Figure 1.1 Schematic diagram of HBV structure: The HBV virion
consists of 3 envelope proteins (S, M and L glycoprotein), a
nucleocapsid protein Core, a partially double-stranded DNA genome
and a polymerase enzyme that transcribes viral RNA from a DNA
template. Figure taken from the Swiss Institute of Bioinformatics
website; http://www.isb-sib.ch
7
Figure 1.2 Schematic diagram of HBV life cycle: The figure was
adopted from Rehermann and Nascimbeni 2006.
HBV binds to an unidentified receptor on the hepatocyte surface and
fuses with the cell membrane. After cell entry, HBV nucleocapsids are
uncoated and released into the cytoplasm. They are transported to the
nucleus, where the covalently closed circular DNA (cccDNA) is formed.
Transcription yields four viral RNAs which are subsequently transported
to the ribosome and translated to form viral proteins as part of virion
formation. Pre-genomic RNA is translated to core and reverse
transcriptase proteins. Nucleocapsids are enveloped with Small, Medium
and Large envelope proteins (HBs) as they pass through the endoplasmic
reticulum (ER) and/or Golgi complex and are then secreted.
Alternatively, nucleocapsids can be recycled back to the nucleus where
their pre-genomic RNA is reverse transcribed to DNA as part of the viral
replication process.
8
Figure 1.3 The course of acute and chronic HBV infection:
shows serology and virology of acute (a) and chronic (b) HBV
infection. Figure adopted from Chang & Lewin 2006.
13
CD8+
T cell
Proliferation
Induction
of apoptosis
DC
IFN-, IL-2
Antigen
Presentation
CD4+
T cell
TNF-α, IL-12
Proliferation
IFN-
IL-2
IL-12,
IL-18
IFN-α/β
Induction
of apoptosis
Virus-infected
cells
IFN-
IFN-α/β
IL-1, IL-6
Inflammation
NK
TNF-α, IL-12
IL-2
Macrophage
Figure 1.4 Anti-viral immune response: Virus-infected cells release
IFN-α/β which activate DC, macrophages and NK cells. The expression
of viral antigens or danger molecules on the surface of infected cells also
activate these cells. NK cell can also be indirectly activated by DC- or
macrophage-derived cytokines. Activated, NK cells can kill infected
cells and secrete IFN- and TNF-α which can exert direct anti-viral
effects, or IL-2 which can promote T cell proliferation. The activated DC
can present antigens to CD4+ and CD8+ T cells and also produces IFN-,
TNF-α and IL-2 which provide signals that stimulate clonal expansion
and subsequent cytotoxic T cell responses. The production of IFN- and
IL-2 by CD4+ T cells can sustain and amplify the anti-viral immune
responses. Macrophages can also produce IL-1 and IL-6 to initiate
inflammation.
20
1.5
% V  1 + CD3 +
lymphocytes
Concentration
(μg / ml)
1.0
1
0.5
0.25
0.1
0.05
0.5
0.0
Donor 1
Donor 2
Figure 2.2: The optimal concentration of anti-V1 mAb is
0.25μg/ml. Scatterplot showing the different frequencies of V1+ T
cells from 2 healthy donors, quantified for each anti-V1 mAb
concentrations used.
61
A
B
C
1.5
R1
R6
IL-12
1.0
0.5
0
101 102 103 104 105
FL3
MFI
D
E
F
G
H
I
J
K
L
M
FL2
Figure 2.8: Analysis of cytometric bead array standards. A, dot plot
of antibody coated beads with electronic gate R1 surrounding the
antibody coated beads and excluding the debris. B, dot plot of all
populations of cytokine beads, previously gated in R1 and now, divided
by their fluorescence intensity in the FL3 and FL4 channels. IL-12
conjugated beads are gated in R6. C, IL-12 standard curve with MFI of
anti-IL-12 coated bead population on X-axis and IL-12 concentration on
Y-axis in ng/ml. D-M, dot plots showing IL-12 conjugated beads in ten
standard tubes, from bottom tube with no IL-12 content (D) to the top
tube with the highest IL-12 content (M).
79
A
E
R5
R1
B
F
R2
R6
C
G
R3
R7
H
D
R4
R8
Figure 2.9: Analysis of cytometric bead array. A, representative dot
plots of antibody coated beads with electronic gate R1 surrounding the
antibody coated beads and excluding the debris (left) and representative
dot plot of all populations of cytokine beads, previously gated in R1 to
exclude debris and now, divided by their fluorescence intensity in the FL3
and FL4 channels (right). B, C, D, E, F, G, (left), representative dot plots
of all populations of cytokine beads showing gating of conjugated beads
for identification of IL-4 in R2 (B), IL-6 in R3 (C), IL-10 in R4 (D), IFN in R5 (E), IL-12 in R6 (F) and IL-13 in R7 (G). B, C, D, E, F, G,
(right), representative dot plot showing only IL-4, IL-6, IL-10, IFN-, IL12 or IL-13 conjugated beads, respectively, because the plot has been
gated on R2, R3, R4, R5, R6 or R7. The MFI of each single bead
population in FL2 indicates the level of cytokine expression in the sample.
H, representative dot plots showing TGF-β1 conjugated beads only. The
TGF-β1 conjugated beads are the only beads present in this sample and
are gated in R8 (left). The MFI values of the populations in FL2
represents the TGF-β1 concentration of the sample (right).
80
B
50
*
40
30
20
10
0
Control Chronic HBV
NT Cells
NK
Cells
PE – CD56 Log
D
% CD56+CD3+ lymphocytes
PE - IgG1 Log
C
% CD56+CD3- lymphocytes
T
Cells
PECy5 – CD3 Log
PE-Cy5 – IgG1 Log
A
50
***
40
30
20
10
0
Control Chronic HBV
% CD56+ lymphocytes
E
50
***
40
30
20
10
0
Control Chronic HBV
Figure 3.1: Frequencies of circulating NK, NT and total CD56+ cells
are significantly higher in HBV patients than in control subjects. A
and B, representative dot plots of whole PBMC, gated on lymphocytes
and A, stained with matched isotype controls PE- and PE-Cy5-labelled
anti-IgG1 mAb or B, stained with PE-labelled anti-CD56 mAb and PECy5-labelled anti-CD3 mAb for identification of NK, NT and total
CD56+ cell populations. C, D and E, scatterplots showing the
frequencies of circulating CD56+CD3-, CD56+CD3+ and total CD56+
cells from each of the 66 control subjects (red) and each of the 62 HBV
patients (blue). * p<0.05,*** p<0.0005.
96
PE=Cy5 – CD3 Log
A
CD56DIM NK Cells
CD56BRIGHT NK Cells
PE - CD56 Log
CD56DIM cells
(% of CD56+ cells)
100
**
**
**
C
CD56DIM cells
(% of lymphocytes)
B
80
60
40
20
0
50
*
**
*
40
30
20
10
0
NK NT CD56+
Cells Cells Cells
NK
NT CD56+
Cells Cells Cells
CD56BRIGHT cells
(% of CD56+ cells)
100
**
** ***
80
60
40
20
0
NK
NT CD56+
Cells Cells Cells
CD56BRIGHT cells
(% of lymphocytes)
E
D
5
4
3
2
1
0
NT CD56+
NK
Cells Cells Cells
Figure 3.2: The frequencies of the CD56DIM, but not the CD56BRIGHT,
NK, NT and total CD56+ cells, are significantly higher in HBV
infection. A, B, C and D, scatterplots showing the frequencies of
CD56DIM cells (B, C) and CD56BRIGHT cells (D, E) within the
CD56+CD3-, CD56+CD3+ and total CD56+ cell populations as a
percentage of each CD56+ population (B, D) and as a percentage of
lymphocytes (C, E) from each of the 66 control subjects (red) and each
of the 62 HBV patients (blue). * p<0.05, ** p<0.009, ***p<0.0007.
99
A
B
%  + CD3 + cells
PE-Cy5 - CD3 Log
40
***
30
20
10
0
FITC -  Log
Control Chronic HBV
Figure 3.3: The frequencies of + T cells are significantly higher in
HBV infection. A, representative dot plot of PBMC, gated on
lymphocytes and stained with FITC-labelled anti--TCR mAb and PECy5-labelled anti-CD3 mAb for the identification of + T cells. B,
scatterplot showing the frequency of +CD3+ lymphocytes in 23 control
subjects (red) and 20 HBV patients (blue). *** p=0.0009.
101
FITC - Vα24 Log
% Vα24+Vβ11+ T cells
B
PE - Vβ11 Log
A
D
% 6B11+ T cells
PE-Cy5 - CD3 Log
C
1.0
0.8
0.6
0.4
0.2
0.0
Control
Chronic HBV
Control
Chronic HBV
4
3
2
1
0
PE - 6B11 Log
Figure 3.4: There are no significant differences in the frequencies of
invariant NKT (iNKT) cells between controls subjects or HBV
patients. A, C, representative dot plots of PBMC, gated on lymphocytes
and, stained with FITC-labelled anti-Va24 mAb and PE-labelled antiVb11 mAb (A) or PE-labelled anti-6B11 mAb and PE-Cy5-labelled antiCD3 mAb (C). B, D, scatterplots showing frequencies of iNKTs in 23
controls (red) and 20 HBV patients (blue).
102
PE - CD1d Log
D
PE-Cy5 - CD14 Log
PE - CD1d Log
100
80
60
40
20
0
CD1a
CD1b CD1c CD1d
CD1a
CD1b CD1c CD1d
100
% CD1 + CD14 + cells
C
% CD1 + CD19 + lymphocytes
B
APC - CD19 Log
A
80
60
40
20
0
Figure 3.5: There are no significant differences in the frequencies of
CD1+ cells between controls subjects or HBV patients. A and C,
representative dot plots of PBMC, gated on lymphocytes (A) or
monocytes (C) and, stained with PE-labelled anti-CD1d mAb and APClabelled anti-CD19 mAb (A) or PE-labelled anti-CD1d mAb and PECy5-labelled anti-CD14 mAb (C). B, D, scatterplots showing
frequencies of CD1a+, CD1b+, CD1c+ and CD1d+ B cells (B) or
monocytes (D) in 18 controls (red) and 18 HBV patients (blue).
