nk, t and nk t -cells in ageing, coeliac disease and ......nk cells in untreated and treated coeliac...
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NK, T and NK T-cells in ageing, coeliac disease and IBD Randall Grose
i
NK, T and NK T-cells in ageing,
coeliac disease and inflammatory
bowel disease
BY
RANDALL HILTON GROSE B.Biotech (Hons)
A thesis submitted to the University of Adelaide as the requirement for the
degree of Doctor of Philosophy
The Department of Medicine, the University of Adelaide;
The Basil Hetzel Institute for Medical Research and the Department of
Gastroenterology and Hepatology, The Queen Elizabeth Hospital
March 2008
Chapter 5: NK, T and NK T cells in coeliac disease Randall Grose
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5 CHAPTER 5 NK, T and NK T-CELLS IN COELIAC
DISEASE
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5.1 INTRODUCTION Coeliac disease is mediated by an inappropriate reaction of intestinal T-cells to
dietary wheat derived-gluten causing intestinal damage in genetically
susceptible individuals. Gliadin peptides are deamidated by intestinal tTG
enzyme and are presented by DQ2 (or DQ8) dendritic cells to mucosal T-cells.
This inappropriate T-cell activity has been attributed to lack of immunological
oral tolerance (Mowat et al., 1987), but there is limited evidence of loss of
immunological suppression in coeliac disease. NK T-cells are recognized as
important immunoregulatory cells that are deficient in several autoimmune
diseases.
Previous studies have shown a relative deficiency in the number of intestinal
and peripheral NK cells in coeliac disease (Hadziselimovic et al., 1992). Di
Sabatino et al. (1998b) briefly investigated and showed a deficiency of CD16+
NK T-like cells in coeliac subjects. Nevertheless, these studies did not
investigate the number or function of Vα24+ T-cells or iNK T-cells in subjects
with coeliac disease. A report by van der Vliet et al. (2001) investigated a
variety of diseases characterised by autoreactive tissue damage, including
coeliac disease, and showed that Vα24+ Vβ11+ T-cells were not deficient in
ten coeliac subjects.
5.2 AIMS AND HYPOTHESIS The aim of this Chapter was to investigate the number of circulating NK cells,
T-cells, NK T-like and iNK T-cells in coeliac disease. Cytokine production by
Vα24+ T-cells and iNK T-cells after in vitro anti-CD3 stimulation were
examined and compared to cytokine production by CD3+ T-cells after in vitro
anti-CD3 and gluten fraction 3 stimulation in normal subjects and in subjects
with coeliac disease.
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The hypothesis of this Chapter was that NK cells, immunoregulatory Vα24+ T-
cells, NK T-like cells and/or iNK T-cells are deficient in subjects with coeliac
disease.
5.3 MATERIALS AND METHODS
5.3.1 Subjects Coeliac subjects were recruited from patients attending the Department of
Gastroenterology and Hepatology at The Queen Elizabeth Hospital. Additional
volunteers were recruited through the Coeliac Society of South Australia who
responded to a notice in their newsletter. All coeliac subjects had been
diagnosed by intestinal biopsy and clinical response to a GFD. Coeliac subjects
were generally reviewed at 12 monthly intervals as part of this study. They
were strongly encouraged to adhere to a GFD and had repeat serology tests to
ascertain this. Upon sample collection, details regarding adherence to and
duration of diet were recorded. Control subjects were recruited from those
attending for endoscopy for non-ulcer dyspepsia or iron deficiency in the
Department of Gastroenterology and Hepatology at The Queen Elizabeth
Hospital, in whom no major pathology was identified. The Human Ethics
Committee of The Queen Elizabeth Hospital approved this study.
5.3.2 Flow cytometry Peripheral blood lymphocytes were collected and stained using antibodies
directed against CD56, CD57, CD94 or CD161 NK markers, CD3, CD4,
Vα24, Vβ11 or Vβ13 T-cell, Vα24 6B11 and Vα24 α-GalCer/CD1d tetramer
iNK T-cell markers as previously described in Chapter 2.
5.3.3 In vitro anti-CD3 stimulation of peripheral blood T-cells Peripheral blood lymphocytes were stimulated in vitro for 4 h and 24 h with
anti-CD3 antibody as previously described in Chapter 2.
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5.3.4 Depletion of Vα24+ NK T-cells by magnetic beads
Vα24+ cells were depleted from blood of normal healthy control subjects using
magnetic beads as previously described in Chapter 2.
5.3.5 In vitro gluten fraction 3 stimulation of peripheral blood T-
cells Blood was collected in lithium heparin tubes and mononuclear cells isolated on
a density gradient. Cells were washed and resuspended in RPMI 1640 (Gibco,
Life Technologies, Melbourne, Australia) supplemented with 10% foetal calf
serum (CSL Ltd, Melbourne, Australia), 0.3 mg/ml L-glutamine, 0.12 mg/ml
benzylpenicillin and 10 µg/ml gentamicin. Cells were stimulated in a 6 or 12
well plate with 100 µg/ml gluten fraction 3 for 24 h at 37ºC, in 5% CO2. 10
µg/ml brefeldin A (Sigma Chemical Co, St Louis, MO) was added to the
cultures 4 h prior to cell harvest. Three-colour flow cytometry was used to
determine intracellular cytokine production by classical CD3+ T-cells. Cells
were incubated for 10 minutes at room temperature with permeabilizing
solution (Becton Dickinson, San Jose, CA). Aliquots of approximately 1-2 x106
cells were incubated with saturating concentrations of anti-IL-4, anti-IFN-γ or
isotype control PE-labelled antibodies. Antibodies to cell surface markers CD3,
or Vα24 (either Cy5 or FITC conjugated) were added. The samples were
incubated at 4°C for 30 minutes then washed twice. Labelled cells were
analysed on a flow cytometer (Becton Dickinson) after selecting a lymphocyte
gate based on forward and side-scatter characteristics. The number of IL-4+
CD3+ and IFN-γ+ CD3+ cells was calculated from the complete blood
examination.
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5.4 RESULTS
5.4.1 Comparison of NK cells in coeliac disease Total numbers of circulating CD56+, CD57+ and CD94+ NK cells from
coeliac subjects were similar to the levels in blood of normal control subjects
(Figure 5.1). In contrast, the number of circulating CD161+ NK cells were
reduced in coeliac subjects when compared to normal control subjects (Figure
5.1). The mean±SEM number of CD161+ NK cells in normal subjects was
5.9±0.3 x105 compared to 4.4±0.3 x105 cells per ml for coeliac subjects (Figure
5.1). Circulating CD94+ NK cells were reduced in untreated coeliac subjects
compared to those on a GFD. The mean±SEM number of circulating CD94+
NK cells in untreated and treated coeliac subjects was 3.6±0.5 x105 and
5.5±0.6 x105 cells per ml, respectively (Figure 5.2). This difference in the
number of circulating CD94+ NK cells and diet was not observed for CD56+,
CD57+ or CD161+ NK cells. Thus, NK cells were generally not affected in
coeliac disease, except for CD161 and CD94 NK cells.
As in previous Chapters, total NK cells were divided into bona fide NK cells
and NK T-like cells by their CD3 expression. Coeliac subjects had an increased
number of circulating CD56+ NK T-like cells. The mean±SEM number of
CD56+ NK T-like cells in normal subjects and subjects with coeliac disease
was 1.0±0.1 x105 and 1.6±0.2 x105 cells per ml, respectively (Figure 5.1, filled
bars). There was no significant difference in the number of circulating bona
fide CD56+, CD57+ or CD94+ NK cells or CD57+ or CD94+ NK T-like cells
when comparing coeliac subjects with normal healthy subjects. In contrast, the
entire CD161 linage was affected in coeliac disease. Bona fide CD161+ and
CD161+ NK T-like cells were deficient in subjects with coeliac disease
compared to normal control subjects. The mean±SEM number of bona fide
CD161+ NK cells was 3.4±0.3 x105 and 2.7±0.2 x105 cells per ml (Figure 5.1,
open bars), while the number of circulating CD161+ NK T-like cells was
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2.5±0.2 x105 and 1.7±0.2 x105 cells per ml (Figure 5.1, filled bars), for normal
subjects and subjects with coeliac disease, respectively.
Figure 5.1 Comparison of circulating (A) CD56+, (B) CD57+, (C) CD94+ and
(D) CD161+ NK cells in normal control subjects and subjects with coeliac
disease. Total circulating NK cells have been divided into NK T-like cells
(filled bars) and bona fide NK cells (open bars). Data are given as the
mean±SEM x105 cells/ml (n=number of subjects).
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Figure 5.2 Comparison of circulating CD94+ NK cells in normal control
subjects, coeliac subjects untreated and treated, respectively. Data are given as
the mean±SEM x105 cells/ml (n=number of subjects).
As detailed above (Figure 5.1 and Figure 5.2), there are various changes in the
number of circulating total NK cells, bona fide NK cells and NK T-like cells
when comparing normal subjects with subjects with coeliac disease.
• The number of circulating CD161 NK cells were reduced in subjects
with coeliac disease. The decrease in number of circulating CD161+
NK cells was due to the deficiency of both bona fide and NK T-like
subset.
• The number of circulating CD94+ NK cells was dependant upon
disease state, as untreated coeliac subjects had lower numbers of
CD94+ NK cells compared to treated coeliac subjects and normal
healthy control subjects.
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5.4.2 Comparison of CD3+, CD4+, Vα24+, Vβ11+ and Vβ13+ T-
cells in coeliac disease
There was no significant difference in the mean±SEM number of circulating
CD3+ or CD4+ T-cells when comparing normal and coeliac subjects (Figure
5.3), nor was there any difference with respect to diet. (Figure 5.3).
TCR Vα24+ T-cells were deficient in coeliac subjects (Figure 5.4). Vα24+
cells in normal subjects and subjects with coeliac disease represented
0.35±0.01% and 0.11±0.01% of circulating lymphocytes, respectively. Vα24+
cells made up 0.48±0.02% of circulating CD3+ T-cells in normal control
subjects, compared to 0.18 ±0.01% for coeliac subjects. The mean±SEM
numbers of circulating Vα24+ T-cells in normal and coeliac subjects was
8.8±0.4 x103 and 2.4±0.1 x103 cells per ml, respectively (Figure 5.4). As
shown in Chapter 4, the mean number of circulating Vα24+ T-cells decreased
with age in normal subjects, though there was no significant change in Vα24+
T-cell numbers with age in coeliac disease (Figure 5.4). Furthermore, there was
no significant relationship between diet, or length on a GFD and the number of
circulating Vα24+ T-cells (Figure 5.5).
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Figure 5.3 Comparison of circulating (A) CD3+ and (B) CD4+ T-cells in
normal and coeliac subjects. Coeliac subjects were divided into untreated and
treated coeliac subjects, respectively (C and D). Data are given as the
mean±SEM x106 cells/ml (n=number of subjects).
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Figure 5.4 Comparison of total Vα24+ T-cells in normal subjects and subjects
with coeliac disease. Representative two-colour dot plots comparing Vα24+
CD3+ T-cells from (A) a normal subject and (B) a subject with coeliac disease.
(C) Comparison of total number of Vα24+ T-cells between normal subjects and
subjects with coeliac disease. (D) Comparison of total Vα24+ T-cells for
normal (●) and coeliac (○) subjects with age. Data are given as the mean±SEM
x103 cells/ml (n=number of subjects).
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Figure 5.5 Comparison of total Vα24+ T-cells for (A) untreated and treated
coeliac subjects. The number of Vα24+ T-cells for coeliac subjects in contrast
to (B) length on GFD was examined. Data are given as the mean±SEM x103
cells/ml (n=number of subjects).
Only Vα24+ T-cells were deficient in coeliac disease, whereas the general CD3
and CD4 markers were unaffected. The decrease in number of circulating
Vα24 T-cells was specific to coeliac disease as it was not affected by dietary
status and therefore presumably not affected by inflammation.
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The numbers of circulating Vβ11+, but not Vβ13+, T-cells were decreased in
coeliac subjects compared to normal control subjects (Figure 5.6). There was
no significant relationship between age, diet or length of GFD and number of
circulating Vβ11+ or Vβ13+ T-cells in coeliac disease.
Figure 5.6 Comparison of total (A) Vβ11+ and (B) Vβ13+ T-cells in normal
subjects and subjects with coeliac disease. Data are given as the mean±SEM
x103 cells/ml (n=number of subjects).
5.4.3 Comparison of CD56+, CD57+, CD94+ and CD161+ Vα24+
NK T-cells in coeliac disease
The proportion of Vα24+ T-cells that co-expressed NK markers CD56, CD57,
CD94 and CD161 were examined. As shown in Chapter 3 Vα24+ T-cells had
low expression of CD56, CD57 and CD94 NK markers. The CD161 NK
marker was expressed on the majority of Vα24+ T-cells. While there was no
significant difference in the proportion of Vα24+ cells that were CD56+,
CD57+, CD94+ or CD161+ when comparing normal subjects and subjects with
coeliac disease, there was a significant reduction in the number of circulating
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Vα24+ T-cells that co-expressed CD56, CD57, CD94 and CD161 NK markers
(Figure 5.7).
