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Regulation of immunity in Multiple Sclerosis: CD4+ T cells and the influence of natalizumab
Linköping University, Department of Clinical and Experimental Medicine, Division of Inflammation Medicine. Linköping University, Faculty of Health Sciences.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2014. , 146 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1413
National Category
Neurology Immunology in the medical area
Identifiers
URN: urn:nbn:se:liu:diva-108910DOI: 10.3384/diss.diva-108910ISBN: 978-91-7519-272-7 (print)OAI: oai:DiVA.org:liu-108910DiVA: diva2:733849
Public defence
2014-08-27, Berzeliussalen, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2014-07-11 Created: 2014-07-11 Last updated: 2016-06-22Bibliographically approved
List of papers
1. Transcriptional characteristics of CD4+ T cells in multiple sclerosis: relative lack of suppressive populations in blood
Open this publication in new window or tab >>Transcriptional characteristics of CD4+ T cells in multiple sclerosis: relative lack of suppressive populations in blood
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2011 (English)In: Multiple Sclerosis, ISSN 1352-4585, E-ISSN 1477-0970, Vol. 17, no 1, 57-66 p.Article in journal (Refereed) Published
Abstract [en]

Background:Multiple sclerosis (MS) is hypothetically caused by autoreactive Th1 and Th17 cells, whereas Th2 and regulatory T cells may confer protection. The development of Th subpopulations is dependant on the expression of lineage-specific transcription factors.

Objective:The aim of this study was to assess the balance of CD4+T cell populations in relapsing-remitting MS.

Methods:Blood mRNA expression of TBX21, GATA3, RORC, FOXP3 and EBI3 was assessed in 33 patients with relapsing-remitting MS and 20 healthy controls. In addition, flow cytometry was performed to assess T lymphocyte numbers.

Results:In relapsing-remitting MS, diminished expression of FOXP3 (Treg) was found (p < 0.05), despite normal numbers of CD4+CD25hiTreg. Immunoregulatory EBI3 and Th2-associated GATA3 ([a-z]+) was also decreased in MS (p < 0.005 and p < 0.05, respectively). Expression of TBX21 (Th1) and RORC (Th17) did not differ between patients and controls. Similar changes were observed when analysing beta-interferon treated (n = 12) or untreated (n = 21) patients. Analysis of transcription factor ratios, comparing TBX21/GATA3 and RORC/FOXP3, revealed an increase in the RORC/FOXP3 ratio in patients with relapsing-remitting MS (p < 0.005).

Conclusion:Our findings indicate systemic defects at the mRNA level, involving downregulation of beneficial CD4+phenotypes. This might play a role in disease development by permitting activation of harmful T cell populations.

Place, publisher, year, edition, pages
Sage Publications, 2011
Keyword
EBI3, FOXP3, multiple sclerosis, RORC, T cells, transcription factors
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-64758 (URN)10.1177/1352458510381256 (DOI)000285867200006 ()20847001 (PubMedID)
Available from: 2011-02-04 Created: 2011-02-04 Last updated: 2017-12-11
2. Regulatory T cells in Multiple Sclerosis – Indications of impaired function of suppressive capacity and a role for chemokines
Open this publication in new window or tab >>Regulatory T cells in Multiple Sclerosis – Indications of impaired function of suppressive capacity and a role for chemokines
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2014 (English)Manuscript (preprint) (Other academic)
Abstract [en]

BACKGROUND Regulatory T cells (Treg) are critical for immune regulation and homeostasis. In multiple sclerosis (MS), the function of these cells has been shown to be impaired, although the underlying mechanism has yet to be shown. In the current study, we aimed to characterize and assess the phenotypical, functional and transcriptional characteristics of memory and naïve Treg in MS patients and controls.

MATERIAL AND METHODS 27 patients with relapsing-remitting disease were included, along with 29 healthy controls. Flow cytometry was used for detailed phenotyping of Treg subpopulations CD4+CD45RA+/- and CD4dimCD25++ and their expression of FOXP3, CD39 and HELIOS. CFSE (proliferation marker) and CD69 (activation marker) were used to investigate the functional capacity of Treg. A microarray was employed for genome-wide transcriptional characterization of isolated Treg.

RESULTS CD4+CD45RA–CD25++ activated Treg displayed a higher expression of FOXP3 and CD39 than resting CD4+CD45RA+CD25+ Treg, while no significant phenotypical differences were observed in Treg subpopulations between patients and controls. However, a lower anti-proliferative capacity was observed in activated Treg of MS patients compared with those of controls (p<0.05), while suppression of activation was similar to controls. Gene set enrichment analysis (GSEA) of microarray data revealed enrichment for the GO gene set ‘chemokine receptor binding’ in MS Treg.