104
B
30
20
10
0
Low
% CD56 + CD3 - of lymphocytes
% CD56 + CD3 - of lymphocytes
A
30
20
10
0
High
< 40
ALT
D
30
20
10
0
Male
Female
Gender
% CD56 + CD3 - of lymphocytes
C
% CD56 + CD3 - of lymphocytes
Viral load
> 40
30
20
10
0
19 - 35 35 - 55
Age
Figure 3.6: There are no significant differences in the frequencies of
NK cells between patient groups within the study cohort. A,
scatterplot showing frequencies of NK cells in 41 HBV patients with
viral load between 10 and 10,000 copies / ml and 8 HBV patients with
viral load between 300,000 and 5x108 copies / ml. B, scatterplot showing
frequencies of NK cells in 39 HBV patients with ALT below 40 IU/ml
and 13 HBV patients with ALT above 40 IU/ml. C, scatterplot showing
frequencies of NK cells in 27 male HBV patients and 28 female HBV
patients. D, scatterplot showing frequencies of NK cells for 28 HBV
patients between the age of 19 and 35 years and 27 HBV patients
between the age of 35 and 55 years.
108
B
50
40
30
20
10
0
Low
High
% CD56 + CD3 + of lymphocytes
% CD56 + CD3 + of lymphocytes
A
50
40
30
20
10
0
< 40
ALT
D
50
40
30
20
10
0
Male
Female
Gender
% CD56 + CD3 + of lymphocytes
% CD56 + CD3 + of lymphocytes
Viral load
C
> 40
50
40
30
20
10
0
19 - 35
35 - 55
Age
Figure 3.7: There are no significant differences in the frequencies of
NT cells between patient groups within the study cohort. A,
scatterplot showing frequencies of NT cells in 40 HBV patients with
viral load between 10 and 10,000 copies / ml and 9 HBV patients with
viral load between 300,000 and 5x108 copies / ml. B, scatterplot showing
frequencies of NT cells in 38 HBV patients with ALT below 40 IU/ml
and 13 HBV patients with ALT above 40 IU/ml. C, scatterplot showing
frequencies of NT cells in 26 male HBV patients and 28 female HBV
patients. D, scatterplot showing frequencies of NT cells for 33 HBV
patients between the age of 19 and 35 years and 21 HBV patients
between the age of 35 and 55 years.
109
B
40
30
20
10
0
Low
%  + CD3 + of lymphocytes
%  + CD3 + of lymphocytes
A
40
30
20
10
0
High
< 40
30
20
+
10
0
Male
Female
Gender
%  + CD3 + of lymphocytes
ALT
D
40
+
C
%  CD3 of lymphocytes
Viral load
> 40
40
30
20
10
0
19 - 35
35 - 55
Age
Figure 3.8: There are no significant differences in the frequencies of
 T cells between patient groups within the study cohort. A,
scatterplot showing frequencies of  T cells in 40 HBV patients with
viral load between 10 and 10,000 copies / ml and 9 HBV patients with
viral load between 300,000 and 5x108 copies / ml. B, scatterplot showing
frequencies of δ T cells in 38 HBV patients with ALT below 40 IU/ml
and 13 HBV patients with ALT above 40 IU/ml. C, scatterplot showing
frequencies of  T cells in 26 male HBV patients and 28 female HBV
patients. D, scatterplot showing frequencies of δ T cells for 33 HBV
patients between the age of 19 and 35 years and 21 HBV patients
between the age of 35 and 55 years.
111
G
0.2
0.1
% 6B11 + CD3 +
lymphocytes
E
% V a 24+ V b 11 + CD3 +
lymphocytes
0.2
0.1
% 6B11 + CD3 +
lymphocytes
% V a 24 + V b 11 + CD3 +
lymphocytes
C
% V a 24 + V b 11 + CD3 +
lymphocytes
0.2
0.1
% 6B11 + CD3 +
lymphocytes
% V a 24 + V b 11 + CD3 +
lymphocytes
A
0.2
0.1
% 6B11 + CD3 +
lymphocytes
0.3
B
0.3
Low
0.3
0.3
0.0
25 - 35 35 - 50
Age (years)
117
4
0.0
D
0.0
F
0.0
H
3
2
1
< 40 > 40
ALT
0
< 40
> 40
ALT
4
3
2
1
High
0
Viral load
Male Female
Gender
Low
High
Viral load
4
3
2
1
0
Male Female
Gender
4
3
2
1
0
25 - 35 35 - 50
Age (years)
Figure 3.9: There are no significant differences in the frequencies of
iNKT cells between patient groups within the study cohort.
A, scatterplot showing frequencies of Vα24+Vβ11+CD3+ lymphocytes in
12 HBV patients with ALT below 40 IU/ml and 5 HBV patients with
ALT above 40 IU/ml. B, scatterplot showing frequencies of
Vα24+Vβ11+CD3+ lymphocytes in 15 HBV patients with viral load
below 10,000 copies / ml and 2 HBV patients with viral load between
300,000 and 5x108 copies / ml. C, scatterplot showing frequencies of
Vα24+Vβ11+CD3+ lymphocytes in 9 male HBV patients and 8 female
HBV patients. D, scatterplot showing frequencies of Vα24+Vβ11+CD3+
lymphocytes for 7 HBV patients under the age of 35 years and 10 HBV
patients between the age of 35 and 55 years. E, scatterplot showing
frequencies of 6B11+CD3+ lymphocytes in 12 HBV patients with ALT
below 40 IU/ml and 6 HBV patients with ALT above 40 IU/ml. F,
scatterplot showing frequencies of 6B11+CD3+ lymphocytes in 15 HBV
patients with viral load below 10,000 copies / ml 3 HBV patients with
viral load between 300,000 and 5x108 copies / ml. G, scatterplot showing
frequencies of 6B11+CD3+ lymphocytes in 9 male HBV patients and 9
female HBV patients. H, scatterplot showing frequencies of 6B11+CD3+
lymphocytes for 8 HBV patients under the age of 35 years and 10 HBV
patients between the age of 35 and 55 years.
118
% CD1+CD14+
monocytes
20
0
F
% CD1+CD14+
monocytes
60
40
20
0
G
% CD1+CD14+
monocytes
60
40
20
0
H
% CD1+CD14+
monocytes
100
80
% CD1+CD19+
lymphocytes
D
40
100
80
% CD1+CD19+
lymphocytes
C
60
100
80
% CD1+CD19+
lymphocytes
B
E
100
80
% CD1+CD19+
lymphocytes
A
60
40
20
0
a
b c d
(CD1 isoform)
100
80
ALT
< 40
> 40
60
40
20
0
100
80
Viral load
Low
High
60
40
20
0
100
80
Gender
Male
Female
60
40
20
0
100
80
Age (yrs)
< 35
35 - 55
60
40
20
0
a b c d
(CD1 isoform)
Figure 3.10: There are no significant differences in the frequencies of
CD1a, b, c or CD1d+ B cells or monocytes between patient groups within
the study cohort. A, E, scatterplots showing frequencies of CD1+CD19+
lymphocytes (A) or CD1+CD14+ monocytes (E) in 12 HBV patients with
ALT below 40 IU/ml and 6 HBV patients with ALT above 40 IU/ml. B, F,
scatterplots showing frequencies of CD1+CD19+ lymphocytes (B) or
CD1+CD14+ monocytes (F) in 15 HBV patients with viral load below 10,000
copies / ml and 3 HBV patients with viral load between 300,000 and 5x108
copies / ml. C, G, scatterplots showing frequencies of CD1+CD19+
lymphocytes (C) or CD1+CD14+ monocytes (G) in 9 male HBV patients and
9 female HBV patients. D, H, scatterplots showing frequencies of
CD1+CD19+ lymphocytes (D) or CD1+CD14+ monocytes (H) in 8 HBV
patients under the age of 35 years and 10 HBV patients between the age of 35
and 55 years.
119
NK cells
% of lymphocytes
A
20
African
Caucasian
Asian
15
10
5
0
C
CD56DIM NK cells
(% of lymphocytes)
CD56DIM NK Cells
(% of NK cells)
B
100
90
80
70
60
15
10
5
0
E
CD56BRIGHT NK cells
(% of lymphocytes)
CD56BRIGHT NK Cells
(% of NK cells)
D
20
40
30
20
10
0
1.5
1.0
0.5
0.0
Figure 3.11: Frequencies of circulating total NK cells, CD56DIM NK cells
and CD56BRIGHT NK cells are similar in african, caucasian and asian
healthy control subjects. A, scatterplot showing the frequencies of circulating
NK cells in 15 african (green), 15 caucasian (blue) and 10 asian (pink) healthy
control subjects. B, C, D and E, scatterplots showing the frequencies of
circulating CD56DIM (B,C) and CD56BRIGHT NK cells (D,E), as a percentage of
NK cells (B,D) and as a percentage of lymphocytes (C,E) in 15 african
(green), 15 caucasian (blue) and 10 asian (pink) healthy control subjects.
124
B
***
NT cells
(% of lymphocytes)
NT cells
(% of lymphocytes)
A
5
4
3
2
1
0
D
CD56DIM NT cells
(% of lymphocytes)
CD56DIMNT Cells
(% of NT cells)
C
100
95
90
85
***
6
4
2
0
F
CD56BRIGHT NT cells
(% of lymphocytes)
E
CD56BRIGHT NT cells
(% of NT cells)
50
40
30
20
10
0
10
8
6
4
2
0
0.2
0.1
0.0
African
Caucasian
Asian
Figure 3.12: Differences in the frequencies of circulating total NT cells
between african and caucasian control subjects but not between african
and caucasian patients. A, scatterplots showing the frequencies of circulating
NT cells in 15 african (green), 15 caucasian (blue) and 10 asian (pink) healthy
control subjects (A) and, in 21 african (green), 21 caucasian (blue) and 7 asian
(pink) HBV-infected subjects (B). C, D, E and F, scatterplots showing the
frequencies of circulating CD56DIM (C, D) and CD56BRIGHT NT cells (E, F), as
a percentage of NT cells (C, E) and as a percentage of lymphocytes (D, F) in
15 african (green), 15 caucasian (blue) and 10 asian (pink) healthy control
subjects. (*p=0.05,**p=0.008).
