If
this
theory
were
applied
to
our
model,
it
would
suggest
a

possible
negative
feedback
mechanism
between
IL-‐2,
Tregs
and
CD4+
iNKT
cells.

Hua
et
al
showed
that
in
CD1d
deficient
mice
have
a
reduced
hepatic
population
of

Tregs
[23],
further
demonstrating
that
the
interactions
between
NKT
cells
and
Tregs

may
play
a
critical
role
in
liver
homeostasis.
Additionally,
in
vitro,
CD4+CD25+
Tregs

have
also
been
shown
to
inhibit
Vα24
iNKT
cells,
but
the
mechanism
was
not

elucidated
[4].

A
recent
study
documented
that
the
frequency
of
Tregs
is
positively

correlated
with
IL-‐10
and
TGFβ
and
is
negatively
correlated
with
IFNγ
in
patients

with
HCC
[33].

39

In
a
Con-‐A
induced
model
of
hepatitis,
it
was
found
that
the
presence
of
IL-‐17A

negatively
regulates
the
function
of
NKT
cells
and
their
IFNγ
production
[73].

Although
named
proinflammatory,
TH17
cells
have
been
shown
to
enhance
the

survival
of
virally
infected
cells
[71].
Yang
et
al
found
that
increased
IL-‐17
levels

contribute
to
the
aggravation
of
CHB
infection
in
patients
[68].

More
evidence
for

the
detrimental
effects
of
IL-‐17
production
were
exemplified
in
patients
with
HCC,

demonstrating
an
increased
frequency
of
TH-‐17
cells
in
tumor
infiltrating

lymphocytes,
which
correlated
with
reduced
survival
[33].
Recently,
it
has
been

demonstrated
that
any
iNKT
population
can
be
polarized
towards
TH-‐17
type
cells

and
secrete
IL-‐17.
This
polarization
can
occur
in
the
presence
of
TGFβ
[40],
whose

production
is
elevated
in
the
early
stages
of
the
HBV
infection.
Additionally,
the

induction
of
IL-‐17
iNKT
cells
involves
the
downregulation
of
their
NK1.1
and
CD4+

expression
[40].
Taken
together,
these
results
could
explain
through
that
IFNα

signaling,
at
least
in
the
mice
with
lower
viral
titer,
could
cause
an
upregulation
of

IL-‐17
production.
IL-‐17
and
the
HBV
induced
TFGβ
production
could
alter
the
iNKT

phenotype,
and
ensue
their
NK1.1
and
CD4
downregulation.
Furthermore,
while

Tregs
can
inhibit
NKT
cells
and
IFNγ
production,
they
have
not
been
shown
to

suppress
the
proinflammatory
functions
of
TH-‐17
cells,
they
actually
help
stabilize

these
populations
[71].

3.
Future
Directions

This
study
also
provided
us
with
a
tool
to
study
the
role
of
iNKT
cells
in
a
human-‐
like
model
of
chronic
hepatitis
B,
and
the
anti-‐viral
responses
in
HBV
pathogenesis.

40

There
are
other
possible
pathways
to
explore
in
investigating
the
reason
behind
the

iNKT
cell
decline.
Without
PD-‐1
expression,
the
iNKT
cells
could
still
have
been

undergoing
apoptosis.
Cell
staining
with
Annexin
V
identify
cells
undergoing

apoptosis,
regardless
of
the
pathway
induced.

Additionally,
staining
cells
for

chemokine
receptors
could
identify
if
the
iNKT
cells
are
homing
to
other
tissues.
It

will
be
very
interesting
to
examine
the
Annexin
V
expression
in
iNKT
cells
in
our

model.

Our
study
gives
us
a
snapshot
in
chronic
stage
of
HBV
infection
at
one
particular

point.
It
would
therefore
be
useful
to
assess
some
other
characteristics
that
could
be

influencing
the
changes
in
these
cell
populations.
Viral
titer
is
a
very
important

measurement
that
could
provide
explanations
for
variations
of
cell
populations
and

PD-‐1
expression.
Although
viral
titer
is
relatively
constant
through
out
the
mouse

lifespan,
females
are
known
to
have
a
reduced
viral
load,
compared
to
male
mice

[57].
Additionally,
we
first
bred
mice
through
the
use
of
both
HBV
positive
parents,

which
made
some
progeny
sicker.
Also,
it
is
then
possible
that
some
of
these
mice

had
two
copies
of
the
HBV
transgene,
and
some
had
one,
which
would
also
influence

the
viral
titer.

Our
collaborators
in
Dr.
James
Ou’s
group
have
found
that
in
low
HBV

DNA
conditions,
IFNα/β
signaling
results
in
the
increase
of
HBV
replication
[57].

Therefore,
variations
in
viral
titer
can
result
in
different
spectrum
of
cytokine

activation.
It
would
thus
be
beneficial
to
examine
the
relationship
between
cell

populations
and
viral
titer.

