Regulation of immunity in Multiple Sclerosis CD4+ T cells and the influence of natalizumab
Abstract: Multiple sclerosis (MS) is an autoimmune disease targeting the central nervous system (CNS) and the most common neurological cause of disability in young adults. In most cases, the disease course is characterised by the cycling of relapses and remissions, so called relapsing-remitting MS (RR-MS). Although extensively studied, the underlying mechanisms are not fully elucidated, yet CD4+ T cells have been shown to be of importance in disease pathology. A range of treatments are available; the most effective to date being natalizumab, a monoclonal antibody directed against the adhesion molecule VLA-4 on the lymphocyte surface, thereby preventing entry into the CNS.The aim of this thesis was to assess the nature of lymphocyte populations in MS. This was achieved by studying CD4+ T helper cells (TH) and regulatory T cells (TREG) in peripheral blood. In addition, the influence of natalizumab was also investigated, both regarding the effect of the drug on the composition of the peripheral lymphocyte compartment as well as its effects on CD4+ T cells in vitro.We showed an imbalance in the mRNA expression of CD4+ T helper cell lineage specific transcription factors in peripheral blood. While TH1 and TH17 associated TBX21 and RORC expression was comparable in MS and healthy individuals, the TH2 and TREG associated GATA3 and FOXP3 expression was decreased in RR-MS. Given the reciprocally inhibitory nature of TH subsets, this might imply not only diminished function of TH2 and TREG cells but also a permissive state of harmful TH1 and TH17 cells. The size of the peripheral TREG population was unaltered in RR-MS. When analysed in detail, activated and resting TREG were distinguished, showing clear differences in FOXP3 and CD39 expression. Furthermore, when investigating these subpopulations functionally, the ability of activated TREG to suppress proliferation of responder T cells was found to be decreased in RR-MS patients compared to controls. To further investigate this defect, the global gene expression of TREG was compared between patients and controls. Gene set enrichment analysis revealed an enrichment (over-expression) of chemokine receptor signalling genes in RR-MS TREG, possibly suggesting a role for chemokines in TREG function.A sizable effect of natalizumab treatment was seen in the composition of peripheral lymphocyte populations after one year of treatment. While the number of lymphocytes increased over all, the largest increase was seen in the NK and B cell compartments. Furthermore, T cells from patients with MS displayed decreased responsiveness towards antigens and mitogens in vitro. Natalizumab treatment was able to normalise the responsiveness in blood, an effect not solely dependent on the increased number of cells.The importance of CD4+ T cells in human disease, including MS, was shown by a systems biology approach; using GWAS data, genes associated with CD4+ T cell differentiation were enriched for many, not only immunerelated, diseases. Furthermore, global CD4+ T cell gene expression (by microarray) could discriminate between patients and controls. Lastly, using in vitro treated CD4+ T cells, we could show that natalizumab perturbated gene expression differently in patients responding to the drug compared to those not responding.In conclusion, our results demonstrate an imbalance of peripheral CD4+ T cells in MS, along with a functional deficiency in the case of TREG. Taken together, these aberrations might result in differentiation and activation of harmful TH1 and TH17 cells, resulting in CNS tissue damage. The importance of CD4+ T cells was further demonstrated by the finding that genes associated with CD4+ T cell differentiation constitute a pleiotropic module common to a number of diseases. Investigation of natalizumab revealed drastic changes in the peripheral lymphocyte compartment caused by treatment. It also appears as treatment might influence the responsiveness of peripheral T cells to antigens. In addition, by using CD4+ T cell transcriptomics after in vitro drug exposure, prediction of treatment outcome may be possible.
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