Bentum, R. van (Renée) (2016) The effects of blocking Kv1.3 potassium channels by ShK-186 on dysfunctional regulatory T cells in granulomatosis with polyangiitis in vitro. thesis, Medicine.
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Abstract
Introduction – Granulomatosis with polyangiitis (GPA) is characterized by the presence of anti-neutrophil cytoplasmic autoantibodies (ANCA). However, several observations support the involvement of T cells and especially effector memory T cells (TEM cells) in the pathogenesis as well. Comprising the T cell lineage are also regulatory T cells (Tregs), a T cell subset that has been shown to have impaired suppressive function in GPA patients. It is hypothesized that this dysfunction might be caused by their capacity to differentiate into IL-17-producing Tregs in the context of a pro-inflammatory cytokine environment. Tregs are characterized by the expression of the transcription factor FoxP3 of which at least one isoform exists that lacks the second exon ((FoxP3Δ2). Previous research suggests that FoxP3Δ2 Tregs produce higher levels of IL-17 and that the proportion of these cells is increased in GPA patients. Recently, a new immune modulating therapy has been developed directed at blocking Kv1.3 potassium channels to inhibit activation of (pathogenic) TEM cells and their IL-17 production. No data is available on the effects of Kv1.3 blockade on IL-17 production of Tregs and their suppressive function. It has been shown that deletion of the Kv1.3 gene biases CD4+ T cells towards an immunoregulatory phenotype. Therefore, this study investigated the effects of inhibiting Kv1.3-channels on the phenotype and suppressive function of Tregs in GPA patients. Materials and methods – Phenotyping of Tregs was performed on whole blood samples of 8 GPA patients in remission, 1 active GPA patient and 10 healthy controls. Samples were stimulated with and without the Kv1.3-channel blocker ShK-186 and phenotypes were determined using flowcytometry. Samples were stained for surface markers (CD4, CD8, CD45RO) and intracellularly for IL-17, and the transcription factors (FoxP3, FoxP3exon2). Additionally, isolated peripheral blood mononuclear cells (PBMCs) were cultured in a Th17-polarizing environment containing IL-6 and IL-23 and analysed at day 1, 4 and 6. To study the suppressive capacity, sorted Tregs and responder T cells (Tresp) were co-cultured in different ratios with various stimulation techniques. Results – IL-17 production of memory CD4+ T cells in GPA patients was significantly reduced after incubation with ShK-186 and normalized to the level of healthy controls after stimulation. Differences in phenotype of Tregs were found between healthy controls and GPA-patients in remission, with significantly less full-length FoxP3 and higher amounts of FoxP3Δ2, albeit statistically not significant. FoxP3Δ2 Tregs were significantly more prone to IL-17 production, that could be effectively reduced after incubation with ShK-186. ShK-186 also diminished IL-17 production in PBMCs cultured in Th17 promoting conditions. No functional assay could be established to test the suppressive capacity of Tregs. Conclusion – ShK-186 effectively reduced IL-17 production in both total memory CD4+ T cells as well as memory Tregs from GPA patients in vitro, in which IL-17 production was preferentially found in memory Tregs expressing FoxP3Δ2.
| Item Type: | Thesis (Thesis) |
|---|---|
| Supervisor name: | Heeringa PhD. Prof. PhD. P. and Abdulahad W.H. MSc. and Lintermans, L.L. and Department: Pathology and Medical Biology, section Medical B |
| Faculty: | Medical Sciences |
| Date Deposited: | 25 Jun 2020 11:02 |
| Last Modified: | 25 Jun 2020 11:02 |
| URI: | https://umcg.studenttheses.ub.rug.nl/id/eprint/2199 |
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