Figure 5.7 Comparison of total Vα24+ T-cells that were (A) CD56+, (B)
CD57+, (C) CD94+ and (D) CD161+ (filled bars) from blood of normal and
coeliac subjects. Data are given as the mean±SEM x103 cells/ml (n=number of
subjects).
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5.4.4 Comparison of Vα24+ SP T-cells in coeliac disease
The CD4+ subset of Vα24+ T-cells was investigated to see if there was any
selective SP subset deficiency in coeliac disease. No significant difference in
the proportion of circulating Vα24+ SP subset was apparent when comparing
normal subjects and subjects with coeliac disease. The SP population made up
around 65% of the total Vα24+ T-cells for both normal subjects and subjects
with coeliac disease. The actual numbers of circulating Vα24+ CD4+ were
decreased in coeliac subjects compared to normal control subjects. The
mean±SEM numbers of Vα24+ CD4+ T-cells in normal and coeliac subjects
was 5.3±0.4 x103 and 1.6±0.2 x103 cells per ml, respectively (Figure 5.8).
Figure 5.8 Comparison of total Vα24+ that were CD4+ (filled bars) from
blood of normal and coeliac subjects. Data are given as the mean±SEM x103
cells/ml (n=number of subjects).
5.4.5 Comparison of Vα24+ Vβ11+ and Vα24+ Vβ13+ T-cells in
coeliac disease
There was a significant reduction in the proportion and number of Vα24+ T-
cells that co-expressed Vβ11 β-chain in subjects with coeliac disease compared
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to normal controls. TCR Vβ11 was found to pair with 45% and 17% of Vα24+
T-cells in normal and coeliac subjects, respectively. The mean±SEM numbers
of Vα24+ Vβ11+ T-cells in normal and coeliac subjects was 3.9±0.4 x103 and
0.6±0.08 x103 cells per ml, respectively (Figure 5.9). As shown in Chapter 3,
the TCR Vβ13 was rarely found paired with TCR α-chain Vα24. There was no
significant difference mean number of Vα24+ Vβ13+ T-cells when comparing
normal control and coeliac subjects (Figure 5.9).
Figure 5.9 Comparison of total Vα24+ T-cells that were (A) Vβ11+ and (B)
Vβ13+ T-cells in normal subjects and subjects with coeliac disease. Data are
given as the mean±SEM x103 cells/ml (n=number of subjects).
5.4.6 Comparison of Vα24+ 6B11+ and Vα24+ Vβ11+ α-
GalCer/CD1d tetramer+ iNK T-cells in coeliac disease
The proportion of Vα24+ T-cells that co-expressed 6B11 iNK T-cell
phenotypic marker was 55±4% and 38±4% for normal subjects and subjects
with coeliac disease, respectively. The mean±SEM numbers of Vα24+ 6B11+
iNK T-cells in normal and coeliac subjects was 5.4±1.0 x103 and 1.6±0.4 x103
cells per ml, respectively (Figure 5.10).
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Figure 5.10 Comparison of circulating Vα24+ T-cells that were 6B11+ (iNK
T-cells, filled bars) in normal control subjects and subjects with coeliac disease.
Data are given as the mean±SEM x103 cells/ml (n=number of subjects).
The number of total circulating α-GalCer/CD1d tetramer+ cells were reduced
in subjects with coeliac disease when compared to normal healthy control
subjects. The mean number of total circulating α-GalCer/CD1d tetramer+ cells
for normal and coeliac subjects was 4.3±0.4 x103 and 1.3±0.5 x103 cells per
ml, respectively (Figure 5.12). Vα24+ α-GalCer/CD1d tetramer+ ‘Type I NK
T-cells’ were deficient in the blood of coeliac subjects compared to normal
control subjects. The mean number of circulating Vα24+ α-GalCer/CD1d
tetramer+ ‘Type I NK T-cells’ for normal and coeliac subjects was 3.8±0.4 x103
and 0.6±0.2 x103 cells per ml, respectively (Figure 5.12 and Table 5.1). The
number of circulating Vα24+ Vβ11+ α-GalCer/CD1d tetramer+ iNK T-cells
were reduced in subjects with coeliac disease when compared to normal
control subjects. 79% of Vα24+ Vβ11+ cells also bind α-GalCer/CD1d
tetramer+ for normal healthy control subjects, which compared to 22% for
subjects with coeliac disease (Figure 5.11). The mean±SEM number of Vα24+
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Vβ11+ α-GalCer/CD1d tetramer+ iNK T-cells in normal control subjects and
subjects with coeliac disease was 3.3±0.5 x103 and 0.3±0.1 x103 cells per ml,
respectively (Figure 5.12 and Table 5.1).
Figure 5.11 Sample dot plot of Vα24+ Vβ11+ T-cells (gate R2) and histogram
showing the proportion that bind α-GalCer/CD1d tetramer.
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Figure 5.12 Comparison of circulating (A) α-GalCer/CD1d tetramer+ and the
proportion that were (B) Vα24+ α-GalCer/CD1d tetramer+ (filled bars), (C)
Vβ11+ α-GalCer/CD1d tetramer+ (filled bars) and (D) Vα24+ Vβ11+ α-
GalCer/CD1d tetramer+ (filled bars) iNK T-cells in normal control subjects and
subjects with coeliac disease. Data are given as the mean±SEM x103 cells/ml
(n=number of subjects).
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Table 5.1 Comparison of numbers of circulating Vα24+ T-cells and iNK T-
cells for normal subjects and subjects with coeliac disease.
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5.4.7 Comparison of Vα24+ T-cells in the intestine using
immunofluorescence
Intestinal Vα24+ T-cells were reduced in the mucosa of coeliac subjects when
compared to normal control subjects. The mean number of intestinal Vα24+ T-
cells for normal and coeliac subjects was 248±37 and 39±14 cells per mm2,
respectively (Figure 5.13).
5.4.8 Intestinal Vα24 T-cell mRNA expression in coeliac disease
Intestinal Vα24 T-cells mRNA expression was investigated using relative PCR.
Specific expression was relative to 18S ribosomal RNA as an internal control.
Vα24 mRNA from subjects with coeliac disease was decreased when
compared to the intestine of control subjects (Figure 5.14). The Vα24: 18S
PCR product band net intensity ratio was 2.4±0.3 and 0.4±0.1 for normal and
coeliac subjects, respectively. The deficiency of Vα24 mRNA expression
within the mucosa of coeliac subjects was confirmed using RT-PCR. The
Vα24:GAPDH (log10 (Vα24:GAPDH+1)) was from 9.6±3.6 x10-2 and 0.5±0.3
x10-2 for normal subject and subjects with coeliac disease, respectively (Figure
5.15). The deficiency of Vα24 noted within the mucosa by relative and RT-
PCR and immunofluorescence reinforces the systemic deficiency of Vα24 T-
cells in blood assessed by flow cytometry.
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Figure 5.13 Comparison of intestinal Vα24+ T-cells in normal subjects and in
subjects with coeliac disease. Data are given as the mean±SEM cells/mm2
(n=number of subjects).
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Figure 5.14 Comparison of intestinal mRNA expression for Vα24+ T-cells in
normal subjects and those with coeliac disease. Representative agarose gel (A)
containing liver control (lane 1), normal (lanes 2-5) and coeliac (lanes 6-11)
18S (upper band) and Vα24 (lower band) PCR products. Comparison of
Vα24:18S PCR product ratio for normal and coeliac subjects (B). Data are
given as the ratio of net intensity of Vα24 and internal 18S band (n=number of
subjects).
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Figure 5.15 Comparison of intestinal mRNA expression for Vα24+ T-cells in
normal subjects and those with coeliac disease as assessed by RT-PCR.
Comparison of log10 (Vα24:GAPDH+1) RT-PCR product ratio for normal and
coeliac subjects. Data are given as log10 ((Vα24(ng/ul):GAPDH(ng/ul))+1)
(n=number of subjects).
In summary, Vα24+ T-cells were deficient systemically in blood as was the
invariant subset of α-GalCer and 6B11+ iNK T-cells. Moreover Vα24+ cells
and Vα24 mRNA were deficient in the intestinal mucosa. Therefore, by various
measures the work of this Chapter has shown that Vα24 T-cells were deficient
systemically and in the intestinal mucosa of coeliac subjects.
5.4.9 Comparison of CD3+ T-cell cytokine production in coeliac
disease In vitro activation of peripheral blood was investigated. Circulating CD3+ T-
cells produced detectable levels of IL-4 and IFN-γ cytokines after 4 and 24 h in
vitro anti-CD3 and gluten fraction 3 stimulation.
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IL-4 and IFN-γ intracellular production by CD3+ T-cells of normal and coeliac
subjects after 4 h and 24 h in vitro anti CD3 stimulation was investigated.
There was a significant increase in IL-4 producing CD3+ T-cells for normal
subjects, while a significant increase in IFN-γ producing CD3+ T-cells for
coeliac subjects after 4 h in vitro anti-CD3 stimulation. The number of IL-4
producing CD3+ T-cells increased by 2.4±0.7 x104 for normal subjects while
IFN-γ producing cells increased by 0.8±0.2 x104 cells per ml for subjects with
coeliac disease after 4 h stimulation anti-CD3 stimulation (Figure 5.16). After
24 h anti-CD3 stimulation there was as increase in the number of IL-4
producing CD3+ T-cells for both normal subjects and subjects with coeliac
disease. The number of IL-4 producing CD3+ T-cells increased by 2.5±0.8
x104 and 1.0±0.3 x104 cells per ml for normal and coeliac subjects,
respectively (Figure 5.17). The number of IFN-γ producing CD3+ T-cells
increased after 24 h in vitro anti-CD3 stimulation for normal subjects, but no
significant change was noted for coeliac subjects. IFN-γ producing CD3+ T-
cells increased by 1.3±0.5 x104 cells per ml for normal subjects after 24 h anti-
CD3 stimulation (Figure 5.17).
IL-4 and IFN-γ intracellular production by CD3+ T-cells of normal and coeliac
subjects after 4 and 24 h in vitro gluten fraction 3 stimulation was investigated.
There were variable non specific changes in the number CD3+ T-cells that
produced IL-4 and IFN-γ for normal subjects after 4 and 24 h in vitro gluten
fraction 3 stimulation (Figure 5.16). Similarly, there were variable changes in
the number of CD3+ T-cells that produced IL-4 and IFN-γ after 4 h in vitro
gluten fraction 3 stimulation for coeliac subjects (Figure 5.16). There was a no
change in the number of IL-4 producing CD3+ T-cells, but a significant
increase in IFN-γ producing CD3+ T-cells for coeliac subjects after 24 h in
vitro gluten fraction 3 stimulation. The number of CD3+ T-cells that produced
IFN-γ increased by 1.1±0.4 x104 cells per ml for coeliac subjects after 24 h in
vitro gluten fraction 3 stimulation (Figure 5.17).
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Figure 5.16 Intracellular (A) IL-4 and (B) IFN-γ cytokine production CD3+ T-cells after 4 h in vitro anti-CD3 (♦) or gluten fraction 3 (◊) stimulation in normal and coeliac subjects. Data are given as the change in number (graphed) and percentage (Table) of cytokine producing CD3+ T-cells unstimulated (-) or stimulated (+) for 4 h (n=number of subjects).
Figure 5.17 Intracellular IL-4 and IFN-γ cytokine production CD3+ T-cells after 24 h in vitro anti-CD3 (♦) or gluten fraction 3 (◊) stimulation in normal and coeliac subjects. Data are given as the change in number (graphed) and percentage (Table) of cytokine producing CD3+ T-cells unstimulated (-) or stimulated (+) for 24 h (n=number of subjects).
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Thus, short term (4 h) incubation with anti-CD3 increased IL-4+ T-cells only in
normal control subjects, but not in coeliac subjects. However, after longer
incubations (24 h) both normal and coeliac subjects had increased IL-4+
production by T-cells. Gluten fraction 3 antigen stimulation only increased
IFNγ+ T-cells in coeliac subjects.
To investigate whether low numbers of circulating Vα24 cells are sufficient to
prime an appropriate cytokine response, Vα24+ cells were depleted using
magnetic beads from blood of normal healthy control subjects. IL-4 and IFN-γ
production by CD3+ T-cells was investigated from normal subjects with
depleted levels of Vα24+ T-cells. There was no significant change in IL-4 or
IFN-γ producing CD3+ T-cells for normal subjects with depleted levels of
Vα24+ cells after 4 h in vitro anti-CD3 stimulation (Figure 5.18). After 24 h
anti-CD3 stimulation there was as increase in the number of IL-4 producing
CD3+ T-cells for normal subjects with depleted levels of Vα24+ T-cells, but no
significant change in those CD3+ T-cells producing IFN-γ. The number of IL-4
producing CD3+ T-cells increased by 1.2±0.3 x104 (Figure 5.19). There was no
significant change in IL-4 or IFN-γ intracellular production by CD3+ T-cells of
normal subjects with depleted levels of Vα24+ T-cells after 4 or 24 h in vitro
gluten fraction 3 stimulation (Figure 5.18 and Figure 5.19).