125
Total CD56+ cells
(% of lymphocytes)
A
African
Caucasian
Asian
20
15
10
5
0
C
Total CD56DIM cells
(% of lymphocytes)
CD56DIM cells
(% of CD56+ cells)
B
100
90
80
70
60
15
10
5
0
E
Total CD56BRIGHT cells
(% of lymphocytes)
CD56BRIGHT cells
(% of CD56+ cells)
D
20
40
30
20
10
0
1.5
1.0
0.5
0.0
Figure 3.13: Frequencies of circulating total CD56+ cells, CD56DIM cells
and CD56BRIGHT cells are similar in african, caucasians and asian healthy
control subjects. A, scatterplot showing the frequencies of circulating total
CD56+ cells in 15 african (green), 15 caucasian (blue) and 10 asian (pink)
healthy control subjects. B, C, D and E, scatterplots showing the frequencies
of circulating CD56DIM (B,C) and CD56BRIGHT cells (D,E), as a percentage of
total CD56+ cells (B,D) and as a percentage of lymphocytes (C,E) in 15
african (green), 15 caucasian (blue) and 10 asian (pink) healthy control
subjects.
126
% 6B11 + T cells
2.5
2.0
1.5
1.0
0.5
0.0
African
Caucasian
Asian
Figure 3.14: Frequencies of circulating invariant NKT cells are similar in
african, caucasian and asian healthy control subjects. Scatterplot showing
the frequencies of circulating iNKT cells, as a percentage of total T cells, in 15
african (green), 15 caucasian (blue) and 10 asian (pink) healthy control
subjects.
127
A
B
30
% specific lysis
% specific lysis
30
20
10
0
25
5
E/T ratio
1
C
50
25
5
E/T ratio
1
D
50
50
40
40
% specific lysis
% specific lysis
10
0
50
30
20
10
30
20
10
0
0
50
25
5
E/T ratio
1
E
50
25
5
E/T ratio
1
F
30
% specific lysis
30
% specific lysis
20
20
10
0
20
10
0
50
25
E/T ratio
5
130
50
25
5
E/T ratio
1
G
Control
Medium
Chronic HBV
IL-2
*
IFN-α
0
1
2
H
3
4
5
6
% Specific lysis
Medium
IL-2
IFN-α
0
5
10
15
20
% Specific lysis
I
Medium
IL-2
IFN-α
0
5
10
15
20
% Specific lysis
131
25
30
35
Figure 3.15: Consistently higher cytotoxic capabilities of PBMC
from HBV patients.
A, B, plots showing percentages of specific lysis of K562 cells by
unstimulated PBMC from 7 control subjects (A) and 15 HBV patients
(B) at E:T ratios of 50, 25, 5 and 1:1. C, D, plots showing percentages of
specific lysis of K562 cells by IL-2-stimulated PBMC from 7 control
subjects (C) and 15 HBV patients (D) at E:T ratios of 50, 25, 5 and 1:1.
E, F, percentages of specific lysis of K562 cells by IFN-α-stimulated
PBMC from 7 – 55 control subjects (E) and 15 HBV patients (F) at E:T
ratios of 50, 25 and 5:1. G, H, I, bar charts showing mean percentages of
specific lysis of K562 cells by natural, IL-2-induced and IFN-α-induced
cytotoxicity carried out by PBMC from 7 control subjects (red) and 15
HBV patients (blue), at E:T ratios of 5:1 (G) and 25:1 (H), and PBMC
from 55 control subjects (red) and 15 HBV patients (blue) at an E:T ratio
of 50:1 (I) (*p<0.05).
132
B
SSC - Lin
Pe-CY5 – CD3 Log
A
CD56T cells
NT cells
NK
cells
PE – CD56 Log
FSC - Lin
PE – CD56 Log
C
IFN-+ CD56BRIGHT lymphocytes
IFN-+ CD56DIM lymphocytes
FITC – IFN- Log
E
IFN--producing
T and NT cells
FITC – IFN- Log
PE-Cy5 – IgG1 Log
Pe-CY5 – CD3 Log
D
FITC - IgG1 Log
Figure 4.1: Representative dot plots of IFN--producing
NK, NT and CD56- T cells. A, representative dot plot of
whole PBMC with region R1 gated around lymphocytes. B,
C, dot plots of whole PBMC gated on lymphocytes and
showing further gating on NK cell, NT and CD56- T cell
populations (B) and gating on IFN-+ CD56DIM and
CD56BRIGHT NK cell populations (C). D, dot plot of whole
PBMC gated on lymphocytes and showing further gating on
IFN--producing T and NT cell populations. E, dot plot of
isotype control used to gate on IFN-+ population
148
No. of IFN-+ cells
per 100 lymphocytes
IFN-+ cells
A
B
40
10
NK cells
% of NK cells
50
30
20
8
4
10
2
0
0
Medium PMA/I anti-CD3/28
D
**
CD56DIM NK cells
50
40
30
20
10
0
8
*
6
4
2
0
Medium PMA/I anti-CD3/28
Medium PMA/I anti-CD3/28
F
100
*
*
80
60
40
20
0
Medium PMA/I anti-CD3/28
CD56DIM NK cells
% of CD56DIM NK cells
% of CD56BRIGHT NK cells
E
*
6
Medium PMA/I anti-CD3/28
C
*
1.0
0.8
0.6
0.4
0.2
0.0
Medium PMA/I anti-CD3/28
Figure 4.2: Higher frequencies of IFN--producing CD56DIM and
CD56BRIGHT NK cells and total NK cells in HBV infection. A, B,
scatterplots showing frequencies of
IFN--producing NK cells
quantified as a percentage of total NK cells (A) and total lymphocytes
(B) in 23 controls (red) and 32 HBV patients (blue), following incubation
in medium conditioned with and without PMA/I or anti-CD3 and antiCD28 mAb. C, D, E, F, scatterplots showing frequencies of IFN-producing CD56DIM (C, D) and CD56BRIGHT NK cells (E, F) quantified
as a percentage of total CD56DIM NK cells (C) or CD56BRIGHT NK cells
(E) and as a percentage of total lymphocytes (D, F) in 23 controls (red)
and 26 HBV patients (blue), following incubation in medium
conditioned with and without PMA/I or anti-CD3 and anti-CD28 mAb
(*p<0.05, **p<0.005).
149
No. of IFN-+ cells
per 100 lymphocytes
IFN-+ cells
A
***
**
B
10
80
NT cells
% of NT cells
100
60
40
% of lymphocytes
4
0
0
30
20
10
0
40
Medium PMA/I anti-CD3/28
D
***
10
***
8
6
4
2
0
Medium PMA/I anti-CD3/28
E
6
2
CD56- T cells
% of CD56- T cells
C
***
8
20
Medium PMA/I anti-CD3/28
***
*
*
Medium
PMA/I
Medium PMA/I anti-CD3/28
30
20
10
0
Figure 4.3: The frequencies of IFN--producing NT, CD56- T and
total lymphocytes are higher in chronic HBV infection. A, B, C, D,
scatterplots showing frequencies of IFN--producing NT (A, B) and
CD56- T cells (C, D) quantified as a percentage of total NT cells (A) or
total CD56- T cells (C) and as a percentage of total lymphocytes (B, D)
in 23 controls (red) and 26 HBV patients (blue), following incubation in
medium conditioned with and without PMA/I or anti-CD3 and antiCD28 mAb. E, scatterplot showing frequencies of undtimulated and
PMA/I-stimulated IFN--producing lymphocytes in 34-39 controls (red)
and 34-36 HBV patients (blue) (* p<0.05,** p<0.005, ***p<0.0001).
153
FITC CD56 Log
A
IL-10+CD56BRIGHT lymphocytes
IL-10+CD56DIM lymphocytes
PE IL-10 Log
Pe-CY5 - CD3 Log
B
IL-10-producing
NT and T cells
PE IL-10 Log
Figure 4.4: Representative dot plots of IL-10-producing
NK, NT and CD56- T cells. A, representative dot plot of
whole PBMC gated on lymphocytes and showing further
gating on IL-10+ CD56DIM and CD56BRIGHT NK cell
populations. B, dot plot of whole PBMC gated on
lymphocytes and showing further gating on IL-10-producing
T and NT cell populations.
156
No. of IL-10+ cells
per 100 lymphocytes
IL-10+ cells
A
B
*
8
1.0
NK cells
% of NK cells
10
6
4
0.4
0
0.0
Medium PMA/I anti-CD3/28
D
10
**
CD56DIM NK cells
% of CD56DIM NK cells
**
0.6
0.2
8
6
4
2
0
Medium PMA/I anti-CD3/28
0.6
**
***
**
0.4
0.2
0.0
Medium PMA/I anti-CD3/28
F
15
*
CD56DIM NK cells
E
% of CD56BRIGHT NK cells
C
**
0.8
2
Medium PMA/I anti-CD3/28
**
10
5
0
Medium PMA/I anti-CD3/28
0.6
0.4
0.2
0.0
Medium PMA/I anti-CD3/28
Figure 4.5: Higher frequencies of IL-10-producing CD56DIM and
CD56BRIGHT NK cells and total NK cells in HBV infection. A, B,
scatterplots showing frequencies of IL-10-producing NK cells quantified
as a percentage of total NK cells (A) and total lymphocytes (B) in 23
controls (red) and 26 HBV patients (blue), following incubation in
medium conditioned with and without PMA/I or anti-CD3 and antiCD28 mAb. C, D, E, F, scatterplots showing frequencies of IL-10producing CD56DIM (C, D) and CD56BRIGHT NK cells (E, F) quantified
as a percentage of total CD56DIM NK cells (C) or CD56BRIGHT NK cells
(E) and as a percentage of total lymphocytes (D, F) in 23 controls (red)
and 26 HBV patients (blue), following incubation in medium
conditioned with and without PMA/I or anti-CD3 and anti-CD28 mAb
(*p<0.05,**p<=0.005,***p<0.0001).
