NK, T and NK T -cells in ageing, coeliac disease and ......NK cells in untreated and treated coeliac...

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

120

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



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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


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



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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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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)


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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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.

Chapter 6: NK cells, T-cells and NK T-cells in IBD Randall Grose

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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


165

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



166

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


Subjects with ulcerative colitis had comparable numbers of circulating Vα24+

T-cells when compared to normal control subjects. In contrast, the number of


167

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



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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.


169

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


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).


170

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+


172

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


173

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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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



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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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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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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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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



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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


193

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


194

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


195

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


196

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.

Chapter 7: General conclusions Randall Grose

197

7 CHAPTER 7 GENERAL CONCLUSIONS


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-


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


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


201

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


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.


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.


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


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.


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


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.


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:



A http://dx.doi.org/10.1007/s10620-006-9261-7

A


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).


�

� ��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:



A http://dx.doi.org/10.1007/s10620-007-0093-x

A

Chapter 8: References Randall Grose

219

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