The increase in IL-4 producing CD3+ T-cells as shown in Figure 5.16 was not
observed in normal control subjects with depleted levels of Vα24+ T-cells
(Figure 5.18). This suggests that Vα24+ T-cells were the main source of IL-4,
during the initial stimulation of CD3+ T-cells.
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Figure 5.18 Intracellular IL-4 and IFN-γ cytokine production by blood CD3+ T-cells after 4 h in vitro anti-CD3 (♦) or gluten fraction 3 (◊) stimulation in
normal subjects that have been depleted of Vα24+ cells. Data are given as the change in number (graphed) and percentage (Table) of cytokine producing CD3+ T-cells unstimulated (-) or stimulated (+) for 4 h (n=number of subjects).
Figure 5.19 Intracellular IL-4 and IFN-γ cytokine production by blood CD3+ T-cells after 24 h in vitro anti-CD3 (♦) or gluten fraction 3 (◊) stimulation in
normal subjects that have been depleted of Vα24+ cells. Data are given as the change in number (graphed) and percentage (Table) of cytokine producing CD3+ T-cells unstimulated (-) or stimulated (+) for 24 h (n=number of subjects).
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5.4.10 Comparison of Vα24+ T-cells cytokine production in coeliac
disease
In view of previous studies showing impaired IL-4 production by Vα24+ T-
cells in type 1 diabetes (Wilson et al., 1998), production of IL-4, IL-10, IL-13
and IFN-γ by Vα24+ T-cells was investigated for subjects with coeliac disease.
As shown in Chapter 3, intracellular IL-4 and IL-10 increased for Vα24+ T-
cells, while variable changes in IL-13 and IFN-γ cytokine production after 4 h
in vitro anti-CD3 stimulation for normal subjects. No significant change in the
number of IL-4, IL-10, IL-13 or IFN-γ producing Vα24+ T-cells for coeliac
subjects was observed (Figure 5.20). Vα24+ T-cells were not only deficient in
number but had a functional deficiency of IL-4 and IL-10 production in coeliac
disease.
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Figure 5.20 Intracellular IL-4, IL-10, IL-13 and IFN-γ cytokine production by
circulating Vα24+ T-cells after 4 h in vitro anti-CD3 stimulation in normal
subjects and in subjects with coeliac disease. Data are given as the mean±SEM
change in cytokine producing Vα24+ T-cells after 4 h incubation without (-) or
with (+) anti-CD3 stimulation. Also shown (Table below) are the percentages
of total Vα24+ T-cells that produced cytokines before and after stimulation for
normal subjects and subjects with coeliac disease. Data are given as the change
of cytokine producing Vα24+ T-cells x103 cells/ml and percentage (n=number
of subjects).
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5.4.11 Comparison of iNK T-cells cytokine production in coeliac
disease.
Intracellular IL-4, IL-10, IL-13 and IFN-γ cytokine production by 6B11+ and
Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells was examined after 4 h in vitro
anti-CD3 stimulation for normal subjects and subjects with coeliac disease.
As shown in Chapter 3, intracellular IL-4, IL-10 and IL-13, but not IFN-γ
cytokine production increased for 6B11+ iNK T-cells after in vitro anti-CD3
stimulation for normal subjects. Like circulating Vα24+ T-cells, there was no
significant change in the number of IL-4, IL-10, IL-13 or IFN-γ cytokine
producing 6B11+ iNK T-cells for coeliac subjects after 4 h in vitro anti CD3-
stimulation (Figure 5.21).
Cytokine production by Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells was
detectable even in normal control subjects with low numbers of circulating
Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells (Figure 5.22). There was a
significant increase in the mean number of IL-4 and IL-10 producing Vα24+
α-GalCer/CD1d tetramer+ iNK T-cells of normal subjects after 4 h in vitro
anti-CD3 stimulation as shown in Chapter 3. There was no significant change
in the production of IL-4 or IL-10 by Vα24+ α-GalCer/CD1d tetramer+ iNK
T-cells in subjects with coeliac disease after 4 h in vitro anti-CD3 stimulation.
There was no significant change in IL-13 or IFN-γ production by Vα24+ α-
GalCer/CD1d tetramer+ iNK T-cells in both normal subjects and subjects with
coeliac disease (Figure 5.23).
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Figure 5.21 Intracellular IL-4, IL-10, IL-13 and IFN-γ cytokine production by
circulating 6B11+ iNK T-cells after 4 h in vitro anti-CD3 stimulation in normal
subjects and in subjects with coeliac disease. Data are given as the mean±SEM
change in cytokine producing 6B11+ iNK T-cells after 4 h in vitro incubation
without (-) or with (+) anti-CD3. Also shown (boxed below) are the
percentages of total 6B11+ iNK T-cells that produced cytokines before and
after 4 h in vitro stimulation for normal subjects and subjects with coeliac
disease. Data are given as the change of cytokine producing 6B11+ iNK T-cells
x103 cells/ml and percentage (n=number of subjects).
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Figure 5.22 Comparison of circulating Vα24+ IL-4+ cells that were α-
GalCer/CD1d tetramer+/- for a normal subject with low number of circulating
Vα24 cells and a subject with coeliac disease after 4 h in vitro anti CD3
stimulation.
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Figure 5.23 Intracellular IL-4, IL-10, IL-13 and IFN-γ cytokine production by circulating Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells after 4 h in vitro anti-CD3 stimulation in normal subjects and in subjects with coeliac disease. Data are given as the mean±SEM change in cytokine producing Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells after 4 h in vitro incubation without (-) or with (+) anti-CD3. Also shown (boxed below) are the percentages of total Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells producing cytokines before and after stimulation. Data are given as the change of cytokine producing Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells x103 cells/ml and percentage (n=number of subjects).
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Principal findings of this Chapter
• Coeliac subjects have reduced numbers of circulating:
o CD161+ NK cells (total, bona fide and NK T-like).
o Vα24+ T-cells.
o Vβ11+ T-cells.
o Vα24+ CD4+ T-cells.
o Vα24+ Vβ11+ T-cells.
o Vα24+ CD161+ NK T-cells.
o Vα24+ 6B11+ and Vα24+ Vβ11+ α-GalCer/CD1d tetramer+
iNK T-cells.
• The number of circulating CD94+ NK cells was dependant upon
disease state, as untreated coeliac subjects had lower numbers of
circulating CD94+ NK cells compared to treated coeliac subjects and
normal healthy control subjects.
• Intestinal Vα24+ T-cells were deficient in coeliac disease.
• Coeliac subjects had impaired IL-4 production and increased IFN-γ
production by CD3+ T-cells after 4 h in vitro anti-CD3 stimulation.
• Coeliac subjects had increased in IFN-γ production by CD3+ T-cells
after 24 h in vitro gluten fraction 3 stimulation.
• Coeliac subjects had impaired IL-4, IL-10 and IL-13 production by
Vα24+ T-cells, 6B11+ and Vα24+ α-GalCer/CD1d tetramer+ iNK T-
cells after 4 h in vitro anti-CD3 stimulation.
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5.5 DISCUSSION This Chapter has shown that circulating CD161 NK cells were deficient in
coeliac disease, reduced to approximately 75% of the levels from blood of
normal healthy subjects. Coeliac subjects at diagnosis have reduced numbers of
circulating CD94+ NK cells compared to normal subjects and coeliac subjects
on a GFD. The number of circulating CD94+ NK cells in untreated coeliac
subjects was reduced to 65% of the numbers present in coeliac subjects on a
GFD, yet the number of circulating CD56, CD57 and CD161 NK cells was
independent of diet. The decreased number of circulating CD94+ NK cells in
untreated coeliac subjects can be explained by their localization to the mucosa
as described by Jabri et al. (2000).
Circulating CD56+ NK T-like cells were increased in subjects with coeliac
disease by approximately 60% the levels of normal control subjects. The entire
CD161+ NK lineage (i.e.; total, bona fide and NK T-like) was affected in
coeliac disease. The number of circulating bona fide CD161+ and CD161+ NK
T-like cells were reduced to approximately 80% and 68%, respectively, of the
levels of normal control subjects. Unlike CD94, the deficiency of CD161 was
independent of diet. Chen et al. (1997) have shown that NK1.1 expression is
lost on Vα14+ T-cells after prolonged in vitro stimulation. The work of this
Chapter was unable exclude a similar process for coeliac disease although; re-
suppression in treated coeliac subjects who still had NK T-cell deficiency
would be expected. The functional significance of other NK T-like cells,
especially CD161+ NK T-like cells remains unknown, although this Chapter
has shown they were deficient in coeliac disease. The decrease of circulating
CD161+ NK cells, bona fide CD161+ and CD161+ NK T-like cells may be in
keeping with the increased prevalence of malignancy in coeliac disease (Di
Sabatino et al., 1998a)
Although there was no deficiency in the number of circulating CD3+, CD4+ or
Vβ13+ T-cells there was a selective deficiency of circulating Vα24+ and
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Vβ11+ T-cells in coeliac disease. The mean number of circulating Vα24+ T-
cells was reduced in blood of coeliac subjects to approximately 27% of the
number present in normal subjects. The deficiency of Vα24+ T-cells was
independent of diet, duration of the GFD and was present at all ages. Thus the
deficiency was unlikely to be due to inflammation alone in coeliac disease. In
contrast, Vα24+ T-cells declined with age in control subjects (Chapter 4). The
deficiency of Vα24+ T-cell was not confined to the circulating lymphocytes,
but was present in the small intestine mucosa as shown by decreased Vα24
mRNA expression and immunofluorescence staining.
The number of the SP subset was reduced to 30% of the numbers present in
normal subjects. The co-expression of Vα24 and Vβ11 was also reduced in
coeliac disease. Vα24+ Vβ11+ T-cells were markedly deficient in coeliac
subjects, reduced to 15% of the numbers present in normal subjects. These
results contrast with that of Van der Vliet et al. (2001), who investigated
Vα24+ Vβ11+ T-cells in blood from 10 coeliac subjects and concluded these
cells were not deficient. Their data were distributed in the lower end of their
range for normal subjects. They did not have α-GalCer/CD1d tetramers
available, which are regarded as the gold standard for identifying iNK T-cells,
nor did they investigate the cytokine production by these cells. The work
presented within this Chapter further defined iNK T-cells by the co-expression
of Vα24 and 6B11 as well as Vα24, Vβ11 and α-GalCer/CD1d tetramer+
markers. Invariant NK T-cells were reduced in coeliac subjects to
approximately 9-30% of the numbers present in normal subjects. The loss of
these immunoregulatory iNK T-cells could partly explain the inappropriate
activation of gluten response T-cells that result in intestinal damage in coeliac
disease. The deficiency of Vα24+ T-cells and iNK T-cells in coeliac disease
was independent of age, diet or duration of gluten free diet. I acknowledge that
it is difficult to assess compliance with a gluten-free diet and to ensure full
exclusion of gluten.
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Previous studies have investigated cytokine profiles of both circulating and
intestinal lymphocytes after polyclonal stimulation. While Kerttula et al.
(1999) and Lahat et al. (1999) were able to detect cytokine changes within the
intestinal lymphocytes they found it difficult to determine cytokine profiles in
coeliac subjects (both treated and untreated coeliac subjects). In vitro activation
of peripheral blood was investigated and this demonstrated that circulating
CD3+ T-cells of coeliac subjects produced detectable levels of IL-4 and IFN-γ
cytokines after in vitro anti-CD3 stimulation. This work has shown that
classical CD3+ T-cells from coeliac subjects, although depleted in Vα24+ T-
cells and iNK T-cells, were still able to produce cytokines IL-4 and IFN-γ at
levels, which were lower yet comparable to the levels of normal subjects.
As well as being deficient in coeliac disease, the work of this Chapter has
shown that Vα24+ T-cells, Vα24+ 6B11+ and Vα24+ α-GalCer/CD1d
tetramer+ iNK T-cells were functionally defective after in vitro anti-CD3
stimulation, unlike equivalent cells from normal subjects. A negligible cytokine
response was observed in Vα24+ T-cells, 6B11+ and Vα24+ α-GalCer/CD1d
tetramer+ iNKT-cells from coeliac subjects, although some IL-4, IL-10, IL-13
and IFN-γ intracellular staining was evident prior to stimulation for both
normal and coeliac subjects. Multiple differences in gene expression of IL-4-
null Vα24+ T-cell clone from a human monozygotic twin affected with type I
diabetes has been identified compared to an IL-4 intact Vα24+ T-cell clone
from the other unaffected twin (Wilson et al., 2000). The same may be present
in Vα24+ T-cells and iNK T-cells from coeliac subjects.