CONCLUSION Although numerical phenotypical assessment of resting and activated Tregs did not reveal any significant difference between patients and controls, functional co-culturing experiments showed an impaired function in activated Treg of MS patients. Furthermore, GSEA revealed immune-related gene sets overexpressed in Treg of MS patients, possibly containing clues to the functional impairment. In particular over-activity in chemokine signalling in Treg would be of interest for further investigation.

Keyword
EBI3, FOXP3, multiple sclerosis, RORC, T cells, transcription factors
National Category
Clinical Medicine Basic Medicine
Identifiers
urn:nbn:se:liu:diva-108908 (URN)
Available from: 2014-07-11 Created: 2014-07-11 Last updated: 2015-04-10Bibliographically approved
3. An Increase in B cell and Cytotoxic NK cell Proportions and Increased T cell Responsiveness in Blood of Natalizumab-treated Multiple Sclerosis Patients
Open this publication in new window or tab >>An Increase in B cell and Cytotoxic NK cell Proportions and Increased T cell Responsiveness in Blood of Natalizumab-treated Multiple Sclerosis Patients
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 12, e81685Article in journal (Refereed) Published
Abstract [en]

Background

Changes in the peripheral blood lymphocyte composition probably both mediate and reflect the effects of natalizumab treatment in multiple sclerosis, with implications for treatment benefits and risks.

Objectives

To assess changes in circulating lymphocyte subpopulation compositions and T-cell responses during natalizumab treatment.

Material and methods

A broad panel of markers for blood lymphocyte populations, including states of activation and co-stimulation as well as T-cell responses to recall antigens and mitogens, was assessed by flow cytometry in 40 patients with relapsing multiple sclerosis before and after one-year natalizumab treatment.

Results

Absolute numbers of all major populations of lymphocytes increased after treatment, most markedly for NK- and B-cells. The fraction of both memory and presumed regulatory B-cell subsets increased, as did CD3-CD56dim cytotoxic NK-cells, whereas CD3-CD56bright regulatory NK-cells decreased. Treatment was also associated with a restored T-cell responsiveness to recall antigens and mitogens.

Conclusions

Our data confirms that natalizumab treatment increases the number of lymphocytes in blood, likely mirroring the expression of VLA-4 being highest on NK- and B-cells. This supports reduction of lymphocyte extravasation as a main mode of action, although the differential composition of lymphocyte subpopulations suggests cell-signalling effects may also be operative. The systemic increase in T-cell responsiveness reflects the increase in numbers, and while augmenting anti-infectious responses systemically, localized responses become correspondingly decreased.

Place, publisher, year, edition, pages
San Francisco, USA: Public Library of Science, 2013
Keyword
Multiple sclerosis, natalizumab, flow cytometry, T-cells, NK-cells, B-cells, lymphocyte proliferation
National Category
Neurology Immunology in the medical area
Identifiers
urn:nbn:se:liu:diva-84268 (URN)10.1371/journal.pone.0081685 (DOI)000327944500088 ()24312575 (PubMedID)2-s2.0-84891420120 (Scopus ID)
Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2017-12-07Bibliographically approved
4. Integrated genomic and prospective clinical studies show the importance of modular pleiotropy for disease susceptibility, diagnosis and treatment
Open this publication in new window or tab >>Integrated genomic and prospective clinical studies show the importance of modular pleiotropy for disease susceptibility, diagnosis and treatment
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2014 (English)In: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 6, no 17Article in journal (Refereed) Published
Abstract [en]

Background: Translational research typically aims to identify and functionally validate individual, disease-specific genes. However, reaching this aim is complicated by the involvement of thousands of genes in common diseases, and that many of those genes are pleiotropic, that is, shared by several diseases. Methods: We integrated genomic meta-analyses with prospective clinical studies to systematically investigate the pathogenic, diagnostic and therapeutic roles of pleiotropic genes. In a novel approach, we first used pathway analysis of all published genome-wide association studies (GWAS) to find a cell type common to many diseases. Results: The analysis showed over-representation of the T helper cell differentiation pathway, which is expressed in T cells. This led us to focus on expression profiling of CD4(+) T cells from highly diverse inflammatory and malignant diseases. We found that pleiotropic genes were highly interconnected and formed a pleiotropic module, which was enriched for inflammatory, metabolic and proliferative pathways. The general relevance of this module was supported by highly significant enrichment of genetic variants identified by all GWAS and cancer studies, as well as known diagnostic and therapeutic targets. Prospective clinical studies of multiple sclerosis and allergy showed the importance of both pleiotropic and disease specific modules for clinical stratification. Conclusions: In summary, this translational genomics study identified a pleiotropic module, which has key pathogenic, diagnostic and therapeutic roles.

Place, publisher, year, edition, pages
BioMed Central, 2014
National Category
Clinical Medicine Basic Medicine
Identifiers
urn:nbn:se:liu:diva-106873 (URN)10.1186/gm534 (DOI)000334631300002 ()
Available from: 2014-05-28 Created: 2014-05-23 Last updated: 2017-12-05

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