157
No. of IL-10+ cells
per 100 lymphocytes
IL-10+ cells
% of NT cells
50
B
**
5
40
30
20
3
2
1
10
0
0
Medium PMA/I anti-CD3/28
D
10
8
6
4
2
0
% of lymphocytes
8
6
4
2
0
Medium PMA/I anti-CD3/28
E
Medium PMA/I anti-CD3/28
CD56- T cells
% of CD56- T cells
C
**
4
NT cells
A
Medium PMA/I anti-CD3/28
15
10
5
0
Medium
PMA/I
Figure 4.6: The frequencies of IL-10-producing NT cells are slightly
higher in HBV infection but frequencies of CD56- T cells and total
lymphocytes are unchanged. A, B, C, D, scatterplots showing
frequencies of IL-10-producing NT (A, B) and CD56- T cells (C, D)
quantified as a percentage of total NT cells (A) or total CD56- T cells (C)
and as a percentage of total lymphocytes (B, D) in 23 controls (red) and
26 HBV patients (blue), following incubation in medium conditioned
with and without PMA/I or anti-CD3 and anti-CD28 mAb. E, scatterplot
showing frequencies of undtimulated and PMA/I-stimulated IL-10producing lymphocytes in 21-26 controls (red) and 28 HBV patients
(blue) (**p<0.005).
160
B
IL-13-producing
NK cells
PE – IL-13 Log
Pe-Cy5 – CD3 Log
FITC – CD56 Log
A
IL-13producing
NT and T cells
PE – IL-13 Log
Figure 4.7: Representative dot plots of IL-13-producing
NK, NT and CD56- T cells. A, representative dot plot of
whole PBMC gated on lymphocytes and showing further
gating on IL-13+ NK cells. B, representative dot plot of
whole PBMC gated on lymphocytes and showing further
gating on IL-13-producing T and NT cell populations.
162
No. of IL-13+ cells
per 100 lymphocytes
IL-13+ cells
% of NK cells
40
B
*
NK cells
A
30
20
10
4
3
2
1
0
0
Medium PMA/I anti-CD3/28
Medium PMA/I anti-CD3/28
Figure 4.8: Responses of IL-13-producing NK cells to in vitro
stimulation are lower in HBV infection. A, B, scatterplots showing
frequencies of IL-13-producing NK cells quantified as a percentage of
total NK cells (A) and total lymphocytes (B) in 18-22 controls (red) and
11-19 HBV patients (blue), following incubation in medium conditioned
with and without PMA/I or anti-CD3 and anti-CD28 mAb (*p<0.05).
163
No. of IL-13+ cells
per 100 lymphocytes
IL-13+ cells
B
40
1.0
30
0.8
NT cells
% of NT cells
A
20
10
0.6
0.4
0.2
0
0.0
Medium PMA/I anti-CD3/28
D
20
*
**
CD56- T cells
% of CD56- T cells
C
Medium PMA/I anti-CD3/28
15
10
5
% of lymphocytes
8
***
***
8
6
4
2
0
0
Medium PMA/I anti-CD3/28
E
10
**
Medium PMA/I anti-CD3/28
*
6
4
2
0
Medium
PMA/I
Figure 4.9: Altered frequencies of IL-13-producing CD56- T cells and
total lymphocytes in HBV infection. A, B, C, D, scatterplots showing
frequencies of IL-13-producing NT (A, B) and CD56- T cells (C, D)
quantified as a percentage of total NT cells (A) or CD56- T cells (C) and
as a percentage of total lymphocytes (B, D) in 18-22 controls (red) and
11-19 HBV patients (blue), following incubation in medium conditioned
with and without PMA/I or anti-CD3 and anti-CD28 mAb. E, scatterplot
showing frequencies of unstimulated and PMA/I-stimulated IL-13producing lymphocytes in 15-17 controls (red) and 11 HBV patients
(blue) (*p<0.05, **p<0.005, ***P<=0.0008).
166
FITC – CD56 Log
IL-17producing
NK cells
PE – IL-17 Log
Pe-Cy5 – CD3 Log
B
A
IL-17producing
NT and T cells
PE – IL-17 Log
Figure 4.10: Representative dot plots of IL-17-producing
NK, NT and CD56- T cells. A, representative dot plot of
whole PBMC gated on lymphocytes and showing further
gating on IL-17+ NK cells. B, representative dot plot of
whole PBMC gated on lymphocytes and showing further
gating on IL-17-producing T and NT cell populations.
168
No. of IL-17+ cells
per 100 lymphocytes
IL-17+ cells
A
B
30
NK cells
% of NK cells
40
20
10
0
4
3
2
1
0
Medium PMA/I anti-CD3/28
Medium PMA/I anti-CD3/28
Figure 4.11: Frequencies of IL-17-producing NK cells are similar in
HBV patients and control subjects. A, B, scatterplots showing
frequencies of IL-17-producing NK cells quantified as a percentage of
total NK cells (A) and total lymphocytes (B) in 19-22 controls (red) and
11-19 HBV patients (blue), following incubation in medium conditioned
with and without PMA/I or anti-CD3 and anti-CD28 mAb.
169
No. of IL-17+ cells
per 100 lymphocytes
IL-17+ cells
B
80
4
60
3
NT cells
% of NT cells
A
40
20
2
1
0
0
Medium PMA/I anti-CD3/28
D
10
*
CD56- T cells
% of CD56- T cells
C
Medium PMA/I anti-CD3/28
8
6
4
2
0
% of lymphocytes
8
6
4
2
0
Medium PMA/I anti-CD3/28
E
10
Medium PMA/I anti-CD3/28
6
4
2
0
Medium
PMA/I
Figure 4.12: Slightly higher frequencies of IL-17-producing CD56- T
cells and total lymphocytes in HBV infection. A, B, C, D, scatterplots
showing frequencies of IL-17 producing NT (A, B) and CD56- T cells
(C, D) quantified as a percentage of total NT cells (A) or CD56- T cells
(C) and as a percentage of total lymphocytes (B, D) in 18-22 controls
(red) and 11-19 HBV patients (blue), following incubation in medium
conditioned with and without PMA/I or anti-CD3 and anti-CD28 mAb.
E, scatterplot showing frequencies of unstimulated and PMA/Istimulated IL-17-producing lymphocytes in 17-19 controls (red) and 14
HBV patients (blue) (*p<0.01).
172
β-actin Ct Value
A
20
19
18
17
16
Control Chronic HBV
Ct Value
B
40
30
20
10
0
y = -3.4x + 42.5
0
5
10
Copy Number 10 n
Copy numbers
y = -3.2x + 40.6
0
5
10
Copy Number 10n
C
D
40
30
20
10
0
10
8
6
4
2
0
y = -0.3x + 12.4
0
10
20
30
10
8
6
4
2
0
40
y = -0.31x + 12.6
0
10
20
30
40
Ct Value
4
10
**
3
10
2
10
1
10
0
10
Control
Chronic HBV
Figure 4.13: The levels of IFN- RNA are higher in PBMC from HBV
patients. Scatterplots showing (A) β-actin Ct values in control subjects
(red) and HBV patients (blue). C, D, line graphs showing standard
curves for β-actin (top) and IFN- (bottom) with Ct Values on the y-axis
and copy numbers on the x-axis (C) and, with copy numbers on the yaxis and Ct Values on the x-axis (D). E, scatterplot showing the copy
numbers of IFN- RNA in 10 control subjects (red) and 10 HBV patients
(blue), (** p=0.008).
174
β-actin Ct Value
A
20
19
18
17
16
Control Chronic HBV
Ct Value
B
y = -3.4x + 42.8
40
30
20
10
0
0
5
10
y = -3.2x + 38.6
0
5
10
Copy Number 10n
C
Copy Number 10 n
40
30
20
10
0
10
8
6
4
2
0
y = -0.3x + 12.4
0
10
20
30
10
8
6
4
2
0
40
y = -0.31x + 11.8
0
10
20
30
40
Ct Value
Copy numbers
D
4
10
3
10
2
10
1
10
0
10
Control
Chronic HBV
Figure 4.14: No significant difference in the level of IL-10 RNA in
PBMC from HBV patients. A, Scatterplot showing β-actin Ct values in
control subjects (red) and HBV patients (blue). B, C, line graphs
showing standard curves for β-actin (top) and IL-10 (bottom) with Ct
Values on the y-axis and copy numbers on the x-axis (B) and, with copy
numbers on the y-axis and Ct Values on the x-axis (C). D, scatterplot
showing the copy numbers of IL-10 RNA in 10 control subjects (red) and
10 HBV patients (blue).
176
A
β-actin Ct Value
20
19
18
17
16
Control
Chronic HBV
Ct Value
B
40
30
20
10
0
0
y = -3.5x + 43.5 40
30
20
10
0
0
5
10
y = -3.2x + 41.9
5
10
Copy Number 10n
Figure 4.15: Undetectable levels of IL-17A RNA in PBMC from HBV
patients and healthy control subjects. A, Scatterplot showing β-actin
Ct values in control subjects (red) and HBV patients (blue). B, line
graphs showing standard curves for β-actin (top) and IL-17A (bottom)
with Ct Values on the y-axis and copy numbers on the x-axis.
178
IFN-+ cells
8
80
% of NK cells
NK cells
A
No. of IFN-+ cells
per 100 lymphocytes
60
40
20
0
% of NK cells
NK cells
60
40
20
0
2
6
ALT
< 40
> 40
4
2
0
% of NK cells
8
60
40
20
4
2
0
80
8
60
40
20
Gender
Male
Female
6
0
% of NK cells
NK cells
4
8
C 80
NK cells
6
0
B 80
D
Viral load
Low
High
Age (yrs)
< 35
35 - 55
6
4
2
0
0
Medium PMA/I anti-CD3/28
Medium PMA/I anti-CD3/28
Figure 4.16: Frequencies of IFN--producing NK cells in subgroups
of HBV patients. A, B, C, D (left), scatterplots showing frequencies of
IFN--producing NK cells as a percentage of the total NK cell population
in 13-17 HBV patients with viral load below 10,000 copies / ml and 5-8
patients with viral load between 100,000 and 3.2x108 copies / ml (A), 1318 HBV patients with ALT below 40 IU/ml and 5-7 patients with ALT
above 40 IU/ml (B), 10-14 male HBV patients and 8-11 female patients
(C) and, 10 HBV patients under the age of 35 years and 8 patients
between the age of 35 and 55 years (D). A, B, C, D (right), scatterplots
showing frequencies of IFN--producing NK cells as a percentage of
total lymphocytes in 13-17 HBV patients with viral load below 10,000
copies / ml and 5-8 patients with viral load between 100,000 and 3.2x108
copies / ml (A), 13-18 HBV patients with ALT below 40 IU/ml and 5-7
patients with ALT above 40 IU/ml (B), 10-14 male HBV patients and 811 female patients (C) and, 10 HBV patients under the age of 35 years
and 8 patients between the age of 35 and 55 years (D).