Vα24+ T-cells are believed to be immunoregulatory because they direct a Th2
immune response, rather then a Th1 outcome that is associated with coeliac
disease. The Th1 bias was seen in our studies of gluten-stimulation of
conventional CD3 T-cells from coeliac subjects, as production of IFN-γ (a Th1
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cytokine) increased, whereas IL-4 (a Th2 cytokine) increased in similar cells
from normal healthy subjects. The IL-4 produced by normal Vα24+ T-cells
presumably suppresses activation of gluten-stimulated CD3 T-cells in vivo. An
additional mechanism is that Vα24+ T-cells are cytotoxic to antigen-presenting
dendritic cells which otherwise would induce a Th1 response (Nicol et al.,
2000a). Thus, Vα24+ T-cells may be important in preventing development of
coeliac disease in those who are genetically predisposed. What remains
unexplained is how natural glycolipid antigenic stimulation of Vα24+ iNK T-
cells occurs. It is possible that damaged epithelial cells from a viral infection in
the gastrointestinal tract may provide such stimulation. Van der Vliet et al.
(2001) suggest that Vα24+ Vβ11+ immunoregulatory cells are unable to
differentiate and/or proliferate adequately in response to T-cell activation or
cytokine stimulation. This defect might result from either exhaustion or
replicative senescence due to overstimulation, exogenous factors such as viral
infections, since these have repeatedly been implicated in the pathogenesis of
coeliac disease (van der Vliet et al., 2001).
In summary, Vα24 T-cells are deficient in animal models and human
autoimmune disease. It has been shown that autoimmune disease increases
with the duration of coeliac disease from 5.1% at diagnosis of less than 2 years
to 34% at diagnosis at greater than 20 years (Ventura et al., 1999). Ventura et
al. (1999) found that the prevalence of autoimmune disease in all coeliac
subjects was 14% compared to 3% in normal control subjects. This raises the
possibility that both coeliac and autoimmune diseases share a common disease
pathway (i.e., genetic predisposition, Vα24+ T-cell deficiency) or that gluten
exposure in coeliac disease predisposes to autoimmune disease. In relation to
this present Chapter, a possibility might be that gluten exposure causes
progressive Vα24+ T-cell and iNK T-cell deficiency, however this was not
evident. Vα24+ T-cells and iNK T-cells did not decline with age in coeliac
subjects, though they did decrease in normal subjects. There was no significant
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difference in numbers of circulating Vα24 T-cells or iNK T-cells with respect
to diet. Vα24 T-cell and iNK T-cell deficiency was present at the time of
diagnosis and thus likely contributed to the pathogenesis rather than be caused
by coeliac disease. Vα24 T-cell and iNK T-cell deficiency was not confined to
the circulating lymphocytes but also observed at the site of the disorder, within
the small intestine of coeliac subjects. This work shows an association of
coeliac disease and autoimmune disease through a common deficiency of
Vα24+, Vα24+ Vβ11+ T-cells, Vα24+ 6B11+ and Vα24+ Vβ11+ α-
GalCer/CD1d tetramer+ iNK T-cells.
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6 Chapter 6 NK, T and NK T-cells in IBD
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6.1 INTRODUCTION The pathogenesis of ulcerative colitis and Crohn’s disease seems to be due
primarily due to a loss of immunoregulation resulting from a combination of
genetic and environmental factors. There is loss of immunoregulation to
luminal bacterial antigens in both Crohn’s disease and ulcerative colitis
(Duchmann et al., 1995b; Kraus et al., 2004a; Kraus et al., 2004b; MacDonald,
1995). The inflammation involved with Crohn’s disease is due to an
inappropriate T-cell response to endogenous bacterial antigens (Duchmann et
al., 1995a; Lodes et al., 2004; MacDonald, 1995; Targan et al., 2005). In
Crohn’s disease there is an exaggerated response to bacterial flagellin antigens
(Lodes et al., 2004), with an increased expression of Th1 cytokines by lamina
propria cells (Cobrin and Abreu, 2005) although recent data indicate that IL-17
may also be pro-inflammatory (Seiderer et al., 2007). Inflammation in
ulcerative colitis is more complex and involves both T-cell and neutrophil
mediated responses (Olives et al., 1997; Targan, 1998). The immune response
in ulcerative colitis is less well defined but includes an atypical Th2 response
from a non-invariant NK T-cell producing IL-13, possibly mixed with an
Arthus reaction with immune complex activation and neutrophil recruitment
(Fuss et al., 2004; Heller et al., 2005; Mayer, 2005). An unexplained feature of
both Crohn’s disease and ulcerative colitis is low or absent mucosal expression
of IL-4 (Karttunnen et al., 1994).
The basis of this work originated from the deficiency of Vα24 T-cells in
autoimmune diseases (Baxter et al., 1997; Maeda et al., 1999; Sumida et al.,
1995; Wilson et al., 1998). A preliminary study by van der Vliet et al (2001)
showed that Vα24+ Vβ11+ T-cells are deficient in Crohn’s disease and
ulcerative colitis. This deficiency of NK T-cells could contribute to the loss of
immunoregulation of the gut-associated immune response to commensal
bacteria. The deficiency of immunoregulatory NK T-cells could identify new
targets for IBD therapy.
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6.2 AIMS AND HYPOTHESIS The aim of this Chapter was to investigate the number of circulating NK cells,
T-cells, NK T-like and iNK T-cells in IBD. Cytokine production by Vα24+ T-
cells and iNK T-cells after in vitro anti-CD3 and PMA:ionomycin stimulation
were examined and compared to cytokine production by CD3+ T-cells after in
vitro anti-CD3 and PMA:ionomycin stimulation in normal subjects and
subjects with IBD.
The hypothesis of this Chapter is that a deficiency of NK cells,
immunoregulatory T-cells, NK T-like cells and/or iNK T-cells could explain
loss of immunoregulation in IBD.
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6.3 MATERIALS AND METHODS
6.3.1 Subjects Subjects with IBD were recruited from those attending the Department of
Gastroenterology and Hepatology, The North West Adelaide Health Service at
The Queen Elizabeth and Lyell McEwin Hospitals, as well as from those who
responded to an invitation in the newsletter of the Crohn’s and Colitis
Association of South Australia. Only those with a verified diagnosis of either
Crohn’s disease or ulcerative colitis were recruited. A total of 97 subjects with
Crohn’s disease, 68 subjects with ulcerative colitis and 156 subjects who were
healthy (apart from non-ulcer dyspepsia) were recruited. Crohn’s patients were
divided into those who only had disease of the small intestine and those who
had large intestinal disease (either alone or with small intestinal involvement).
Where known, disease activity was assessed by bowel frequency, pain, quality
of life and extra intestinal features or by erythrocyte sedimentation rate and C-
reactive protein levels. Blood was collected for flow cytometry and for a
complete blood examination. Members of the normal control group were also
used in previous Chapters. This study had ethical permission from the Human
Ethics’ Committee of the North West Adelaide Health Service.
6.3.2 Flow cytometry Peripheral blood lymphocytes were collected and stained using antibodies
directed against the CD56, CD57, CD94 and CD161 NK markers and CD3,
CD4, Vα24, Vβ11, Vβ13 T-cells or 6B11 iNK T-cell markers as previously
described in Chapter 2. α-GalCer/CD1d tetramer binding to ligand Vα24+
Vβ11+ was examined as previously described in Chapter 2.
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6.3.3 In vitro anti-CD3 and PMA/ionomycin stimulation of
peripheral blood T-cells Peripheral blood lymphocytes were stimulated in culture for 4 h and 24 h with
anti-CD3 antibody or PMA and ionomycin as previously described in Chapter
2.
6.3.4 Statistics
Data were summarized as the mean±SEM. Means of multiple groups were
compared for significance using Peritz’ multiple comparison F test (Harper,
1984). Slopes of linear regression lines were compared for Vα24+ T-cells
versus age using GraphPad InStat version 3.00 for Windows 95 (GraphPad
Software, San Diego California). Data of the ratio of copy number of
Vα24:GAPDH mRNA were log (x+1) transformed to normalise the data and
stabilise the variance before analysis.
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6.4 RESULTS
6.4.1 Comparison of circulating NK cells in IBD The number of circulating CD56+, CD57+, CD94+ and CD161+ NK cells
were reduced in subjects with ulcerative colitis and Crohn’s disease when
compared to normal control subjects (Figure 6.1). The mean±SEM number of
circulating CD56+, CD57+, CD94+ and CD161+ NK cells was 2.2±0.5x105,
2.3±0.3 x105, 2.6±0.3 x105 and 3.5±0.3 x105 cells per ml, in subjects with
ulcerative colitis and 1.9±0.4 x105, 2.3±0.3 x105, 2.9±0.5 x105 and 3.4±0.5
x105 cells per ml in subjects with Crohn’s disease, respectively (Figure 6.1).
As in previous Chapters, circulating NK cells were divided into bona fide NK
cells and NK T-like cells by their CD3 co-expression. Bona fide CD56+,
CD57+, CD94+ and CD161+ NK cells were reduced in subjects with
ulcerative colitis and Crohn’s disease when compared to normal control
subjects. The mean±SEM number of circulating bona fide CD56+, CD57+,
CD94+ and CD161+ NK cells was 1.3±0.2 x105, 0.6±0.1 x105, 1.2±0.2 x105
and 1.7±0.2 x105 cells per ml in subjects with ulcerative colitis and 0.9±0.2
x105, 0.7±0.1 x105, 1.4±0.3 x105 and 1.2±0.2 x105 cells per ml in subjects with
Crohn’s disease, respectively (Figure 6.1). There was no significant difference
in the number of circulating CD56+, CD57+ or CD94+ NK T-like cells in
subjects with subjects with ulcerative colitis or Crohn’s disease when compared
to normal control subjects. Nevertheless, there was a significant decrease in the
number of circulating CD161+ NK T-like cells in Crohn’s disease, but not
ulcerative colitis when compared to the numbers present in normal healthy
subjects. The mean±SEM number of circulating CD161+ NK T-like cells in
subjects with Crohn’s disease was 2.2±0.3 x105 cells per ml (Figure 6.1).
In summary, the number of circulating CD56+, CD57+, CD94+ and CD161+
NK cells were reduced in both ulcerative colitis and Crohn’s disease. The
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deficiencies of CD56+, CD57+ and CD94+ NK cells were due to the bona fide
non T-cell subset. As a consequence there was no difference in the number of
circulating CD56+, CD57+ or CD94+ NK T-like cells when comparing normal
subjects with subjects with ulcerative colitis and Crohn’s disease. In contrast,
circulating CD161+, bona fide CD161+ and CD161+ NK T-like cells were
deficient in Crohn’s disease.
Figure 6.1 Comparison of circulating (A) CD56+, (B) CD57+, (C) CD94+ and
(D) CD161+ NK cells in normal control subjects and subjects ulcerative colitis
and Crohn’s disease. Circulating NK cells were divided by their CD3 co-
expression. Total circulating NK cells have been divided into NK T-like cells
(filled bars) and bona fide NK cells (open bars). Data are given as the
mean±SEM x105 cells/ml (n=number of subjects).
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6.4.2 Comparison of CD3+, CD4+, Vα24+, Vβ11+ and Vβ13+ T-
cells in IBD There was no significant difference in the number of circulating CD3+ or
CD4+ T-cells when comparing normal subjects and subjects with ulcerative
colitis or Crohn’s disease (Figure 6.2). Ulcerative colitis and Crohn’s subjects
were further characterized by activity and site of disease. Disease status
referred to whether ulcerative colitis or Crohn’s disease was active or in
remission at the time of peripheral blood collection. Disease site correlated to
portion of bowel affected in Crohn’s disease (Crohn’s and Crohn’s colitis).
There was no significant difference in the number of circulating CD3+ or
CD4+ T-cells when comparing ulcerative colitis and Crohn’s subjects with
active disease or when in remission, nor was there any significant difference
when comparing small and large bowel Crohn’s disease.
Figure 6.2 Comparison of blood (A) CD3+ and (B) CD4+ T-cells in normal,
ulcerative colitis and Crohn’s subjects, respectively. Data are given as the
mean±SEM x106 cells/ml (n=number of subjects).
Subjects with ulcerative colitis had comparable numbers of circulating Vα24+
T-cells when compared to normal control subjects. In contrast, the number of
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circulating Vα24+ T-cells were significantly reduced in subjects with Crohn’s
disease. The mean±SEM number of circulating Vα24+ T-cells in ulcerative
colitis and Crohn’s subjects was 8.2±0.5 x103 and 3.0±0.2 x103 cells per ml,
compared to 8.9±0.4 x103 cells per ml in normal healthy control subjects
(Figure 6.3). There was no significant difference in the mean number of
circulating Vα24+ T-cells when comparing disease activity in subjects with
ulcerative colitis or Crohn’s disease. Equally, the systemic deficiency of
Vα24+ T-cells was independent of site of Crohn’s disease (Crohn’s disease of
the small intestine and Crohn’s colitis). As shown in Chapter 4, there was a
dramatic decrease in the number of circulating Vα24+ T-cells with age in
normal healthy subjects. A similar trend was present in ulcerative colitis, but
not in Crohn’s disease (Figure 6.4). In contrast to that observed for normal
healthy subjects, the decrease with age in ulcerative colitis patients was solely
due to the reduction of circulating Vα24+ T-cells in male subjects (Figure 6.4).
