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Angela Bikker

Wednesday 22 August 2012

Processing of MHC Class II in dendritic cells

‘Interleukin-7 and its receptor in T cell and B cell-driven immunity in primary Sjögren’s syndrome’

Promotor: Prof.dr FPJG Lafeber
Defence: 13 September 2012

The importance of IL-7 and its receptor has been investigated in several (rheumatic) autoimmune diseases, most extensively in RA. Based on growing evidence, it is believed that activation of the IL-7/IL-7R pathway leads to a cascade of immune-enhancing and proinflammatory responses both in vivo and in vitro in relation to these autoimmune diseases. In this thesis, we set out to identify a crucial role for IL-7 and its receptor in the immunopathology of primary Sjögren’s Syndrome. Summarized below are the main findings reported in this thesis.

Chapter 2: Increased expression of interleukin-7 in labial salivary glands of patients with primary Sjögren’s syndrome correlates with increased immunopathology.

• IL-7 producing cells in LSG of pSS patients as compared to nSS-sicca patients are increased.
• The main source of IL-7 in the LSG of pSS patients are cells with a fibroblast-like morphology and endothelial cells.
• Salivary IL-7 levels are increased in pSS patients compared with healthy controls, while serum IL-7 levels are comparable.
• Glandular IL-7 levels correlate with local (LFS, and % IgA+ plasma cells) and systemic parameters of disease.
• In vitro stimulation of PBMCs from pSS patients with IL-7 significantly induced several important pro-inflammatory (Th1/Th17 predominance over Th2) cytokines and chemokines.

Chapter 3: Increased IL-7Rα expression in salivary glands of patients with primary Sjögren’s Syndrome is restricted to T cells and correlates with IL-7 expression as well as lymphocyte numbers and activity.

• IL-7Rα+ cells are increased in the LSG of pSS patients compared to nSS-sicca patients.
• Glandular IL-7Rα expression correlates with several immune cells present in the salivary gland (CD3, CD20, CD1a, CD208, CD209 DCs), proliferating cells as well as IL-7 expression and local and peripheral parameters of disease.
• Flow cytometry analysis of LSG biopsy specimen confirmed the increased IL-7Rα expression and identified CD4 and CD8 T cells as the main cell types expressing the IL-7R.
• Relative expression of IL-7Rα+ CD4 T cells was significantly lower in pSS patients as compared to nSS-sicca patients, reflecting a significant increased percentage of IL-7Rα-CD25+ CD4 T cells, similar to the expression found in the peripheral blood of pSS patients compared to HCs.

Chapter 4: IL-7 drives Th1 and Th17 cytokine production in patients with primary Sjögren’s Syndrome despite an increase in CD4 T cells lacking the IL-7Rα.

• IL-7Rα+ CD4 T cells are highly proliferating cells which can be suppressed by IL-7Rα-CD25+ CD4 T cells.
• Anergic/suppressive FoxP3-expressing IL-7RαCD25+ and IL-7RαCD25- CD4  cells are increased in pSS patients.
• IL-7 strongly induces Th1/Th17 activity in pSS patients despite an increased number of CD25IL-7RαFoxP3high Tregs.

Chapter 5: IL-7 correlates with an increased number of activated lymphocytes in the salivary gland and induces T cell-dependent B cell activation in patients with primary Sjögren’s syndrome.

• Proliferating cells are increased in the LSG of pSS patients compared with nSS-sicca patients and significantly correlate with the IL-7 expression.
• IL-7 induces both CD4 T cell and CD19 B cell activation and proliferation in PBMCs isolated from pSS patients, despite the lack of the surface expression of IL-7Rα on B cells.
• Isolated B cells show no response to IL-7, however in a CD4 T/CD19 B cell co-culture, IL-7-induced CD4 T cell-dependent B cell activation is observed.

Chapter 6: Inhibition of the IL-7R pathway selectively inhibits TLR7-induced B cell activation in primary Sjögren’s Syndrome.

• Stimulation through TLR7 induces a profound lymphocytic proliferation in CD4 T/CD19 B cell co-cultures, however only of B cells and not of CD4 T cells, and this seems to be less pronounced in pSS patients.
• Monocytes/macrophages significantly enhance this effect on B cells when added to culture.
• In isolated B cells TLR7 activation leads to the intracellular expression of IL-7 and IL-7Rα.
• Upon inhibition of the IL-7/IL-7R pathway by shuIL-7Rα and a fully human anti-IL-7 antibody a significant reduction in TLR7-induced B cell activation is observed.

Chapter 7: Synergistic B cell and T cell activation by interleukin-7 and Toll-like receptor 7 is enhanced by myeloid cells.

• IL-7 cooperates with TLR7 activation to induce a synergistic proliferation and activation of CD4 T and CD19 B cells in co-culture.
• When monocytes/macrophages are introduced into the co-cultures, the synergistic B cell proliferation and activation is amplified.
• Furthermore, for the synergistic IL-7/TLR7-induced production of immunoglobulins (IgG, IgM, free-light chains) the presence of monocytes/macrophages in a CD4 T/CD19 B cell co-culture is required.
• A synergistic induction of Th1 and Th17-related cytokines and B cell activating cytokine IL-10 is seen upon IL-7 and TLR7 stimulation, again only in the presence of monocytes/macrophages.

Chapter 8: Clinical efficacy of leflunomide in primary Sjögren’s syndrome is associated with regulation of T cell activation and up regulation of IL-7 receptor α expression.

• In clinical responders to leflunomide a significant decrease of IFNγ and TNFα production is observed.
• Significant correlations between increased sialometry values and decreased IFNγ and TNFα production are found.
• Leflunomide reduces levels of inflammatory serum cytokines and CD40L expression, whereas it up-regulates IL-7Rα expression on CD4 T cells with persistent serum IL-7 concentrations.