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An Increase in B cell and Cytotoxic NK cell Proportions and Increased T cell Responsiveness in Blood of Natalizumab-treated Multiple Sclerosis Patients
Linköping University, Department of Clinical and Experimental Medicine, Neurology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Local Health Care Services in Central Östergötland, Department of Neurology.
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
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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. Vol. 8, no 12, e81685
Keyword [en]
Multiple sclerosis, natalizumab, flow cytometry, T-cells, NK-cells, B-cells, lymphocyte proliferation
National Category
Neurology Immunology in the medical area
Identifiers
URN: urn:nbn:se:liu:diva-84268DOI: 10.1371/journal.pone.0081685ISI: 000327944500088PubMedID: 24312575Scopus ID: 2-s2.0-84891420120OAI: oai:DiVA.org:liu-84268DiVA: diva2:558409
Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Immunological Mechanisms and Natalizumab Treatment in Multiple Sclerosis: Studies on lymphocytes, inflammatory markers and magnetic resonance spectroscopy
Open this publication in new window or tab >>Immunological Mechanisms and Natalizumab Treatment in Multiple Sclerosis: Studies on lymphocytes, inflammatory markers and magnetic resonance spectroscopy
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Multiple sclerosis (MS) is a chronic inflammatory, demyelinating disease of the central nervous system (CNS), and a frequent cause of neurological disability among young adults. In addition to focal inflammatory demyelinated lesions, diffuse white matter pathology as well as a neurodegenerative component with accumulating axonal damage and gliosis have been demonstrated and contribute to MS disease characteristics. The inflammatory component is considered autoimmune and mediated by auto-reactive T lymphocytes together with other cell populations of the immune system and their respective products like cytokines and chemokines. Treatment with natalizumab, a monoclonal antibody directed against the α4β1-integrin (VLA-4), reduces migration of potential disease-promoting cells to the CNS. The efficacy of natalizumab in reducing relapses and MRI activity is evident, however associated effects on the immune response and the neurodegenerative component in MS are not clear.

Methods: In total 72 MS patients were included, distributed among paper I-IV. We investigated effects associated with one-year natalizumab treatment in 31 MS patients regarding cytokine and chemokine levels in CSF and blood using multiplex bead assay analyses (paper I), as well as treatment effects on blood lymphocyte composition in 40 patients using flow cytometry, including functional assays of lymphocyte activation (paper II). Normal appearing white matter (NAWM) metabolite concentrations were assessed with proton magnetic resonance spectroscopy (1H-MRS) in 27 MS patients before and after one year of treatment (paper III). We also evaluated the balance between circulating T helper (Th) subsets in 33 MS patients using gene expression analyses of the CD4+ T cell related transcription factors in whole blood (paper IV).

Results: One-year natalizumab treatment was associated with a marked decline in pro-inflammatory cytokines (IL-1β and IL-6) and chemokines (CXCL8, CXCL9, CXCL10 and CXCL11) intrathecally. Circulating plasma levels of some cytokines (GM-CSF, TNF, IL-6 and IL-10) also decreased after treatment. Natalizumab treatment was further associated with an increase in lymphocyte numbers of major populations in blood (total lymphocytes, T cells, T helper cells, cytotoxic T cells, NK cells and B cells). In addition, T cell responsiveness to recall antigens and mitogens was restored after treatment. As to 1H-MRS metabolite concentrations in NAWM, no change in levels were detected post-to pretreatment on a group level. However, correlation analyses between one-year change in metabolite levels (total creatine and total choline) and levels of pro-inflammatory IL-1β and CXCL8 showed a pattern of high magnitude correlation coefficients (r=0.43-0.67). Gene expression analyses demonstrated a systemically reduced expression of transcription factors related to immunoregulatory T cell populations (regulatory T cells and Th2) in relapsing MS compared with controls.

Conclusions: Our findings support that an important mode of action of natalizumab is reducing lymphocyte extravasation, although cell-signalling effects through VLA-4 also may be operative. Correlation analyses between changes 1H-MRS metabolite concentrations and inflammatory markers possibly point towards an association between intrathecal inflammation and gliosis development in NAWM. Finally, gene expression analyses indicate a systemic defect at the mRNA level in relapsing MS, involving downregulation of beneficial CD4+ phenotypes.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. 83 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1332
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84274 (URN)978-91-7519-787-6 (ISBN)
Public defence
2012-11-02, Berzeliussalen, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2012-10-24Bibliographically approved
2. Regulation of immunity in Multiple Sclerosis: CD4+ T cells and the influence of natalizumab
Open this publication in new window or tab >>Regulation of immunity in Multiple Sclerosis: CD4+ T cells and the influence of natalizumab
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:nbn:se:liu:diva-108910 (URN)10.3384/diss.diva-108910 (DOI)978-91-7519-272-7 (ISBN)
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

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