Figure 6.3 Comparison of total number of circulating Vα24+ T-cells in
normal, ulcerative colitis and Crohn’s subjects. Data are given as mean±SEM
x103 cells/ml (n=number of subjects).
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Figure 6.4 Comparison of total number of circulating Vα24+ T-cells in relation
to age for (A) ulcerative colitis and Crohn’s disease. (B) Ulcerative colitis
subjects were divided by gender. Data are given as the mean change in Vα24+
T-cells with age x103 cells/ml (n=number of subjects).
The numbers of circulating Vβ11+ and Vβ13+ T-cells present in Crohn’s
disease and ulcerative colitis are given in Figure 6.5. Circulating Vβ11+ and
Vβ13+ T-cells were not deficient in ulcerative colitis or in Crohn’s disease
compared to the levels of normal subjects.
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Figure 6.5 Comparison of circulating (A) Vβ11+ and (B) Vβ13+ T-cells in
normal, ulcerative colitis and Crohn’s subjects, respectively. Data are given as
mean±SEM x103 cells/ml (n=number of subjects).
6.4.3 Comparison of CD56+, CD57+, CD94+ and CD161+ Vα24+
NK T-cells in IBD
CD56, CD57 and CD97 NK marker expression was low on circulating Vα24+
T-cells for subjects with ulcerative colitis and Crohn’s disease, consistent to
what was observed in normal control subjects (Figure 6.6). The CD161 NK
marker was co-expressed on the majority of Vα24+ T-cells for subjects with
ulcerative colitis and Crohn’s disease. There was a significant decrease in the
mean number of circulating CD161+ Vα24+ NK T-cells in subjects with
Crohn’s disease, when compared to normal control subjects. The mean±SEM
number of Vα24+ CD161+ NK T-cells in subjects with ulcerative colitis and
Crohn’s disease was 4.0±0.5 x103 and 1.3 ±0.1 x103 cells per ml, respectively,
compared to 4.4±0.4 x103 cells per ml for normal control subjects (Figure 6.6).
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Figure 6.6 Comparison of circulating Vα24+ T-cells that co-express NK cell
markers (A) CD56+, (B) CD57+, (C) CD94+ and (D) CD161+ in normal,
ulcerative colitis and Crohn’s subjects. Circulating Vα24+ T-cells were divided
by their NK cell marker co-expression. Filled bars correspond to the co-
expression of the particular NK cell marker, while open bars correspond to
Vα24+ T-cells that are negative for the particular NK-cell marker. Data are
given as mean±SEM x103 cells/ml (n=number of subjects).
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6.4.4 Comparison of Vα24+ CD4+ (SP) T-cells in IBD
The SP subset of Vα24+ T-cells was investigated because they have been
shown to be potent producers of IL-4 and therefore important in the promotion
of a normal Th2 immune response. The mean number of circulating Vα24+ SP
T-cells was deficient in subjects with Crohn’s disease but not ulcerative colitis
when compared to normal control subjects. The mean±SEM number of Vα24+
CD4+ T-cells in ulcerative colitis and Crohn’s subjects was 5.2±0.6 x103 and
2.3±0.2 x103 cells per ml, respectively, compared to 5.3±0.4 x103 cells per ml
for normal healthy control subjects (as shown in Chapter 3) (Figure 6.7).
Figure 6.7 Comparison of circulating Vα24+ CD4+ T-cells in normal,
ulcerative colitis and Crohn’s subjects. Circulating Vα24+ T-cells where
divided by their CD4 T-cell marker co-expression. Filled bars correspond to the
SP subset Vα24+ CD4+, while open bars correspond to the Vα24+ CD4-
population. Data are given as mean±SEM x103 cells/ml (n=number of
subjects).
6.4.5 Comparison of Vα24+ Vβ11+ and Vα24+ Vβ13+ T-cells in
IBD
The pairing of TCR α-chain Vα24 with either β-chain Vβ11 or Vβ13 were
examined and is given in Figure 6.8. Circulating Vα24+ Vβ11+ but not Vα24+
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Vβ13+ T-cells were deficient in both ulcerative colitis and Crohn’s subjects
when compared to normal control subjects. The TCR Vβ11 was paired with 8%
and 15% of circulating Vα24+ T-cells for ulcerative colitis and Crohn’s
subjects, respectively, compared to 43% for normal healthy control subjects.
The mean±SEM number of Vα24+ Vβ11+ T-cells in ulcerative colitis and
Crohn’s subjects was 0.5±0.1 x103 and 0.7±0.2 x103 cells per ml, compared to
3.9±0.4 x103 for normal healthy control subjects (Figure 6.8). There was no
significant difference in the proportion or number of Vα24+ Vβ13+ T-cells
when comparing normal subjects with subjects with ulcerative colitis and
Crohn’s disease (Figure 6.8).
Figure 6.8 Comparison of circulating (A) Vα24+ Vβ11+ and (B) Vα24+
Vβ13+ T-cells in normal, ulcerative colitis and Crohn’s subjects. Filled bars
correspond to the proportion of Vα24+ T-cells that co-express (A) Vβ11 and
(B) Vβ13. Data are given as mean±SEM x103 cells/ml (n=number of subjects).
6.4.6 Comparison of Vα24+ 6B11+ and Vα24+ Vβ11+ α-
GalCer/CD1d tetramer+ iNK T-cells in IBD
The number of circulating Vα24+ 6B11+ iNK T-cells are given in Figure 6.9
and Table 6.1, while the number of circulating Vα24+ Vβ11+ α-GalCer/CD1d
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tetramer+ iNK T-cells are shown in Figure 6.10, Figure 6.11 and summarised in
Table 6.1.
The mean number of circulating Vα24+ 6B11+ iNK T-cells were reduced in
subjects with ulcerative colitis and Crohn’s disease to 13% and 11%,
respectively, of the levels of normal healthy subjects that were autoantibody
screen negative. The mean±SEM number of circulating Vα24+ 6B11+ iNK T-
cells in normal control, ulcerative colitis and Crohn’s subjects was 5.4±1.0
x103, 0.7±0.2 x103 and 0.6±0.3 x103 cells per ml, respectively (Figure 6.9).
Figure 6.9 Comparison of circulating Vα24+ 6B11+ iNK T-cells in normal
subjects and in subjects with ulcerative colitis, Crohn’s disease, respectively.
Filled bars correspond to Vα24+ 6B11+ iNK T-cells, while open bars
correspond to circulating Vα24+ that were 6B11-. Data are given as the
mean±SEM cells/ml x103 (n=number of subjects).
Total circulating α-GalCer/CD1d tetramer+ cells were reduced in subjects with
ulcerative colitis and Crohn’s disease when compared to normal healthy
subjects (autoantibody screen negative). The mean±SEM number of circulating
α-GalCer/CD1d tetramer+ cells in normal control, ulcerative colitis and
Crohn’s subjects was 4.3±0.4 x103, 1.6±0.6 x103 and 1.0±0.2 x103 cells per ml,
respectively (Table 6.1). The mean number of circulating Vα24+ Vβ11+ that
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bound α-GalCer/CD1d tetramer were reduced in subjects with ulcerative colitis
and Crohn’s disease to 6% and 2%, respectively, of the levels of normal control
subjects. The mean±SEM number of Vα24+ Vβ11+ α-GalCer/CD1d tetramer+
iNK T-cells in normal control, ulcerative colitis and Crohn’s subjects was
3.3±0.5 x103, 0.2±0.06 x103 and 0.05±0.01 x103 cells per ml, respectively
(Figure 6.10, Figure 6.11 and Table 6.1).
Thus, this data shows a specific deficiency of iNK T-cells in ulcerative colitis
that was not evident by the Vα24 T-cell marker alone. Whereas both Vα24 T-
cells and iNK T-cells were deficient in Crohn’s disease.
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Figure 6.10 (A) Representative dot plots and histograms. Dot plots show
Vα24+ Vβ11+ T-cells (upper right quadrant) for normal subjects and subjects
with ulcerative colitis and Crohn’s disease. Histograms show the percentage of
Vα24+ Vβ11+ cells that were α-GalCer/CD1d tetramer+ for normal subjects
and subjects with ulcerative colitis and Crohn’s disease. Data are given as the
mean percentage of positive cells per quadrant or gate.
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Figure 6.11 Comparison of circulating (A) α-GalCer/CD1d tetramer+ and the
proportion that were (B) Vα24+ α-GalCer/CD1d tetramer+ (filled bars), (C)
Vβ11+ α-GalCer/CD1d tetramer+ (filled bars) and (D) Vα24+ Vβ11+ α-
GalCer/CD1d tetramer+ (filled bars) iNK T-cells in normal subjects and in
subjects with ulcerative colitis and Crohn’s disease, respectively. Data are
given as the mean±SEM x103 cells/ml (n=number of subjects).
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Table 6.1 Comparison of circulating T-cells and iNK T-cells in normal subjects
and in subjects with ulcerative colitis, Crohn’s disease, respectively. Data are
given as the mean±SEM cells/ml x103 (n=number of subjects, range).
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6.4.7 Comparison of Vα24+ T-cells in the intestine of IBD using
immunofluorescence
Intestinal Vα24+ T-cells were reduced in the mucosa of Crohn’s subject when
compared to normal control subjects. The mean number of intestinal Vα24+ T-
cells for normal subjects and subjects with ulcerative colitis and Crohn’s
disease were 248±37, 101±45 and 56±17 cells per mm2, respectively (Figure
6.12).
6.4.8 Intestinal Vα24+ T-cell mRNA expression in IBD
Intestinal Vα24 T-cells mRNA expression was investigated. Vα24 mRNA from
subjects with ulcerative colitis and Crohn’s disease was decreased to
approximately 29% and 15%, respectively, of the levels present in the intestine
of control subjects (Figure 6.13). The Vα24:18S PCR product band net
intensity ratio was 2.2±0.4, 0.6±0.2 and 0.3±0.1 for normal subjects, subjects
with ulcerative colitis and Crohn’s disease, respectively. The deficiency of
intestinal Vα24 T-cells mRNA expression was confirmed by RT-PCR. There
was no significant difference in intestinal GAPDH mRNA expression when
comparing normal subjects with subjects with ulcerative colitis or Crohn’s
disease. RT-PCR confirmed a reduction of Vα24:GAPDH mRNA in subjects
with ulcerative colitis and Crohn’s disease to 9% and 7%, of levels in control
subjects, respectively. This decrease in mucosal Vα24 mRNA as determined by
both relative and RT-PCR agrees with the systemic deficiency observed in
Crohn’s disease, as well as showing a mucosal deficiency of Vα24+ T-cells in
ulcerative colitis that was not evident in blood.
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Figure 6.12 Comparison of immunostained intestinal Vα24+ T-cells in (A and
B) normal subjects and in subjects with (C) ulcerative colitis and (D) Crohn’s
disease and. Data are given as the mean±SEM cells/mm2 (n=number of
subjects). Inserts are isotype control antibody stains.
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Figure 6.13 Comparison of intestinal mRNA expression for Vα24+ T-cells in
normal control subjects and in subjects with ulcerative colitis and Crohn’s
disease. (A) Representative gel containing samples from normal subjects (lanes
1-3), subjects with Crohn’s disease (lanes 4-6) and ulcerative colitis (lanes 7-9)
with 18S rRNA internal control (upper band) and Vα24 (lower band) PCR
products. Comparison of (B) Vα24:18S rRNA ratio and (C) Vα24:GAPDH
mRNA copy number from normal control subjects and subjects with ulcerative
colitis and Crohn’s disease. Data are given as the net intensity of Vα24:18S
rRNA band and ratio of Vα24:GAPDH mRNA copy number, respectively
(n=number of subjects).
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6.4.9 Comparison of cytokine production from Vα24 T-cells in
IBD
Cytokine production by Vα24+ T-cells was examined after 4 h in vitro anti-
CD3 stimulation in subjects with ulcerative colitis and Crohn’s disease. This
was investigated because invariant NK T-cells have been shown to have
defective cytokine production in type 1 diabetes (Wilson et al., 1998).