182
IFN-+ cells
No. of IFN-+ cells
per 100 lymphocytes
A 100
4
% of NT cells
NT cells
80
60
40
20
0
% of NT cells
NT cells
2
1
4
80
60
40
20
0
ALT
< 40
> 40
3
2
1
0
C 100
4
% of NT cells
NT cells
3
0
B 100
80
60
40
20
0
Gender
Male
Female
3
2
1
0
D 100
4
% of NT cells
80
NT cells
Viral load
Low
High
60
40
20
0
Age (yrs)
< 35
35 - 55
3
2
1
0
Medium PMA/I anti-CD3/28
Medium PMA/I anti-CD3/28
Figure 4.17: Frequencies of IFN--producing NT cells in subgroups
of HBV patients. A, B, C, D (left) showing frequencies of IFN-producing NT cells as a percentage of the total NT cell population in 1215 HBV patients with viral load below 10,000 copies / ml and 5-7 HBV
patients with viral load between 100,000 and 3.2x108 copies / ml (A), 1115 HBV patients with ALT below 40 IU/ml and 6 HBV patients with
ALT above 40 IU/ml (B), 10-13 male HBV patients and 7-8 female HBV
patients (C) and, 10-12 HBV patients under the age of 35 years and 6-9
HBV patients between the age of 35 and 55 years (D). A, B, C, D
(right), scatterplots showing frequencies of IFN--producing NT cells as
a percentage of total lymphocytes in 12-15 HBV patients with viral load
below 10,000 copies / ml and 5-7 HBV patients with viral load between
100,000 and 3.2x108 copies / ml (A), 11-15 HBV patients with ALT
below 40 IU/ml and 6 HBV patients with ALT above 40 IU/ml (B), 1013 male HBV patients and 7-8 female HBV patients (C) and, 10-12 HBV
patients under the age of 35 years and 6-9 HBV patients between the age
of 35 and 55 years (D).
186
20
10
0
30
*
20
10
0
30
20
10
0
CD56- T cells
D
30
20
10
0
Medium PMA/I anti-CD3/28
% of CD56- T cells
CD56- T cells
C
*
10
% of CD56- T cells
CD56- T cells
B
30
10
% of CD56- T cells
CD56- T cells
A
No. of IFN-+ cells
per 100 lymphocytes
10
% of CD56- T cells
IFN-+ cells
10
8
6
Viral load
Low
High
4
2
0
8
6
ALT
< 40
> 40
4
2
0
8
6
Gender
Male
Female
4
2
0
8
6
Age (yrs)
< 35
35 - 55
4
2
0
Medium PMA/I anti-CD3/28
Figure 4.18: Frequencies of IFN--producing CD56- T cells in
subgroups of HBV patients. A, B, C, D (left), scatterplots showing
frequencies of IFN--producing CD56- T cells as a percentage of the total
CD56- T cell population in 12-17 HBV patients with viral load below
10,000 copies / ml and 5-8 HBV patients with viral load between
100,000 and 3.2x108 copies / ml (A), 11-17 HBV patients with ALT
below 40 IU/ml and 6-8 HBV patients with ALT above 40 IU/ml (B), 914 male HBV patients and 8-11 female HBV patients (C) and, 11-15
HBV patients under the age of 35 years and 6-10 HBV patients between
the age of 35 and 55 years (D). A, B, C, D (right), scatterplots showing
frequencies of IFN--producing CD56- T cells as a percentage of total
lymphocytes in 12-17 HBV patients with viral load below 10,000 copies
/ ml and 5-8 HBV patients with viral load between 100,000 and 3.2x108
copies / ml (A), 11-17 HBV patients with ALT below 40 IU/ml and 6-8
HBV patients with ALT above 40 IU/ml (B), 9-14 male HBV patients
and 8-11 female HBV patients (C) and, 11-15 HBV patients under the
age of 35 years and 6-10 HBV patients between the age of 35 and 55
years (D) (*p<0.05).
190
A
FBS
Control serum
HBV serum
HBsAg 1ug
HBsAg 3ug
IFN-+ NK cells
(% of lymphocytes)
5
4
3
2
1
0
1
B
IFN-+ NT cells
(% of lymphocytes)
6
4
2
0
IFN-+ T cells
(% of lymphocytes)
C
1
0.8
0.6
0.4
0.2
0
Figure 4.19: Higher frequencies of IFN--producing NK and NT cells
in the presence of HBsAg. A, B, C, barcharts showing frequencies of
IFN--producing NK (A), NT (B), and CD56- T cells (C) from 3 healthy
donors, following a 24 hour incubation in medium supplemented with
foetal bovine serum (FBS) (black), human serum from a healthy donor
(purple), human serum from a HBV patient (off white), 1μg of HBsAg
(red) and 3μg of HBsAg (blue) (p>0.05).
193
V9+V2+
T cells
Pe - V2 Log
PeCy5 - CD3 Log
B
FITC - V9 Log
A
T cells
V2+T cells
Pe - V2 Log
C
V2 T cells
(% of total T cells)
25
***
20
15
10
5
0
Control
Chronic HBV
Figure 5.1: Frequencies of circulating V2 T cells are significantly
higher in HBV-infected subjects than in control subjects. A,
representative dot plot of whole PBMC, gated on lymphocytes and
stained with FITC-labelled anti-V9 mAb, PE-labelled anti-V2 mAb
and PE-Cy5-labelled anti-CD3 mAb for identification of V9+V2+ T
cells. Since most cells expressing V9 also expressed V2, analysis of
V2 expression alone by CD3+ cells (B) was sufficient. C, scatterplot
showing the frequencies of circulating V2 T cells in 55 control subjects
(red) and 33 HBV patients (blue) (***p<0.0001).
212
B
T cells
V1+T cells
PE - V1 Log
V1 T cells
(% of total T cells)
PE-Cy5 - CD3 Log
A
5
*
4
3
2
1
0
Control Chronic HBV
Figure 5.2: Frequencies of circulating V1 T cells are significantly
higher in HBV-infected subjects than in control subjects. A,
representative dot plot of whole PBMC, gated on lymphocytes and
stained with PE-labelled anti-mouse IgG and unconjugated anti-V1
mAb, together with PE-Cy5-labelled anti-CD3 mAb for identification of
V1 T cells. B, scatterplot showing the frequencies of circulating V1 T
cells in 21 control subjects (red) and 23 HBV patients (blue) (*p=0.02).
214
FITC - CD56 Log
A
CD56+V2+
T cells
PE - V2 Log
% of V2 T cells
100
80
60
40
20
0
Control Chronic HBV
% of total T cells
C
CD56+ V2 T cells
CD56+ V2 T cells
B
8
6
4
2
0
Control Chronic HBV
Figure 5.3: Frequencies of circulating CD56+ V2 T cells are similar
in HBV-infected and control subjects. A, representative dot plots of
whole PBMC, gated on T lymphocytes and stained with FITC-labelled
anti-CD56 mAb and, PE-labelled anti-V2 mAb for the identification of
CD56+ V2 cells. B, C, scatterplots showing the frequencies of
circulating CD56+ V2 as a percentage of V2 T cells (B) and as a
percentage of total T cells (E) in 30 control subjects (red) and 18 HBV
patients (blue).
216
FITC - CD56 Log
A
CD56+V1+
T cells
PE - V1 Log
% of V1 T cells
100
80
60
40
20
0
Control Chronic HBV
% of total T cells
C
CD56+ V1 T cells
CD56+ V1 T cells
B
2.0
*
1.5
1.0
0.5
0.0
Control Chronic HBV
Figure 5.4: Frequencies of circulating CD56+ V1 T cells are higher
in HBV-infected subjects than in control subjects. A, representative
dot plots of whole PBMC, gated on T lymphocytes and stained with
FITC-labelled anti-CD56 mAb and, PE-labelled anti-mouse IgG in
combination with unconjugated anti-V1 mAb for the identification of
CD56+ V1 T cells. B, C, scatterplots showing the frequencies of CD56+
V1 T cells as a percentage of V1 T cells (B) and as a percentage of
total T cells (C) in 15 control subjects (red) and 17 HBV patients (blue)
(*p=0.01).
218
FITC - CD45RA Log
A
TEMRA
NAIVE
TEM
TCM
C
% CD45RA-CD27+ V2 T cells
(Naïve)
100
**
80
60
40
20
0 Control Chronic HBV
E
(TEM )
100
80
60
40
20
0 Control Chronic HBV
% CD45RA+CD27+ V2 T cells
D
% CD45RA-CD27- V2 T cells
B
% CD45RA+CD27+ V2 T cells
APC - CD27 Log
(TCM )
100
*
80
60
40
20
0 Control Chronic HBV
(TEMRA)
100
80
60
40
20
0
*
Control Chronic HBV
Figure 5.5: Effector-memory Vδ2+ T cells are expanded in HBV
infection. A, representative dot plot of whole PBMC stained with FITClabelled anti-CD45RA mAb, PE-labelled anti-Vδ2 mAb, PerCP-labelled
anti-CD3 mAb and APC-labelled anti-CD27 mAb and, gated on T
lymphocytes for the identification of TEM, TCM, naïve and TEMRA V2+ T
cells. B, C, D, E, scatterplots showing the frequencies of circulating
naïve (B), TCM (C), TEM (D) and TEMRA (E) Vδ2+ T cells as a percentage
of V2+ T cells in 40 control subjects (red) and 27 HBV patients (blue)
(*p<0.05, **p = 0.007).
