Impairment of CD4+CD25+ regulatory T-cells in autoimmune liver disease
Introduction
Mechanisms underlying the breakdown of self-tolerance and consequent development of autoimmune disease are unclear, though deficient immune-regulation is likely to be involved. Among T-cells with regulatory function, such as NKT, Th3, Tr1 and CD8+CD28− T-cells, CD4+ lymphocytes constitutively expressing the interleukin-2-receptor (IL-2R) α-chain (CD25) are central to immune-regulation, preventing the activation of autoreactive T-cells [1]. This recently characterised immunoregulatory subpopulation is able to suppress proliferation and effector function of CD4 and CD8 autoreactive T-cells by a T-cell contact mechanism [2], [3] and possibly through the release of inhibitory cytokines [4]. Once stimulated specifically through their T-cell receptor, the CD4+CD25+ T-cell regulatory activity is antigen non-specific [5].
A number of surface markers characterise CD25 T-cells with suppressor function, including CTLA4 [6], glucocorticoid-induced tumour necrosis factor receptor [6], forkhead transcription factor Foxp3 [7] and CD62L [8], [9]. The latter has been recently demonstrated to define T-regs with strong suppressor function amongst T-cells co-expressing CD4 and CD25.
Removal of these T-cells leads to the development of spontaneous autoimmune diseases, such as autoimmune thyroiditis, gastritis and insulin dependent diabetes in experimental animals [10]. CD4+CD25+ T-cells are also present in man, where they represent about 5–10% of peripheral CD4 T-cells and exhibit the same functional characteristics as in animals [11], [12], [13], [14].
In human autoimmune pathology, CD4+CD25+ immunoregulatory T-cells (T-regs) have only recently become a focus of investigation. No study has addressed their role in autoimmune liver disease. Autoimmune hepatitis is an inflammatory disorder, characterised by presence of circulating autoantibodies, hypergammaglobulinaemia, histological evidence of interface hepatitis and response to immunosuppressive treatment [15], [16]. There are two types of autoimmune hepatitis [17]: one positive for antinuclear (ANA) and/or anti-smooth muscle (SMA) antibodies, the other for liver kidney microsomal antibody type-1 (LKM1) [18], [19]. An ANA/SMA positive overlap syndrome between autoimmune hepatitis and sclerosing cholangitis is also observed in young patients [20]. In all three forms of autoimmune liver disease circulating antibodies to soluble liver antigen (anti-SLA) may be present, usually in association with more severe disease [21].
The aims of the current study were to: (1) measure the percentage of CD4+CD25+ T-cells in patients with autoimmune liver disease at presentation and during immunosuppression-induced remission; (2) correlate their frequency with disease activity; (3) investigate their ‘ex vivo’ ability to expand; and (4) investigate their suppressor function by testing their ability to inhibit IFNγ production by CD4+CD25− T-cells.
Section snippets
Patients
Forty-one patients with autoimmune liver disease were studied: 30 ANA/SMA and 11 LKM1+. All had histological features of interface hepatitis; retrograde cholangiography, performed in all, showed bile duct changes characteristic of sclerosing cholangitis in 8 ANA/SMA+, who were diagnosed as having overlap syndrome. Thirty patients were female (20 ANA/SMA positive, 10 LKM1 positive). Median age was 11.6 years, ranging from 2.4 to 26.2 years. Thirty patients were studied at diagnosis and 28 during
Characterisation of CD4+CD25+ T-cells
After purification, the ability of CD4+CD25+ T-cells to suppress CD4+CD25− T-cell proliferation was tested in 3 patients with ANA/SMA+AIH and in three normal subjects. After addition of CD4+CD25+ T-cells at the ratios of 1/16 and 1/8, proliferation of CD4+CD25− T-cells was reduced by 16 and 27% in patients and by 18 and 44% in controls (means of the three subjects studied in each group). CD4+CD25+ T-cells expressed CD62L more frequently than CD4+CD25− T-cells both in patients (68.3%±1.5 vs.
Discussion
Patients with autoimmune liver disease have a reduced number of CD4+CD25+ T-cells at diagnosis and their number increases during remission on immunosuppression, though it never reaches normal values. Having demonstrated that these cells suppress the proliferation of CD4+CD25− T-cells and express the surface marker CD62L, possessing therefore the characteristics of regulatory T-cells, we went on to investigate their functionally more relevant ability to suppress production of IFNγ, a Th1
Acknowledgements
Dr Maria Serena Longhi is supported by the Alex Mowat PhD studentship, King's College Medical Research Trust, London. Dr Yun Ma was a Royal Society Dorothy Hodgkin Fellow when this project was initiated. Dr Dimitrios P. Bogdanos is supported by the Children's Liver Disease Foundation (CLDF), Birmingham, UK and Professor Giorgina Mieli-Vergani is supported by CLDF and WellChild, Cheltenham, UK.
We would like to thank Drs Ragai R. Mitry and Edward T. Davies, King's College Hospital, for their help
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Contributed equally.