As shown in Chapter 3, intracellular IL-4 and IL-10 production by Vα24+ T-
cells increased after 4 h in vitro anti-CD3 stimulation, while there were
variable changes in IL-13 and IFN-γ cytokine production in normal healthy
subjects. In ulcerative colitis, both IL-4 and IFN-γ intracellular cytokine
production increased from Vα24+ T-cells, whereas IL-10 and IL-13 production
showed marginal changes after in vitro anti-CD3 stimulation. There was an
increase of 26%, and 22%, equating to 3.6±0.9 x103 and 2.7±0.6 x103 cells per
ml, in the mean number of IL-4 and IFN-γ producing Vα24+ T-cells after 4 h
in vitro anti-CD3 stimulation in subjects with ulcerative colitis, respectively
(Figure 6.14). In contrast, there was no-significant change in the number of IL-
4, IL-10, IL-13 or IFN-γ producing Vα24+ T-cells from Crohn’s subjects
(Figure 6.14). IL-4, IL-10, IL-13 and IFN-γ production by Vα24 T-cells was
impaired in Crohn’s disease. Seven of twenty two subjects with Crohn’s
disease were not receiving any treatment, and nevertheless had reduced
numbers of Vα24+ T-cells and defective cytokine production. Thus, treatment
was not responsible for either the reduced number of circulating Vα24+ T-cells
or the defective cytokine production.
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Figure 6.14 Intracellular IL-4, IL-10, IL-13 and IFN-γ cytokine production by
blood Vα24+ T-cells after 4 h in vitro anti-CD3 (♦) stimulation in normal
subjects and subjects with ulcerative colitis and Crohn’s disease. Data are given
as the change in number (graphed) and percentage (Table) of cytokine
producing Vα24+ T-cells unstimulated (-) or stimulated (+) for 4 h (n=number
of subjects).
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6.4.10 Comparison of cytokine production from 6B11+ and Vα24+
α-GalCer/CD1D tetramer+ iNK T-cells in IBD
Cytokine production by 6B11+ and Vα24+ α-GalCer/CD1d tetramer+ iNK T-
cells was examined to particularly explore any defect in cytokine production in
ulcerative colitis. There was no significant change in IL-4, IL-10 or IL-13
producing 6B11+ iNK T-cells after 4 h in vitro anti-CD3 stimulation in subjects
with either ulcerative colitis or Crohn’s disease (Figure 6.16), in contrast to the
increase observed in normal subjects (as shown in Chapter 3).
Isolated lymphocytes were also stimulated with PMA:ionomycin to bypass any
potential deflect in receptor mediated signalling. The numbers of 6B11+ iNK
T-cells producing IL-4, IL-10 and IL-13 increased after 4 h in vitro
PMA:ionomycin stimulation for normal subjects, but there was no change in
subjects with ulcerative colitis or Crohn’s disease (Figure 6.16). Thus, the
deficiency in cytokine stimulation was not receptor mediated.
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Figure 6.15 Intracellular IL-4, IL-10, IL-13 and IFN-γ cytokine production by
blood 6B11+ iNK T-cells after 4 h in vitro anti-CD3 (♦) or PMA and
ionomycin (◊) stimulation in normal subjects and subjects with ulcerative
colitis and Crohn’s disease. Data are given as the change in number (graphed)
and percentage (Table) of cytokine producing 6B11+ iNK T-cells unstimulated
(-) or stimulated (+) for 4 h (n=number of subjects).
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Figure 6
IL-4, IL-10, IL-13 and IFN-γ cytokine production by Vα24+ α-GalCer/CD1d
tetramer+ iNK T-cells was examined after 4 h in vitro anti-CD3 stimulation in
subjects with ulcerative colitis and Crohn’s disease and compared to normal
control subjects. There was no significant change in the number of IL-4 or IL-
10 producing Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells for subjects with
Crohn’s disease or ulcerative colitis, unlike the changes observed in normal
control subjects (as shown in Chapter 3), nor was there any significant change
in those that produced IL-13 or IFN-γ (Figure 6.16).
The numbers of IL-4, IL-10, IL-13 and IFN-γ producing Vα24+ α-
GalCer/CD1d tetramer+ iNK T-cells increased after 4 h in vitro
PMA:ionomycin stimulation for normal subjects (Figure 6.16). Like 6B11+
iNK T-cells, there was no significant change in the number of cytokine
producing Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells after 4 h in vitro
PMA:ionomycin stimulation for subjects with ulcerative colitis or Crohn’s
disease. PMA and ionomycin stimulation showed persistent defective cytokine
production by iNK T-cells in both ulcerative colitis and Crohn’s disease.
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Figure 6.16 Intracellular IL-4, IL-10, IL-13 and IFN-γ cytokine production by
blood Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells after 4 h in vitro anti-
CD3 (♦) or PMA and ionomycin (◊) stimulation in normal subjects and
subjects with ulcerative colitis and Crohn’s disease. Data are given as the
change in number (graphed) and percentage (Table) of cytokine producing
Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells unstimulated (-) or stimulated
(+) for 4 h (n=number of subjects).
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6.4.11 Comparison of cytokine production from CD3+ T-cells in
IBD Cytokine production by conventional CD3+ T-cells was investigated in
ulcerative colitis and Crohn’s disease after 24 h in vitro anti-CD3 stimulation.
There was an increase of 1.7% and 1.3% in IL-4 and IFN-γ producing CD3+ T-
cells for normal subjects, equating to 2.4±0.7 x104 and 1.5±0.5 x104 cells per
ml, respectively (Figure 6.17). The defect in IL-4 cytokine production by
Vα24+ T-cells and iNK T-cells in subjects with ulcerative colitis and Crohn’s
disease was not present for classical CD3+ T-cells after 24 h in vitro anti-CD3
stimulation. Peripheral blood from subjects with both ulcerative colitis and
Crohn’s disease showed similar trends to normal subjects in IL-4 cytokine
production by CD3+ T-cells after 24 h in vitro anti-CD3 stimulation. IL-4
cytokine production by CD3+ T-cells increased by 3.3% and 1.9%, in subjects
with ulcerative colitis and Cohn’s disease, equating to an increase of 4.1±1.1
x104 and 1.9±0.9 x104 cells per ml, respectively (Figure 6.17). There was an
increase in the number of IFN-γ producing CD3+ T-cells in subjects with
ulcerative colitis, yet this was not observed in subjects with Crohn’s disease.
The number of IFN-γ producing CD3+ T-cells increased by 0.5% in ulcerative
colitis subjects, which equates to 0.5±0.3 x104 cells per ml (Figure 6.17).
These data show that CD3+ T-cells have no general cytokine production
deficiency, implying that the defective cytokine production by invariant NK T-
cells is very specific to that subset alone.
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Figure 6.17 Intracellular IL-4 and IFN-γ cytokine production by blood CD3+
T-cells after in vitro anti-CD3 stimulation for 24 h in normal subjects and
subjects with ulcerative colitis and Crohn’s disease. Data are given as the
mean±SEM change in intracellular cytokine producing CD3+ T-cells/ml x104
(n=number of subjects).
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Principal findings of this Chapter were:
• Crohn’s subjects have reduced numbers of circulating:
o CD56+, CD57+, CD94+ and CD161+ NK cells (total and bona
fide).
o CD161+ NK T-like cells.
o Vα24+ T-cells.
o Vα24+ CD4+ T-cells.
o Vα24+ CD161+ NK T-cells.
• Crohn’s and ulcerative colitis subjects have reduced numbers of
circulating:
o Vα24+ Vβ11+ T-cells.
o Vα24+ 6B11+ iNK T-cells.
o Vα24+ Vβ11+ α-GalCer/CD1d tetramer+ iNK T-cells.
• Intestinal Vα24+ T-cells were deficient in Crohn’s disease and
ulcerative colitis.
• Crohn’s subjects had a functional deficiency of IL-4 production, while
ulcerative colitis subjects had an increase in IFNγ production by
Vα24+ T-cells after 4 h in vitro CD3 stimulation.
• Crohn’s and ulcerative colitis subjects had impaired IL-4, IL-10, IL-13
and IFNγ production by 6B11+ and Vα24+ α-GalCer/CD1d tetramer+
iNK T-cells after 4 h in vitro anti-CD3 and PMA:ionomycin stimulation.
• The defective cytokine production by invariant NK T-cells is very
specific and was not present in conventional CD3+ T-cells
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6.5 DISCUSSION
The attention of this thesis had originally been drawn to deficiency of Vα14+
T-cells in animal models of autoimmunity and to a deficiency of Vα24+ T-cells
in human autoimmune diseases (Baxter et al., 1997; Illes et al., 2000; Maeda et
al., 1999; Sumida et al., 1995; Wilson et al., 1998). As ulcerative colitis and
Crohn’s disease share similar features of an autoimmune disease, the
hypothesis of this Chapter was that Vα24+ T-cells and iNK T-cells may be
deficient and have defective function in subjects with IBD.
Work presented in this thesis has shown a general decrease in the number of
circulating NK cells in ulcerative colitis and Crohn’s disease. This deficiency
of NK cells was solely due to the decreased number in circulating bona fide
(non T-cell) NK cells. The NK cell deficiency was unexpected and not
previously reported. Surprisingly this NK cell deficiency has not been
previously reported, but in part it is explained by the recent advances in
understanding the surface phenotype of NK cells, whereas older studies have
defined NK cells by their cytotoxic action in vitro. Previous studies have
shown either decreased or normal killer cell activity in Crohn’s disease
(Giacomelli et al., 1999; Okabe et al., 1985). The deficiency of NK cells could
have two possible functional consequences. First, NK cell deficiency could
predispose to viral infection and persistence of some viruses, although
evidence for this is controversial. Second, NK cells themselves could have an
immunoregulatory function that would be compromised in ulcerative colitis
and Crohn’s disease. This persistence may be due to loss of NK cells (Joyce et
al., 1998; Wakefield et al., 1995), NK T-like cells and/or iNK T-cells. The other
functions of NK T-cells include action against bacteria, protozoa, and tumours
(Ambrosino et al., 2008; Godfrey et al., 2000). Similarly, there is an increased
risk of colonic cancer in Crohn’s disease that could possibly be due to deficient
NK, NK T-like and/or iNK T-cells. However, this has yet to be formally
investigated.
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Unlike NK cells, circulating T-cells were not deficient in IBD, nor was there
any deficiency of cells expressing the TCR beta chains Vβ11+ or Vβ13+. Only
the Vα24+ T-cell subset were deficient, and only in Crohn’s disease with
circulating Vα24+ T-cells being reduced to 34% of the levels present in normal
control subjects. This reduction was independent of treatment and site of
disease (small or large bowel). Furthermore, the number of circulating Vα24+
T-cells was independent of disease activity. Mucosal Vα24+ T-cells and Vα24
mRNA expression was also reduced in Crohn’s disease and ulcerative colitis.
Circulating Vα24+ T-cells decreased in number with age in ulcerative colitis
subjects, similar to that observed in normal subjects, as shown in Chapter 4.
Even so, circulating Vα24+ T-cells remained consistently low with age in
Crohn’s subjects. Unlike in normal healthy controls, the decrease in circulating
Vα24 T-cells with age was greater in male ulcerative colitis subjects compared
to females. This decrease in circulating Vα24+ T-cells with age observed in
male subjects might offer and explanation for the even gender distribution,
which is approximately 1:1 in ulcerative colitis. This differs with most other
autoimmune disorders as they predominantly affect females and have ratios of
or greater than 2:1. These include rheumatoid arthritis (Ishizuka et al., 2004;
Krishnan, 2003; Voulgari et al., 2004), systemic lupus erythematosus (Lopez et
al., 2003) and scleroderma (Allcock et al., 2004; Tager and Tikly, 1999).
The SP subset of Vα24+ T-cells was deficient in Crohn’s disease, reduced to
43% of control subjects. In contrast, Vα24+ SP T-cells were intact in ulcerative
colitis. Vα24+ Vβ11+ T-cells (the surface phenotype of immunoregulatory
Vα24Jα18 NK T-cells) constituted about 13% and 20% of total Vα24+ T-cells
for ulcerative colitis and Crohn’s subjects, respectively. A preliminary report
reported that Vα24+ Vβ11+ T-cells were low systemically in Crohn’s disease
(n=15) and ulcerative colitis (n=9) (van der Vliet et al., 2001). The experiments
of this Chapter agree with these data, but the investigators did not report any
NK deficiency in Crohn’s disease or ulcerative colitis or CD161+ NK T-like
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and CD161+ Vα24+ NK T-cell deficiency in Crohn’s disease. They did not
report that subjects with ulcerative colitis had comparable numbers of
circulating Vα24+ T-cells and that it was only the Vα24+ Vβ11+ subset that
were deficient. Mucosal Vα24+ T-cells and cytokine function were not
investigated. More importantly, they did not further investigate and define iNK
T-cells by the co-expression of Vα24 and 6B11 or α-GalCer/CD1d tetramer.