220
B
% CD45RA-CD27+ V1 T cells
% CD45RA+CD27+ V1 T cells
(Naïve)
100
***
80
60
40
20
0 Control Chronic HBV
D
100
80
(TEM )
**
60
40
20
0 Control Chronic HBV
% CD45RA+CD27+ V1 T cells
C
% CD45RA-CD27- V1 T cells
A
(TCM )
100
80
60
40
20
0
100
80
*
Control Chronic HBV
(TEMRA)
*
60
40
20
0 Control Chronic HBV
Figure 5.6: Significantly lower frequencies of naive Vδ1 T cells in
HBV infection. A, B, C, D, scatterplots showing the frequencies of
circulating naive (A), TCM (B), TEM (C) and TEMRA (D) Vδ1 T cells as a
percentage of V1 T cells in 21 control subjects (red) and 17 HBV
patients (blue) (*p<0.05, **p<0.005, ***p<0.0005).
222
% of total T cells
A
B
30
V2 T cells
V2 T cells
30
20
10
0
20
10
0
Low
High
Viral load
V2 T cells
30
*
20
10
0
> 40
ALT
D
30
V2 T cells
C
< 40
20
10
0
Male
Female
Gender
19 - 35 35 - 55
Age
Figure 5.7: The frequencies of Vδ2 T cells in HBV patient subsets
based on viral load, ALT, gender and age. A, scatterplot showing
frequencies of Vδ2 T cells in 23 HBV patients with viral load between 10
and 100,000 copies/ml and 5 HBV patients with viral load between
100,000 and 5x108 copies/ml. B, scatterplot showing frequencies of Vδ2
T cells in 19 HBV patients with ALT below 40 IU/ml and 7 HBV
patients with ALT above 40 IU/ml. C, scatterplot showing frequencies of
Vδ2 T cells in 12 male HBV patients and 17 female HBV patients. D,
scatterplot showing frequencies of Vδ2 T cells in 17 HBV patients
between the age of 19 and 35 years and 12 HBV patients between the
age of 35 and 55 years (*p=0.02).
224
% of total T cells
A
B
4
5
V1 T cells
V1 T cells
5
3
2
1
4
3
2
1
0
0
Low
High
Viral load
C
5
V1 T cells
4
3
2
1
> 40
ALT
D
5
V1 T cells
< 40
4
3
2
1
0
0
Male
Female
Gender
19 - 35 35 - 55
Age
Figure 5.8: There are no significant differences in the frequencies of
Vδ1 T cells between patient groups within the study cohort. A,
scatterplot showing frequencies of Vδ1 T cells in 19 HBV patients with
viral load between 10 and 100,000 copies/ml and 3 HBV patients with
viral load between 100,000 and 5x108 copies/ml. B, scatterplot showing
frequencies of Vδ1 T cells in 13 HBV patients with ALT below 40 IU/ml
and 7 HBV patients with ALT above 40 IU/ml. C, scatterplot showing
frequencies of Vδ1 T cells in 11 male HBV patients and 12 female HBV
patients. D, scatterplot showing frequencies of Vδ1 T cells in 10 HBV
patients between the age of 19 and 35 years and 12 HBV patients
between the age of 35 and 55 years.
226
% of total T cells
B
8
CD56+ V2 T cells
CD56+ V2 T cells
A
6
4
2
0
8
6
4
2
0
Low
High
Viral load
D
8
6
4
2
0
> 40
ALT
CD56+ V2 T cells
CD56+ V2 T cells
C
< 40
8
6
4
2
0
Male
Female
Gender
19 - 35 35 - 55
Age
Figure 5.9: There are no significant differences in the frequencies of
CD56+ Vδ2 T cells between patient groups within the study cohort. A,
scatterplot showing frequencies of CD56+ Vδ2 T cells in 13 HBV
patients with viral load between 10 and 100,000 copies/ml and 4 HBV
patients with viral load between 100,000 and 5x108 copies/ml. B,
scatterplot showing frequencies of CD56+ Vδ2 T cells in 9 HBV patients
with ALT below 40 IU/ml and 9 HBV patients with ALT above 40 IU/ml.
C, scatterplot showing frequencies of CD56+ Vδ2 T cells in 8 male HBV
patients and 10 female HBV patients. D, scatterplot showing frequencies
of CD56+ Vδ2 T cells in 10 HBV patients between the age of 19 and 35
years and 8 HBV patients between the age of 35 and 55 years.
228
% of total T cells
B
2.0
CD56+ V1 T cells
CD56+ V1 T cells
A
1.5
1.0
0.5
0.0
2.0
1.5
1.0
0.5
0.0
Low
High
Viral load
D
2.0
CD56+ V1 T cells
CD56+ V1 T cells
C
< 40
ALT
1.5
1.0
0.5
0.0
> 40
2.0
1.5
1.0
0.5
0.0
Male
Female
Gender
19 - 35 35 - 55
Age
Figure 5.10: There are no significant differences in the frequencies of
CD56+ Vδ1 T cells between patient groups within the study cohort. A,
scatterplot showing frequencies of CD56+ Vδ1 T cells in 13 HBV
patients with viral load between 10 and 100,000 copies/ml and 3 HBV
patients with viral load between 100,000 and 5x108 copies/ml. B,
scatterplot showing frequencies of CD56+ Vδ1 T cells in 9 HBV patients
with ALT below 40 IU/ml and 8 HBV patients with ALT above 40 IU/ml.
C, scatterplot showing frequencies of CD56+ Vδ1 T cells in 7 male HBV
patients and 10 female HBV patients. D, scatterplot showing frequencies
of CD56+Vδ1 T cells in 9 HBV patients between the age of 19 and 35
years and 8 HBV patients between the age of 35 and 55 years.
230
A
% of V2 T cells
% V9+ V2+ T cells
80
*
60
Viral load
Low
High
40
20
% V9+ V2+ T cells
B
% of V2 T cells
0
80
ALT
< 40
> 40
60
40
20
0
C
D
% of V2 T cells
% V9+ V2+ T cells
80
Gender
Male
Female
60
40
20
0
% of V2 T cells
% V9+ V2+ T cells
80
Age (yrs)
< 35
35 - 55
60
40
20
0
Naive TCM TEM TEMRA
Figure 5.11: Differentiation status of circulating Vδ2 T cells in
subgroups of HBV patients. A, B, C, D, scatterplots showing
frequencies of naïve,TCM, TEM and TEMRA Vδ2 T cells as a percentage of
the total Vδ2 T cell population in 12 HBV patients with viral load below
100,000 copies/ml and 5 HBV patients with viral load between 100,000
and 1x108 copies/ml (A), 9 HBV patients with ALT below 40 IU/ml and
8 HBV patients with ALT above 40 IU/ml (B), 7 male HBV patients and
10 female HBV patients (C) and, 10 HBV patients under the age of 35
years and 8 HBV patients between the age of 35 and 55 years (D),
(*p=0.02).
232
% of V1 T cells
A
80
Viral load
Low
High
60
40
20
0
B
C.
% of V1 T cells
% of V1 T cells
80
ALT
< 40
> 40
60
40
20
0
80
Gender
Male
Female
60
40
20
D
% of V1 T cells
0
80
Age (yrs)
< 35
35 - 55
60
40
20
0
Naive TCM TEM TEMRA
Figure 5.12: No correlation between the memory phenotype of Vδ1 T
cells and viral load, disease severity, gender or age in HBV infection.
A, B, C, D, scatterplots showing frequencies of naïve, TCM, TEM and
TEMRA Vδ1 T cells as a percentage of the total Vδ1 T cell population in
12 HBV patients with viral load below 100,000 copies/ml and 5 HBV
patients with viral load between 100,000 and 1x108 copies/ml (A), 9
HBV patients with ALT below 40 IU/ml and 8 HBV patients with ALT
above 40 IU/ml (B), 7 male HBV patients and 10 female HBV patients
(C) and, 10 HBV patients under the age of 35 years and 8 HBV patients
between the age of 35 and 55 years (D).
234
B
6
% V 1+ T cells
% V 2+ T cells
A
4
2
0
10
8
6
4
2
0
African
Caucasian
Asian
Figure 5.13: Frequencies of circulating V2 and V1 cells are similar
in african, caucasian and asian healthy control subjects. A, B,
scatterplots showing the frequencies of circulating V2 T cells (A) and
V1 T cells (B), as a percentage of total T cells, in 13 african, 14
caucasian and 9 asian healthy control subjects.
237
PE - V2 Log
A
FITC - IFN- Log
B
% of V2 T cells
20
*
15
10
5
PMA/I-stimulated
IFN-+ V2 T cells
C
100
% of V2 T cells
Unstimulated
IFN-+ V2 T cells
0
80
60
40
20
0
Control Chronic HBV
E
% of total T cells
2.0
**
1.5
1.0
0.5
0.0
5
% of total T cells
D
Control Chronic HBV
4
3
2
1
0
Control Chronic HBV
Control Chronic HBV
Figure 5.14: Frequencies of circulating IFN--producing V2 T cells
are higher in HBV-infected subjects than in control subjects. A,
representative dot plot of whole PBMC stained with FITC-labelled antiIFN- mAb, PE-labelled anti-V2 mAb and PE-Cy5-labelled anti-CD3
mAb for the identification of IFN--producing Vδ2 T cells. B, C, D, E,
scatterplots showing the frequencies of IFN--producing Vδ2 T cells as a
percentage of total V2 T cells (B,C) and as a percentage of total T
lymphocytes (D,E), following incubation in medium alone (B,D) and
following incubation in medium conditioned with PMA/I (C, E) in 18
control subjects (red) and 10 HBV patients (blue) (*p=0.01, **p=0.005).
239
IFN-+ cells
% of V2 T cells
A
80
Viral load
Very Low
Low - Medium
60
40
20
0
% of V2 T cells
B
% of V2 T cells
C
80
ALT
< 35
> 35
60
40
20
0
80
Gender
Male
Female
60
40
20
0
% of V2 T cells
D
Age (yrs)
< 35
35 - 55
80
60
40
20
0
Medium
PMA/I
Figure 5.15: No correlation between the frequencies of IFN-producing Vδ2 T cells and viral load, disease severity, gender or age
in HBV infection. A, B, C, D, scatterplots showing frequencies of IFN-producing Vδ2 T cells, as a percentage of total Vδ2 T cells, in 5 - 6
HBV patients with viral load below 1,000 copies/ml and 4 HBV patients
with viral load between 1,000 and 100,000 copies/ml (A), 5 - 6 HBV
patients with ALT below 35 IU/ml and 4 HBV patients with ALT above
35 IU/ml (B), 4 male HBV patients and 5 - 6 female HBV patients (C)
and, 6 - 7 HBV patients under the age of 35 years and 3 HBV patients
between the age of 35 and 55 years (D). All frequencies of IFN-producing Vδ2 T cells are shown after a 4 hour incubation in medium
alone and, medium conditioned with PMA/I.