It
would
also
be
beneficial
to
measure
the
amount
of

cytokines
that
are
implicated
in
the
HBV
infection,
such
as
TGFβ,
IL-‐10,
and
IFNs
α/β

41

and
γ,
and
to
test
the
cytotoxic
abilities
of
the
CD8+
T
cells,
to
determine
the
extend

of
the
exhaustion.

Because
of
the
implications
of
IL-‐17
and
Treg
cells
in
liver
homeostasis,
tolerance,

and
activity
during
viral
infections,
it
would
be
beneficial
to
include
the

characterization
of
these
populations
in
this
HBV
tg
hCD1d
model.
Treg
expression

correlates
with
the
CHB
viral
titer
[46].
Tregs
are
also
known
to
suppress
the

functions
of
NKT
cells,
CD4+
and
CD8+
conventional
T
cells,
B
cells,
DCs,
monocytes

and
macrophages
[47],
implicating
their
importance
in
the
pathogenesis
of
HBV.
In

our
characterization,
we
did
not
see
any
change
in
the
population
of
NK
cells.
Tregs

are
known
to
inhibit
NK
cells
by
TGFβ
stimulation
of
NKG2D.
It
is
thus
possible
that

the
lack
of
difference
between
the
populations
shows
a
lack
of
viral
specific

induction
and
proliferation
[47].
It
would
therefore
be
beneficial
to
investigate
the

whether
these
cells
have
been
activated.
Moreover,
it
would
also
be
interesting
to

see
measure
the
levels
of
ALT
and
access
the
liver
damage
in
the
HBVtg
hCD1d-‐KI

mice.

This
HBVtg
hCD1d-‐KI
model
highlights
different
points
of
therapeutic
intervention

in
chronic
HBV
infection.
However,
each
one
of
these
pathways
has
potential

loopholes
making
it
possible
for
viral
persistence.
To
achieve
successful
clearance
of

HBV
different
pathways
of
the
immune
response
should
be
targeted.
For
example,

blocking
PD-‐1
with
simultaneous
stimulation
of
iNKT
cells
could
reverse
CTL

exhaustion
and
synergistically
increase
their
function.
iNKT
IFNγ
production
could

42

also
increase
other
antiviral
mechanisms
and
counteract
Treg
populations.

Matarollo
et
al
proposed
that
in
order
to
increase
the
NKT
efficiency
in
adjuvant

based
tumor
vaccinations,
Treg
populations
should,
at
least
transiently,
be

eliminated
[36].
Additionally,
blocking
the
PD-‐1/PD-‐L1
pathway
has
been
shown
to

restore
CTL
function
in
murine
LCMV
chronic
infection
models
and
ex
vivo

experiments
with
HIV,
chronic
hepatitis
and
HPV
[21].

Finally,
characterizing
the
HBVtg
hCD1d
KI
model
provides
us
with
a
new
platform

for
testing
new
anti-‐HBV
therapeutic
lipids
that
will
be
more
compatible
with

human
immune
conditions.

α-‐Carba-‐GalCer
has
demonstrated
Th-‐1
stimulation
of

Vα24
[56]
cells,
but
other
potential
lipid
antigens
also
need
to
be
investigated.

43

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Abstract (if available)

Abstract Hepatitis B virus gives rise to chronic infection in 350 million people world wide, and infection can result in the development of liver cirrhosis, liver failure or hepatocellular carcinoma. Current therapies merely treat the infection and prevent progression, because there is no cure. There have been promising studies in the clearance of HBV DNA in murine models with the use of iNKT stimulating lipid αGalCer. However, iNKT stimulation of αGalCer has not been an effective measure in anti-viral or anti-cancer clinical trials. It is speculated that these differences between human and mouse reactivity to αGalCer is based on the differences of CD1d/NKT lipid presentation systems between the two species. Previously, our lab generated an hCD1d KI model to support a human like NKT cell environment. In order to investigate the role of invariant NKT cells in HBV and to allow us to test potential therapeutic lipids for HBV in a more human like environment, we generated a novel HBVtg hCD1d-KI model. Thus in this study we seek to characterize the population of iNKT cells in this new HBVtg hCD1d-KI model. As reported in human chronic HBV patients, we found that the iNKT cells are significantly lower in this new HBV-transgenic model. Among the decreased levels of iNKT cells, there was a significant decrease in the population of CD4+ iNKT cells accompanied by a significant increase of CD4- iNKT cells. Similarly, there was an overall significant decrease of total NKT Cells. There was an overall increase in CD8+ conventional T Cells, and a significant increase of total conventional T cells. Our study also found that conventional T cells had and significant upregulation of PD-1, which was restricted to the CD8+ population. In our study, the levels of PD-1 among the iNKT cell populations were not affected

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