Chen and colleagues (1997) have shown a loss of CD161 NK1.1 marker on
mouse Vα14+ T-cells that were expanded and stimulated for 4 days but this is
difficult to relate to physiology and pathology in vivo due to the extreme in
vitro stimulation conditions. One would also expect re-expression in vivo with
Crohn’s disease in remission and also increased rather than decreased IL-
4/IFN-γ basal cytokine expression. This Chapter has reported low numbers of
CD161+ NK cells in Crohn’s subjects regardless of activity. Hammond and
colleagues (Hammond et al., 2001) have shown that some strains of mice have
equivalent Vα14+ T-cells that do not express the NK1.1 marker, showing that
it is not essential for the immunoregulatory T-cell to express this NK marker.
Vα24+ iNK T-cells are defined functionally in vitro or in mice by CD1d
restriction and include a population that reside within Vα14+ T-cells in mice or
Vα24+ T-cells in man. A high proportion of Vα24Jα18+ T-cells (or
Vα14Jα18+ cells in mice) are detected by αGalCer/CD1d tetramers by flow
cytometry, confirming the antigenic specificity of this unique subset. The work
of this Chapter has further shown that Vα24+ 6B11+ and Vα24+ Vβ11+ α-
GalCer/CD1d tetramer+ iNK T-cells were reduced in both ulcerative colitis and
Crohn’s. The deficiency of Vα24+ 6B11+ and Vα24+ Vβ11+ αGalCer/CD1d
tetramer+ iNK T-cells in both ulcerative colitis and Crohn’s disease has not
previously been reported.
Invariant NK T-cells down-regulate immune responses in two ways. Firstly,
iNK T-cells produce immunosuppressive cytokines, secondly, they are
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cytotoxic to dendritic cells, which express CD1d (Nicol et al., 2000b). Lysis of
antigen presenting dendritic cells would abrogate continuing immunological
reactivity, presuming it mainly involves the pro-inflammatory subset. Vuckovic
et al. (2001) have shown that activated intestinal dendritic cells are increased
even at sites of apparently normal mucosa in subjects with Crohn’s disease.
This would at least be consistent with a loss of regulation of dendritic cells in
humans. The first mechanism was the one further investigated in this present
Chapter. Vα24+ T-cells and iNK T-cells promptly produce IL-4, IL-10, IL-13
and IFN-γ cytokines within hours of stimulation, unlike classical T-cells which
take longer to up-regulate cytokine production (Godfrey et al., 2000;
Yoshimoto and Paul, 1994). Studies have shown that Vα24+ (or mouse
Vα14+) NK T-cells have an extreme impairment of IL-4 production in type 1
diabetes mellitus (Mendiratta et al., 1997; Poulton and Baxter, 2001). This was
the basis of the investigation of IL-4 production in IBD. Initial work presented
in this Chapter showed that there was impaired IL-4 production from Vα24+ T-
cells after in vitro anti-CD3 antibody stimulation in Crohn’s disease but not in
ulcerative colitis. Beside an increase in IL-4 producing Vα24+ T-cells in
ulcerative colitis subjects, there was a significant increase in IFN-γ producing
Vα24+ T-cells after in vitro anti-CD3 antibody stimulation. This increase of
IFN-γ producing Vα24+ T-cells was not noted in normal control subjects or
subjects with Crohn’s disease. Increased production of IL-4 and IL-10 by
Vα24+ T-cells in normal subjects after anti-CD3 stimulation is representative
of a Th2 immune response, whereas increases in both IL-4 (a Th2 cytokine)
and IFN-γ (a Th1 cytokine) for ulcerative colitis subjects is representative of a
Th0 immune response. IL-4, IL-10, IL-13 and IFN-γ production by Vα24+ T-
cells was impaired in Crohn’s disease.
Cytokine production by 6B11+ and Vα24+ Vα24+ α-GalCer/CD1d tetramer+
cells was investigated, as they are a more specific marker of iNK T-cells. There
was a functional impairment of IL-4, IL-10 and IL-13 production from 6B11+
iNK T-cells after in vitro anti-CD3 antibody stimulation in subjects with
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Crohn’s disease and ulcerative colitis. Impaired cytokine production was
confirmed after in vitro anti-CD3 stimulation of Vα24+ α-GalCer/CD1d
tetramer+ iNK T-cells not only in Crohn’s disease but also in ulcerative colitis.
This defect in the function of both 6B11+ and Vα24+ α-GalCer/CD1d
tetramer+ iNK T-cells reflected the more selective iNK T-cell deficiency in
ulcerative colitis. No change in IL-13 and IFN-γ cytokine production by
Vα24+ α-GalCer/CD1d tetramer iNK T-cells were found in normal subjects.
This made it difficult to assess any deficiency in subjects with Crohn’s disease
or ulcerative colitis. Further investigations showed that CD1d specific iNK T-
cells failed to produce IL-4, IL-10, IL-13 or IFN-γ in both Crohn’s disease and
ulcerative colitis after in vitro anti-CD3 stimulation. It has been known for
some time that there is low expression of IL-4 in IBD, especially in ulcerative
colitis (Fuss et al., 2004). iNK T-cells are the major source of IL-4 in vivo
(Mendiratta et al., 1997; Yoshimoto and Paul, 1994). In spite of the absence of
IL-4, ulcerative colitis is thought to be a Th2 reaction. This work does not
explain the predominant neutrophil inflammation and the depletion of goblet
cells in crypts that characterize the histology of ulcerative colitis. A study by
Fuss et al. (2004) has proposed that another CD1d restricted non-iNK T-cell
population produces IL-13 that mediates an atypical Th2 response in ulcerative
colitis. This non-iNK T-cell population was not investigated in this Chapter.
Their study investigated a longer period of 48-72 h of stimulation. Kadivar et
al. (2004) have shown that IL-13 production was reduced in ulcerative colitis
patients, at least in intestinal organ cultures. IL-4 in particular prevents a Th1
response and maintains a Th2 immune response (Singh et al., 1999). It is likely
that other Th2 cytokines are also affected and indeed this Chapter has shown a
possible deficiency of IL-10 and IL-13. IL-4 should be only regarded as an
indicator rather than the definite mediator of any Th2 response. Production of
IFN-γ is only short lived and hence IL-4 continues a Th2 bias (Burdin et al.,
1999). The differentiation of naive Th cells towards Th1 or Th2 cells is
regulated by the transcription factors T-box expressed in T-cells (T-bet) and
GATA-binding protein-3 (GATA-3), respectively. Future work would involve
investigation of these transcription factors. The ratio of expression of T-bet and
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GATA-3 reflects changes in the Thl-specific cytokine IFN-γ and Th2-specific
cytokine IL-4. These transcription factors are up-regulated in cells that produce
type 1 and type 2 cytokines and provide a surrogate marker of the Th1/Th2
cytokine balance (Kiwamoto et al., 2006).
Unlike iNK T-cells; there was no impairment in IL-4 cytokine production by
CD3+ T-cells for ulcerative colitis or Crohn’s subjects. Peripheral blood from
both ulcerative colitis and Crohn’s subjects showed similar trends in IL-4
cytokine production by CD3+ T-cells compared to normal subjects (longer 24 h
incubation). Normal subjects as well as subjects with ulcerative colitis and
Crohn’s disease all displayed an increase in IL-4, a Th2 type immune cytokine.
Th2: Th1 (cells/ml) cytokine ratios were 1.6:1, 6.8:1 and 2.5:1 for normal,
ulcerative colitis and Crohn’s subjects, respectively. It was interesting that
CD3+ T-cells did not produce IFN-γ after in vitro anti-CD3 simulation in
Crohn’s disease. Despite this, Fuss et al. (1996) have shown that stimulation of
LP cells via the CD2 receptor does cause IFN-γ release.
The exact relationship between Vα24+ T-cell and iNK T-cell deficiency,
defective function and the bacterial antigens of ulcerative colitis and Crohn’s
disease or of autoantigens in autoimmune disease is still ill defined. It could be
that minor initiating factors (eg, gastrointestinal infection) cause tissue damage,
which is not appropriately down-regulated, due to deficiency of Vα24+ T-cells
and iNK T-cells in IBD. However, other genetic and environmental factors
presumably affect this outcome. Vα24Jα18+ T-cells in humans (or Vα14Jα18+
in mice) are uniquely CD1d restricted and recognize the marine-derived
glycolipid, α-galactosylceramide, as the antigenic determinant of the CD3
receptor. Intestinal epithelial and dendritic cells express CD1d. The α-
galactosylceramide glycolipid was originally isolated from marine sponges and
is presumed to mimic similar glycolipid(s) in vivo because α-
galactosylceramide is not present in mammals (Kawano et al., 1997).
Glycosylphosphatidylinositol was originally suggested as one natural ligand
(Joyce et al., 1998), although a recently identified lysosomal
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glycosphingolipid, isoglobotrihexosylceramide (Zhou et al., 2004) has been
identified. Isoglobotrihexosylceramide, like exogenous marine derived α-
GalCer, is presented by the MHC class I-like CD1d protein (Zhou et al., 2004).
Blumberg (2001) suggests that luminal glycolipid antigens themselves may
either directly stimulate the CD1d molecules on IELs or indirectly through the
presentation of glycolipids to CD1d restricted Vα24+ T-cells. While
glycolipids from bacteria might be thought to be natural ligands of CD1d, this
has not been shown. Only CD1a, b, c (but not CD1d) are known to restrict T-
cells-for example, in mycobacteria infection. It could be that CD1d binds self
intracellular glycolipids that are released during cellular damage and display
these on their surface to down-regulate immune reactivity (Godfrey et al.,
2000).
In summary, the work presented in this Chapter has identified NK cell, Vα24+
T-cell and iNK T-cell deficiency in IBD. This Vα24+ T-cell deficiency was
associated with a CD161+ NK cell deficiency in Crohn’s disease, although this
cannot entirely exclude down-regulation of CD161+ expression. The
deficiency of Vα24+ iNK T-cells is a permissive and absolute requirement for
development of IBD but are not a sufficient condition. Presumably, other
genetic and environmental factors direct the defect into a particular disease
state, such as ulcerative colitis or Crohn’s disease. In ulcerative colitis, there is
a similar number of circulating Vα24+ T-cells but a selective deficiency of
Vα24+ Vβ11+ T-cells and subsiquently iNK T-cells as defined by being
Vα24+ 6B11+ and Vα24+ Vβ11+ α-GalCer/CD1d tetramer+. Vα24+ T-cells
and iNK T-cells are incapable in producing cytokines, which further
compounds the defect in IBD. It is interesting that stimulation of iNK T-cells
by α-galactosylceramide protects mice against experimental colitis
(Saubermann et al., 2000). Equally, the adoptive transfer of α-
galactosylceramide protects mice against experimental colitis (Saubermann et
al., 2000); the same may be possible in humans with ulcerative colitis or
Crohn’s disease, if it can be shown that the defect in cytokine production can
be overcome.
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7 CHAPTER 7 GENERAL CONCLUSIONS
Chapter 7: General conclusions Randall Grose
198
As previously mentioned, the attention of this thesis had originally been drawn
to deficiency of Vα14+ T-cells in animal models of autoimmunity and Vα24+
T-cell deficiency in human autoimmune diseases. When commencing my PhD
candidature, the exact NK T-cell phenotype was not fully defined, especially in
humans. Most work had been done in the mouse. The definition of NK T-cells
changed during my candidature. Chapter 3 of this thesis has confirmed that
Vα24+ T-cells constituted a small proportion of total NK T-like cells, unlike
mouse Vα14 which represents the majority of murine NK1.1+ αβTCR+ cells.
Vα24+ T-cells constitutes only 0.35% of circulating lymphocytes, compared to
NK T-like cells that represent around 4-11% in humans.
Like others (DelaRosa et al., 2002; Jing et al., 2007a; Peralbo et al., 2006), this
work has shown a direct correlation between the number of circulating Vα24+
T-cells and iNK T-cells and age. However, they did not demonstrate that this
decrease was gender specific. The decrease of circulating Vα24+ T-cells with
age was greater in female compared to males. This, in part, may offer an
explanation for the higher female incidence found in many of the autoimmune
diseases, such as rheumatoid arthritis (Ishizuka et al., 2004; Krishnan, 2003;
Voulgari et al., 2004), systemic lupus erythematosus (Lopez et al., 2003) and
scleroderma (Allcock et al., 2004; Tager and Tikly, 1999). The number of
circulating Vα24+ SP subset was unaffected by age. This has since been
confirmed by Jing et al., (2007). TCR Vβ11 is predominantly found paired
with Vα24 α-chain it is not surprising to discover that Vα24+ Vβ11+
decreased with age. Chapter 4 shows for the first time a decrease in the number
of circulating iNK T-cells (defined as Vα24+ 6B11+ and Vα24+ Vβ11+ α-
GalCer/CD1d tetramer+) with age, consistent with that seen with total Vα24+
and Vα24+ Vβ11+ T-cells.