241
IFN-+ V2 T cells
(% of V2 T cells)
A
20
*
15
FBS
Control serum
10
HBV serum
HBsAg 1ug
HBsAg 3ug
5
0
1
Figure 5.16: Lower frequencies of IFN--expressing V2 T cells in
the presence of HBsAg. A, barchart showing the frequencies of
circulating IFN--expressing Vδ2 T cells as a percentage of total V2
T cells in 3 healthy donors following a 24 hour incubation in medium
supplemented with FBS (black), human serum from a healthy donor
(purple), human serum from a HBV patient (off white), 1μg of HBsAg
(red) and 3μg of HBsAg (blue) (*p=0.04).
245
B
100
300
80
NKG2D
(MFI)
NKG2D+ V 2 T cells
(% of V 2 T cells)
A
60
40
200
100
20
0
0
Control HBV
Control HBV
Figure 5.17: Lower NKG2D expression on Vδ2 T cells in HBV
infection. A, scatterplot showing the frequencies of circulating
NKG2D-expressing Vδ2 T cells as a percentage of total V2 T cells in
5 control subjects (red) and 7 HBV patients (blue). B, scatterplot
showing the mean fluorescence intensity (MFI) of NKG2D expression
by Vδ2 T cells in 5 control subjects (red) and 7 HBV patients (blue).
246
A
B
R2
FSC Lin
C
SSC Lin
R4
R3
SSC Lin
R6
R5
PerCP – CD3 Log
FSC Lin
F
PerCP – CD3 Log
E
PerCP – CD3 Log
FSC Lin
D
FITC – BerEP4 Log
SSC Lin
R1
R7
PE – Vd2 Log
R8
R9
PE – Vd2 Log
Figure 6.1: Gating technique for analysis of surface expression on
HMBPP and IL-2-expanded V2 T cells, PHA and IL-2-expanded T
cells and epithelial cells. A, representative dot plot of 14-day old
HMBPP-expanded PBMC, showing lymphocytes are the predominant
living cell type, gated in R1 to exclude dead cell debris. B, representative
dot plot of Hep3B cells, gated in R2 to exclude unwanted cell debris
from subsequent analysis. C, representative dot plot of 14-day old
HMBPP-expanded PBMC (gated in R3) co-cultured with GRM cells
(gated in R4). D, representative dot plot showing that gating on R5 can
exclude the BerEP4+ epithelial cells or that gating in R6 can exclude the
T cells. E, representative dot plot of HMBPP-expanded PBMC
previously gated in R5 to exclude epithelial cells and now gated in R7 to
identify Vδ2 T cells only. F, representative dot plot of PHA and IL-2expanded PBMC showing all T cells in R8 and showing that V2 T cells
gated in R9 are not the predominant T cell population.
263
A
B 200
NKG2A
Counts
MFI
150
100
50
0
V2 T
cells
APC - NKG2A Log
C
NKG2A
D 100
800
MFI
MFI
NKG2A
80
600
400
200
0
V2 T cells +
Hep3B cells
60
40
20
0
V2 T
cells
V2 T cells +
HT29 cells
V2 T
cells
V2 T cells +
GRM cells
Figure 6.2: The surface expression of NKG2A by V2 T cells is not
altered following co-culture with Hep3B, HT29 or GRM cells. A,
representative histogram showing NKG2A expression by Vδ2 T cells in
14-day old HMBPP-expanded PBMC after 24 hours of culture in the
presence (blue) and absence (red) of Hep3B cells. B, C, D, scatterplot
showing the MFI of NKG2A expression by HMBPP-expanded V2 T
cells from 5 donors following a 24 hour incubation in the absence
(circle) and presence (triangle) of Hep3B, HT29 or GRM cells,
respectively.
264
A
B
HLA-DR
250
MFI
Counts
200
150
100
50
0
FITC – HLADR Log
C
HLA-DR
D
200
MFI
MFI
HLA-DR
250
200
150
100
50
0
V2 T V2 T cells +
cells Hep3B cells
150
100
50
V2 T
cells
V2 T cells +
HT29 cells
0
V2 T
cells
V2 T cells +
GRM cells
Figure 6.3: The surface expression of HLA-DR by V2 T cells is not
altered following co-culture with Hep3B, HT29 or GRM cells. A,
representative histogram showing HLA-DR expression by Vδ2 T cells in
14-day old HMBPP-expanded PBMC after 24 hours of culture in the
presence (blue) and absence (red) of HT29 cells. B, C, D, scatterplot
showing the MFI of HLA-DR expression by HMBPP-expanded V2 T
cells from 4-5 donors following a 24 hour incubation in the absence
(circle) and presence (triangle) of Hep3B, HT29 or GRM cells,
respectively.
265
A
B
NKG2D
500
Counts
MFI
400
300
200
100
0
APC – NKG2D Log
C
NKG2D
500
*
400
400
300
300
MFI
MFI
D
NKG2D
500
200
100
0
V2 T V2 T cells +
cells Hep3B cells
*
200
100
0
V2 T V2 T cells +
cells
HT29 cells
E
V2 T
cells
V2 T cells +
GRM cells
NKG2D (MFI)
150
100
50
0
T Cells
F
T Cells +
Hep3B
T Cells +
HT29
NKG2D
100
80
60
40
20
0
MFI
MFI
NKG2D
100
80
60
40
20
0
T Cells +
GRM
PBMC PBMC +
HT29 cells
266
PBMC PBMC +
GRM cells
Figure 6.4: The surface expression of NKG2D by V2 T cells is lower
following co-culture with Hep3B, HT29 or GRM cells. A,
representative histogram showing NKG2D expression by Vδ2 T cells in
14-day old HMBPP-expanded PBMC after 24 hours of culture in the
presence (blue) and absence (red) of Hep3B cells. B, C, D, scatterplot
showing the MFI of NKG2D expression by HMBPP-expanded V2 T
cells from 4-5 donors following a 24 hour incubation in the absence
(circle) and presence (triangle) of Hep3B, HT29 or GRM cells,
respectively (*p<0.05). E, barchart showing NKG2D expression by
PHA and IL-2 expanded T cells from 1-2 donors following a 24 hour
incubation in the absence and presence of Hep3B, HT29 and GRM cells.
F, scatterplots showing the MFI of NKG2D expression by V2 T cells
(black) and αβ T cells (red) in fresh PBMC from 2 donors following a
24 hour incubation in the absence (circle) and presence (triangle) of
HT29 cells (left) or GRM cells (right).
267
A
B
CD54
400
MFI
Counts
300
200
100
0
APC – CD54 Log
C
D
CD54
1500
Hep3B Hep3B cells +
cells
V2 T cells
CD54
1000
*
*
1000
MFI
MFI
800
500
600
400
200
0
0
HT29 HT29 cells +
cells
V2 T cells
E
GRM
cells
GRM cells +
V2 T cells
700
CD54 (MFI)
600
Epithelial cells
500
400
Epithelial cells
and PHA/IL-2
expanded T
cells
300
200
100
0
Hep3B
Cells
F
HT29
Cells
GRM
Cells
CD54
MFI
80
*
*
70
70
60
60
50
50
40
CD54
80
MFI
90
HT29 HT29 cells
cells + PBMC
276
40
GRM GRM cells
cells + PBMC
Figure 6.5: Co-culture with HMBPP-expanded V2 T cells results in
significantly higher levels of CD54 expression by HT29 and GRM
cells, but not by Hep3B cells. A, histogram showing CD54 expression
by BerEP4+ GRM cells culturd alone (red) and cultured in the presence
of HMBPP-expanded Vδ2 T cells (blue). B, C, D, scatterplots showing
the MFI of CD54 expression by Hep3B (B), HT29 (C) and GRM cells
(D) after a 24 hour incubation in the absence (circle) and presence
(triangle) of HMBPP-expanded V2 T cells from 5 donors. E, barchart
showing CD54 expression by Hep3B, HT29 and GRM cells following a
24 hour incubation in the absence (black) and presence (red) of PHA and
IL-2-expanded T cells from 1-2 donors. F, scatterplots showing the MFI
of CD54 expression by HT29 (left) and GRM cells (right) after a 24
hour incubation in the absence (circle) and presence (triangle) of fresh
PBMC from 2 donors (*p<0.05).
277
A
B
HLA-E
20
MFI
Counts
15
10
5
0
Hep3B Hep3B cells +
cells
V2 T cells
PE – HLA-E Log
C
*
D
HLA-E
250
4
200
3
150
MFI
MFI
5
2
100
1
50
0
0
HT29 HT29 cells +
cells
V2 T cells
E
HLA-E
GRM
cells
GRM cells +
V2 T cells
4
HLA-E (MFI)
Epithelial cells
3
2
Epithelial cells
and PHA/IL-2
expanded T
cells
1
0
Hep3B
Cells
MFI
F
2.0
1.9
1.8
1.7
1.6
1.5
1.4
HT29
Cells
GRM
Cells
HLA-E
HT29 HT29 cells
cells + PBMC
278
Figure 6.6: Co-culture with HMBPP-expanded V2 T cells results in
higher levels of HLA-E expression by HT29 , but not by Hep3B or
GRM cells. A, histogram showing HLA-E expression by BerEP4+ HT29
cells culturd alone (red) and cultured in the presence of HMBPPexpanded Vδ2 T cells (blue). B, C, D, scatterplots showing the MFI of
HLA-E expression by Hep3B (B), HT29 (C) and GRM cells (D) after a
24 hour incubation in the absence (circle) and presence (triangle) of
HMBPP-expanded V2 T cells from 3-4 donors. E, barchart showing
HLA-E expression by Hep3B, HT29 and GRM cells following a 24 hour
incubation in the absence (black) and presence (red) of PHA and IL-2expanded T cells from 1-2 donors. F, scatterplot showing the MFI of
HLA-E expression by HT29 cells after a 24 hour incubation in the
absence (circle) and presence (triangle) of fresh PBMC from 2 donors
(*p<0.05).