The work of this thesis has confirmed that Vα24+ T-cells and iNK T-cells are
potent and rapid producers of cytokines. Vα24+ T-cells and iNK T-cells are
capable of promoting both Th1-like (IFN-γ) and Th2-like (IL-4, IL-10, and IL-
Chapter 7: General conclusions Randall Grose
199
13) immune responses. Recent work by Michel et al., (2007) and Niemeyer et
al. (2008) has even suggested that they can also produce IL-17. These cells are
therefore crucial at the onset of an immune response, being either be beneficial
or harmful, depending upon whether they polarize the immune response
towards a Th1, Th2 or Th17 direction. Even so, Vα24+ T-cell and iNK T-cells
do not escape the ageing process as their function was also affected by age. IL-
4 production by Vα24+ T-cells, 6B11+ and Vα24+ α-GalCer/CD1d tetramer+
iNK T-cells was significantly affected with age. Altered cytokine production by
these cells in the elderly might lead to an increased risk of opportunistic
infections, certain autoimmune diseases and cancers.
This work has identified a subpopulation of otherwise normal individuals
whom have normal numbers of circulating Vα24+ T-cells, reduced numbers of
circulating Vα24+ Vβ11+ T-cells and consequently Vα24+ Vβ11+ α-
GalCer/CD1d tetramer+ iNK T-cells. These individuals displayed a normal
phenotype, were not diagnosed with any autoimmune disorder but had one or
more autoimmune antibodies. They have reduced numbers of circulating α-
GalCer/CD1d tetramer+, Vα24+/- α-GalCer/CD1d tetramer+ and Vα24+
Vβ11+ α-GalCer/CD1d tetramer iNK T-cells. It is still unclear whether these
individuals will develop an autoimmune disorder. It would be interesting to
follow these subjects over time. α-GalCer/CD1d tetramer staining could be
utilized as a general early autoimmune marker, often detecting potential
sufferers before symptoms arise. Such a marker would make early treatment
intervention possible.
This is the first study to acknowledge a significant difference in the number of
circulating CD161+ NK cells when comparing breast- and formula-fed 2-
month old infants. There was a significant reduction in the number of
circulating CD161+ NK cells in 2-month-old infants that were exclusively
breast-fed compared to exclusively formula-fed infants. An initial hypothesis of
this Chapter 4 was that breast-feeding of infants increases the number of
circulating Vα24+ and Vα24+ CD4+ immunoregulatory T-cells, which in turn
Chapter 7: General conclusions Randall Grose
200
provide specific immunity and maturation of the infant’s immune system. This
was not so as there was no difference in the number of circulating Vα24+ or
Vα24+ CD4+ T-cells when comparing breast-fed and formula-fed 2-month-old
infants. Nevertheless, it was interesting to find an infant with reduced numbers
of circulating Vα24+ T-cells suggesting that a deficiency of Vα24+ T-cells and
iNK T-cells can occur early in life, possibly during thymus development. The
work presented here has identified a deficiency of Vα24+ T-cells and
ultimately iNK T-cells in apparently healthy control subjects as early as 2
months of age, suggesting that deficiency of iNK T-cells occurs early in life.
Future studies arising from this work should investigate Vα24+ iNK T-cells in
neonates as well as infants pre- and post-weaning that have been exclusively
breast- or formula-fed over several time points, as opposed to just at 2-months
of age. Ongoing work would need to investigate any particular infant or infants
that have low level of circulating iNK T-cells. It is most likely that these infants
would have a predisposition to one or various autoimmune disorders, however
other genetic factors are more than likely involved.
Invariant NK T-cells are deficient in models of animal and human autoimmune
disease. This work has also shown them to be deficient in coeliac disease,
Crohn’s disease and ulcerative colitis. Coeliac disease, Crohn’s disease and
ulcerative colitis share common characteristics of autoimmune disorders. It has
been recently shown that autoimmune disease increases with the duration of
coeliac disease from 5.1% at diagnosis of less than 2 years to 34% at diagnosis
at greater than 20 years (Ventura et al., 1999). Ventura et al. (1999) found that
the prevalence of autoimmune disease in all coeliac subjects was 14%
compared to 3% in normal subjects. Patients with IBD also have an increased
risk of having an autoimmune disorder (Boyles, 2005). This raises the
possibility that coeliac disease, Crohn’s disease, ulcerative colitis and
autoimmune disease share a common disease pathway (i.e., genetic
predisposition, Vα24+ iNK T-cell deficiency). For instance gluten exposure in
genetically susceptible individuals could develop into coeliac disease. Equally,
excess endogenous bacterial antigens may predispose genetically susceptible
Chapter 7: General conclusions Randall Grose
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individuals to IBD. A possibility might be that gluten exposure and/or excess
bacterial antigens causes progressive Vα24+ T-cell and iNK T-cell deficiency,
however this was not evident. Vα24+ T-cells and iNK T-cells did not decline
with age in subjects with coeliac disease or Crohn’s disease, though they did
decrease in normal subjects and subject with ulcerative colitis. There was no
significant difference in numbers of circulating Vα24+ T-cells or iNK T-cells
with respect to diet, disease state or location. Thus, Vα24+ T-cell and iNK T-
cell deficiency was present at the time of diagnosis and thus likely contributed
to the pathogenesis rather than be caused by these disorders. Vα24+ T-cell and
iNK T-cell deficiency was not confined to the circulating lymphocytes but also
observed at the site of the disorder.
As well as being deficient in coeliac disease, Crohn’s disease and ulcerative
colitis, this work has shown that Vα24+ T-cells and iNK T-cells were
functionally defective in cytokine production after in vitro stimulation. This
defective function of iNK T-cells reflected the more selective iNK T-cell
deficiency in ulcerative colitis.
Future work
There are several lines of research arising from this work which should be
pursued, other than the future direction already stated within this thesis. Future
work would involve using HLA DQ2/DQ8 matched controls when comparing
normal and coeliac subjects. We will be recruiting more subjects with a wider
spread of ages with respect to age related changes in iNK T-cell number and
function. We also plan to investigate whether there is a genetic basis for the
deficiency of iNK T-cells in coeliac disease or IBD. First degree relatives will
be investigated. If there is deficiency of iNK T-cells in first degree relatives
this may indicate an independent genetic influence.
We wish to further examine and define iNK T-cells within the intestine of
subjects with coeliac disease, Crohn’s disease and ulcerative colitis. We have
Chapter 7: General conclusions Randall Grose
202
previously tried to stain with α-GalCer/CD1d tetramer and were not successful.
iNK T-cells can be identified by 6B11 staining. 6B11 staining has been
previously used to identify iNK T-cells in bronchial asthma. Future work will
also examine Vα19 mNK T-cells within the intestine of subjects with coeliac
disease, Crohn’s disease and ulcerative colitis. Other regulatory T cells,
including Treg cells (Foxp3+ CD4+ CD25+), Tr1 cells (CD4+ IL-10+) and the
Th17 population will be investigated.
A second line of research, is to investigate whether iNK T-cells are affected by
intestinal CD1d expression in subjects with coeliac disease, Crohn’s disease
and ulcerative colitis. CD1d has a rather restricted tissue distribution and is
though to be very abundant within the intestinal tract. The abundance of
CD1d+ cells in the normal human intestine suggests a possible role for these
cells in the gastrointestinal tract. Initial work by Page et al. (2000) showed that
CD1d expression was increased in the intestine of Crohn’s and ulcerative
colitis subjects. However, a recent report suggested that epithelium of Crohn’s
and ulcerative colitis subjects do not express CD1d (Perera et al., 2007).
Differences in CD1d expression within the intestine of Crohn’s and ulcerative
colitis subjects compared to normal subjects suggests possible NKT cell
involvement in IBD. CD1d expression will be examined in coeliac disease and
IBD.
Finally, we will further investigate the function of iNK T-cells in coeliac
disease and IBD. The work of this thesis has already shown a defect in mitogen
stimulated cytokine production. We will investigate the regulatory function of
iNK T-cells in a mixed lymphocyte culture. We plan to investigate iNK T-cells
with an in vitro suppressor cell assay. We will adopt the suppressor cell assay
described for CD4 CD25 Foxp3 cells or with CD4+ IL-10+ (Tr1) in which a
suppressor cell population is co-cultured with an effector cell population.
Chapter 7: General conclusions Randall Grose
203
The work presented in this thesis has demonstrated a link between coeliac
disease, Crohn’s disease, ulcerative colitis and various autoimmune disorders
through a common deficiency of Vα24+ T-cells. The exact relationship
between Vα24+ T-cell and iNK T-cell deficiency and their defective function
and gluten in coeliac disease is still ill defined. Equally, the relationship
between excessive endogenous bacterial antigens in IBD and autoantibodies in
autoimmunity needs further investigation. It could be that minor initiating
factors like gastrointestinal infection or inflammation that cause tissue damage,
which is not appropriately down regulated, due to deficiency of Vα24+ T-cells
and iNK T-cells. Other genetic and environmental factors presumably affect
this outcome. Genetic and environmental factors may direct the defect into a
particular disease state.
There is potential for α-GalCer, the endogenous glycolipid or analog to
promote a required immune response and therefore be used as a therapeutic
agent. To date most intervention work has been using animal models of
autoimmune disease. α-GalCer administration has been successful in the
treatment of various mouse models including type 1 diabetes, rheumatoid
arthritis, systemic lupus erythematosus, atherosclerosis, experimental
autoimmune encephalomyelitis and IBD (Naumov et al., 2001; Saubermann et
al., 2000; Wang et al., 2001; Yang et al., 2003; Zeng et al., 2003). NK T-cells
exert a protective effect in the TNBS and DSS induced colitis model.
Stimulation of iNK T-cells by α-galactosylceramide has a protective effect and
the adoptive transfet of NK T-cells reduces inflammation in the DSS model of
colitis (Saubermann et al., 2000). Likewise, the adoptive transfer of ex vivo
colitis-extracted protein-pulsed NKT cells reduced inflammation in the TNBS-
induced model (Shibolet et al., 2004). The same may be possible in humans
with coeliac disease, Crohn’s disease, ulcerative colitis and autoimmune
disorders. Saubermann et al., (2000) also showed that CD1d knock out mice
did not benefit from α-galactosylceramide treatment nor did mice with
depleted levels of NK T-cells. Taken together, suggests that NK T-cells may be
a target for future therapies.
Chapter 7: General conclusions Randall Grose
204
The affect iNK T-cell manipulation has on human autoimmune disorders and
inflammatory diseases remains unknown. Further investigations are warranted
in these diseases such as repleating iNKT-cells from normal subjects in an in
vitro suppression cell assay using mixed lymphocyte culture. The manipulation
of iNK T-cells for the therapeutic intervention of coeliac disease, ulcerative
colitis, Crohn’s disease, type 1 diabetes, rheumatoid arthritis, as well as other
autoimmune and inflammatory disorders may be possible, however more
extensive work is required.
Appendix Randall Grose
205
8 APPENDIX
Appendix Randall Grose
206
8.1 APPENDIX 1: Publications arising from this thesis
8.1.1 R H. Grose, A G. Cummins, and F M. Thompson. Deficiency
of invariant NK T-cells in coeliac disease. Gut, 2007; 56:
790-795.
Appendix Randall Grose
207
A Grose, R.H., Cummins, A.G. & Thompson, F.M. (2007) Deficiency of invariant NK T-cells in coeliac disease. Gut, v. 56, pp. 790-795
A NOTE:
This publication is included on pages 207-209 in the print copy of the thesis held in the University of Adelaide Library.
A It is also available online to authorised users at:
A http://dx.doi.org/10.1136/gut.2006.095307
A
Appendix Randall Grose
210
8.1.2 R H. Grose, F M. Thompson, A G. Baxter, D G. Pellicci and
A G. Cummins. Deficiency of invariant NK T-cells in
Crohn’s disease and ulcerative colitis. Dig Dis Sci, 2007; 52:
1415-1422.
Appendix Randall Grose
211
A Grose, R.H., Thompson, F.M., Baxter, A.G., Pellicci, D.G. & Cummins, A.G. (2007) Deficiency of invariant NK T-cells in Crohn’s disease and ulcerative colitis. Digestive Diseases and Sciences, v. 52 (6), pp. 1415-1422
A NOTE:
This publication is included on pages 211-214 in the print copy of the thesis held in the University of Adelaide Library.
A It is also available online to authorised users at:
A http://dx.doi.org/10.1007/s10620-006-9261-7
A
Appendix Randall Grose
215
8.1.3 R H. Grose, A G. Cummins, and F M. Thompson. Deficiency
of 6B11+ invariant NK T-cells in celiac disease (accepted for
publication in Digestive Diseases and Sciences, 2007).
Appendix Randall Grose
�
� ��6�
��
A Grose, R.H., Cummins, A.G. & Thompson, F.M. (2007) Deficiency of 6B11+ Invariant NK T-cells in celiac disease. Digestive Diseases and Sciences, v. 53 (7), pp. 1846-1851
A NOTE:
This publication is included on pages 216-218 in the print copy of the thesis held in the University of Adelaide Library.
A It is also available online to authorised users at:
A http://dx.doi.org/10.1007/s10620-007-0093-x
A
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