279
A
B
40
MICA/B
MFI
Counts
30
20
10
0
APC – MICA/B Log
C
D
MICA/B
200
MICA/B
50
**
*
40
MFI
150
MFI
Hep3B Hep3B cells +
cells
V2 T cells
100
50
30
20
10
0
0
HT29 HT29 cells +
cells
V2 T cells
E
GRM
cells
GRM cells +
V2 T cells
MICA/B (MFI)
50
Epithelial cells
40
30
Epithelial cells
and PHA/IL-2
expanded T
cells
20
10
0
Hep3B
Cells
F
HT29
Cells
GRM
Cells
MICA/B
65
MFI
MFI
70
60
55
HT29 HT29 cells
cells + PBMC
280
135
130
125
120
115
110
105
MICA/B
GRM GRM cells
cells + PBMC
Figure 6.7: Co-culture with HMBPP-expanded V2 T cells results in
lower levels of MICA/B expression by epithelial cells. A, histogram
showing MICA/B expression by BerEP4+ GRM cells cultured alone (red)
and cultured in the presence of HMBPP-expanded Vδ2 T cells (blue). B,
C, D, scatterplots showing the MFI of MICA/B expression by Hep3B
(B), HT29 (C) and GRM cells (D) after a 24 hour incubation in the
absence (circle) and presence (triangle) of HMBPP-expanded V2 T
cells from 3-4 donors. E, barchart showing MICA/B expression by
Hep3B, HT29 and GRM cells following a 24 hour incubation in the
absence (black) and presence (red) of PHA and IL-2-expanded T cells
from 1-2 donors. F, scatterplots showing the MFI of MICA/B expression
by HT29 (left) and GRM cells (right) after a 24 hour incubation in the
absence (circle) and presence (triangle) of fresh PBMC from 2 donors
(*p=0.02, **p=0.002).
281
A
B
R1
C
D
R2
E
IL-4 (pg/ml)
10
8
6
4
2
0
1
2
3
V2
Hep3B
HT29
T cells cells cells
4
5
6
7
GRM
Hep3B
HT29
GRM
cells + V2 + V2 + V2
T cells T cells T cells
Figure 6.8: No significant difference in IL-4 expression following coculture of epithelial cells with HMBPP-expanded V2 T cells. A,
representative dot plot of antibody coated beads with electronic gate R1
surrounding the antibody coated beads and excluding the debris. B, C,
representative dot plots of all populations of cytokine beads, previously
gated in R1 and now, divided by fluorescence intensity in the FL3 and
FL4 channels. IL-4 conjugated beads are gated in R2 (C). D, dot plot
showing IL-4 conjugated beads from a single sample. The plot has been
gated on R2. The MFI of the population in FL2 indicates the level of IL4 expression in the sample. E, barchart showing the concentration of IL4 protein (pg/ml) in supernatants taken from HMBPP-expanded Vδ2 T
cell cultures from 3-5 healthy donors, supernatants taken from Hep3B,
HT29 or GRM cells and supernatants taken from co-cultures containing
both HMBPP-expanded V2 T cells and Hep3B, HT29 or GRM cells.
287
A
B
R1
C
D
R3
E
IL-6 (pg/ml)
3500
3100
2700
2300
1900
1500
1100
700
300
-100
V2
Hep3B
HT29
GRM
Hep3B
HT29
GRM
1
2
3
4
5
6
7
T cells cells cells cells + V2 + V2 + V2
T cells T cellsT cells
Figure 6.9: No significant difference in IL-6 expression following coculture of epithelial cells with HMBPP-expanded V2 T cells. A,
representative dot plot of antibody coated beads with electronic gate R1
surrounding the antibody coated beads and excluding the debris. B, C,
representative dot plots of all populations of cytokine beads, previously
gated in R1 to and now, divided by fluorescence intensity in the FL3 and
FL4 channels. IL-6 conjugated beads are gated in R3 (C). D, dot plot
showing IL-6 conjugated beads only from a single sample, because the
plot has been gated on R3. The MFI of this population in FL2 indicates
the level of IL-6 expression in the sample. E, barchart showing the
concentration of IL-6 protein (pg/ml) in supernatants taken from
HMBPP-expanded Vδ2 T cell cultures from 3-5 healthy donors,
supernatants taken from Hep3B, HT29 or GRM cells and supernatants
taken from co-cultures containing both HMBPP-expanded V2 T cells
and Hep3B, HT29 or GRM cells.
288
A
B
R1
C
D
R4
E
IL-10 (pg/ml)
100
75
*
50
25
0
2
3
V21 Hep3B
HT29
T cells cells cells
4
5
6
7
GRM
Hep3B
HT29
GRM
cells + V2 + V2 + V2
T cells T cells T cells
Figure 6.10: Higher IL-10 expression in epithelial and HMBPPexpanded V2 T cell co-cultures, compared to HMBPP-expanded
V2 T cell cultures. A, representative dot plot of antibody coated beads
with electronic gate R1 surrounding the antibody coated beads and
excluding the debris. B, C, representative dot plots of all populations of
cytokine beads, previously gated in R1 and now, divided by fluorescence
intensity in the FL3 and FL4 channels. IL-10 conjugated beads are gated
in R4 (C). D, dot plot showing IL-10 conjugated beads from a single
sample. The plot has been gated on R4. The MFI of this population in
FL2 indicates the level of IL-10 expression in the sample. E, barchart
showing IL-10 protein concentration (pg/ml) in supernatants of HMBPPexpanded Vδ2 T cell cultures from 3-5 healthy donors, supernatants of
Hep3B, HT29 or GRM cells and supernatants of co-cultures containing
both HMBPP-expanded V2 T cells and Hep3B, HT29 or GRM cells
(*p=0.03).
289
A
B
R1
C
D
R7
E
IL-13 (ng/ml)
4
3
2
1
0
1
2
3
V2
Hep3B
HT29
T cells cells cells
4
5
6
7
GRM
Hep3B
HT29
GRM
cells + V2 + V2 + V2
T cells T cells T cells
Figure 6.11: No significant difference in IL-13 expression following
co-culture of epithelial cells with HMBPP-expanded V2 T cells. A,
representative dot plot of antibody coated beads with electronic gate R1
surrounding the antibody coated beads and excluding the debris. B, C,
representative dot plots of all populations of cytokine beads, previously
gated in R1 and now, divided by fluorescence intensity in the FL3 and
FL4 channels. IL-13 conjugated beads are gated in R7 (C). D, dot plot
showing IL-13 conjugated beads only from a single sample, because the
plot has been gated on R7. The MFI of this population in FL2 indicates
the level of IL-13 expression in the sample. E, barchart showing the
concentration of IL-13 protein (ng/ml) in supernatants of HMBPPexpanded Vδ2 T cell cultures from 3-5 healthy donors, supernatants of
Hep3B, HT29 or GRM cells and supernatants of co-cultures containing
both HMBPP-expanded V2 T cells and Hep3B, HT29 or GRM cells.
290
A
B
R1
C
D
R5
E
IFN- (ng/ml)
4
3
2
1
0
1
2
3
V2
Hep3B
HT29
T cells cells cells
4
5
6
7
GRM
Hep3B
HT29
GRM
cells + V2 + V2 + V2
T cells T cells T cells
Figure 6.12: No significant difference in IFN- expression following
co-culture of epithelial cells with HMBPP-expanded V2 T cells. A,
representative dot plot of antibody coated beads with electronic gate R1
surrounding the antibody coated beads and excluding the debris. B, C,
representative dot plots of all populations of cytokine beads, previously
gated in R1 and now, divided by fluorescence intensity in the FL3 and
FL4 channels. IFN- conjugated beads are gated in R5 (C). D, dot plot
showing IFN- conjugated beads from a single sample. The plot has been
gated on R5. The MFI of this population in FL2 indicates the level of
IFN- expression in the sample. E, barchart showing the concentration of
IFN- protein (ng/ml) in supernatants from HMBPP-expanded Vδ2 T cell
cultures from 3-5 healthy donors, supernatants from Hep3B, HT29 or
GRM cells and supernatants from co-cultures containing both HMBPPexpanded V2 T cells and Hep3B, HT29 or GRM cells.
291
A
B
R1
C
D
R8
E
TGF-β (pg/ml)
250
**
200
*
150
100
50
0
1
2
3
V2
Hep3B
HT29
T cells cells cells
4
5
6
7
GRM
Hep3B
HT29
GRM
cells + V2 + V2 + V2
T cells T cells T cells
Figure 6.13: Lower TGF-β1 expression following co-culture of
epithelial cells with HMBPP-expanded V2 T cells, compared to that
of epithelial cells cultured alone. A, representative dot plot of antibody
coated beads with electronic gate R1 surrounding the antibody coated
beads and excluding the debris. B, C, representative dot plots of single
population of TGF-β1-conjugated beads in single-plex assay tube. TGFβ1 conjugated beads are gated in R8 (C). D, dot plot showing TGF-β1
conjugated beads from a single sample, because the plot has been gated
on R8. The MFI of this population in FL2 indicates the level of TGF-β1
expression in the sample. E, barchart showing the concentration of TGFβ1 protein (pg/ml) in supernatants taken from HMBPP-expanded Vδ2 T
cell cultures from 3-5 healthy donors, supernatants taken from Hep3B,
HT29 or GRM cells and supernatants taken from co-cultures containing
both HMBPP-expanded V2 T cells and Hep3B, HT29 or GRM cells
(*p=0.02, **p=0.005).
292
A
B
R1
R6
FL3
C
FL2
Figure 6.14: Undetectable levels of IL-12 in sample supernatants A,
representative dot plot of antibody coated beads with electronic gate R1
surrounding the antibody coated beads and excluding the debris. B, representative
dot plot of all populations of cytokine beads, previously gated in R1 to exclude
debris and now, divided by their fluorescence intensity in the FL3 and FL4
channels. IL-12 conjugated beads are gated in R6. C, representative dot plots
showing IL-12 conjugated beads only for 6 standard tubes. Dot plot in upper left
represents the tube containing 0 pg/ml IL-12 and the dot plot in the lower right
represents the tube containing 2500 pg/ml IL-12. Most dot plots from sample
tubes were similar to that of the standard blank tube and therefore, IL-12 could
not be detected in the majority of samples.
293