Cytometric profiling in multiple sclerosis uncovers patient population structure and a reduction of CD8

Harvard Medical School/Partners Center for Genomics and Genetics, Brigham & Women's Hospital, 77 Avenue Louis Pasteur, NRB 168C, Boston, MA 02115, USA.
Brain (Impact Factor: 9.2). 06/2008; 131(Pt 7):1701-11. DOI: 10.1093/brain/awn118
Source: PubMed

ABSTRACT As part of a biomarker discovery effort in peripheral blood, we acquired an immunological profile of cell-surface markers from healthy control and untreated subjects with relapsing-remitting MS (RRMS). Fresh blood from each subject was screened ex vivo using a panel of 50 fluorescently labelled monoclonal antibodies distributed amongst 56 pools of four antibodies each. From these 56 pools, we derived an immunological profile consisting of 1018 'features' for each subject in our analysis using a systematic gating strategy. These profiles were interrogated in an analysis with a screening phase (23 patients) and an extension phase (15 patients) to identify cell populations in peripheral blood whose frequency is altered in untreated RRMS subjects. A population of CD8(low)CD4(-) cells was identified as being reduced in frequency in untreated RRMS subjects (P = 0.0002), and this observation was confirmed in an independent sample of subjects from the Comprehensive Longitudinal Investigation of MS at the Brigham & Women's Hospital (P = 0.002). This reduction in the frequency of CD8(low)CD4(-) cells is also observed in 38 untreated subjects with a clinically isolated demyelination syndrome (CIS) (P = 0.0006). We also show that these differences may be due to a reduction in the CD8(low)CD56(+)CD3(-)CD4(-) subset of CD8(low) cells, which have a natural killer cell profile. Similarities between untreated CIS and RRMS subjects extend to broader immunological profiles: consensus clustering of our data suggests that there are three distinct populations of untreated RRMS subjects and that these distinct phenotypic categories are already present in our sample of untreated CIS subjects. Thus, our large-scale immunophenotyping approach has yielded robust evidence for a reduction of CD8(low)CD4(-) cells in both CIS and RRMS in the absence of treatment as well as suggestive evidence for the existence of immunologically distinct subsets of subjects with a demyelinating disease.

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Available from: Ronenn Roubenoff, Sep 27, 2015
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    • "CD8low T cells are less cytotoxic than CD8high T cells, and they express IL-4, IL-10, and interferon-gamma [21]. CD8low lymphocytes, and in particular CD8low NK cells have been shown to be reduced in untreated patients with clinically isolated syndrome and MS [22]. Interestingly, MX appears to restore the frequency of CD8low T cells to the levels observed in healthy individuals (Fig. 1D), suggesting that this regulation may contribute to the efficacy of the treatment in MS. "
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    ABSTRACT: Mitoxantrone is one of the few drugs approved for the treatment of progressive multiple sclerosis (MS). However, the prolonged use of this potent immunosuppressive agent is limited by the appearance of severe side effects. Apart from its general cytotoxic effect, the mode of action of mitoxantrone on the immune system is poorly understood. Thus, to develop safe therapeutic approaches for patients with progressive MS, it is essential to elucidate how mitoxantrone exerts it benefits. Accordingly, we initiated a prospective single-arm open-label study with 19 secondary progressive MS patients. We investigated long-term effects of mitoxantrone on patient peripheral immune subsets using flow cytometry. While we corroborate that mitoxantrone persistently suppresses B cells in vivo, we show for the first time that treatment led to an enrichment of neutrophils and immunomodulatory CD8(low) T cells. Moreover, sustained mitoxantrone applications promoted not only persistent NK cell enrichment but also NK cell maturation. Importantly, this mitoxantrone-induced NK cell maturation was seen only in patients that showed a clinical response to treatment. Our data emphasize the complex immunomodulatory role of mitoxantrone, which may account for its benefit in MS. In particular, these results highlight the contribution of NK cells to mitoxantrone efficacy in progressive MS.
    PLoS ONE 06/2012; 7(6):e39625. DOI:10.1371/journal.pone.0039625 · 3.23 Impact Factor
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    • "Nevertheless, studies using untreated MS patients have mostly detected deficits in NK cells function rather than differences in overall numbers between MS and controls. For instance, a reduction of an NK cell subtype, CD8lowCD56+CD3-CD4-, was observed in untreated, clinical isolated demyelination syndrome (CIS) and in relapse remitting MS (RRMS) patients, suggesting that this decrease in CD8low NK cells is an early event in demyelinating diseases [24]. The action of daclizumab (anti-IL2Rα) may restore to normal levels the CD8lowCD56+CD3-CD4- subset, this expansion correlating with decreased brain inflammation and decreased survival of activated T cells [21]. "
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    ABSTRACT: Pathogenic or regulatory effects of natural killer (NK) cells are implicated in many autoimmune diseases, but evidence in multiple sclerosis (MS) and its murine models remains equivocal. In an effort to illuminate this, we have here analysed expression of the prototypic NK cell marker, NCR1 (natural cytotoxicity triggering receptor; NKp46; CD335), an activating receptor expressed by virtually all NK cells and therefore considered a pan-marker for NK cells. The only definitive ligand of NCR1 is influenza haemagglutinin, though there are believed to be others. In this study, we investigated whether there were differences in NCR1+ cells in the peripheral blood of MS patients and whether NCR1+ cells are present in white matter lesions. We first investigated the expression of NCR1 on peripheral blood mononuclear cells and found no significant difference between healthy controls and MS patients. We then investigated mRNA levels in central nervous system (CNS) tissue from MS patients: NCR1 transcripts were increased more than 5 times in active disease lesions. However when we performed immunohistochemical staining of this tissue, few NCR1+ NK cells were identified. Rather, the major part of NCR1 expression was localised to astrocytes, and was considerably more pronounced in MS patients than controls. In order to further validate de novo expression of NCR1 in astrocytes, we used an in vitro staining of the human astrocytoma U251 cell line grown to model whether cell stress could be associated with expression of NCR1. We found up-regulation of NCR1 expression in U251 cells at both the mRNA and protein levels. The data presented here show very limited expression of NCR1+ NK cells in MS lesions, the majority of NCR1 expression being accounted for by expression on astrocytes. This is compatible with a role of this cell-type and NCR1 ligand/receptor interactions in the innate immune response in the CNS in MS patients. This is the first report of NCR1 expression on astrocytes in MS tissue: it will now be important to unravel the nature of cellular interactions and signalling mediated through innate receptor expression on astrocytes.
    Journal of Neuroinflammation 01/2012; 9(1):1. DOI:10.1186/1742-2094-9-1 · 5.41 Impact Factor
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    • "Reports of reduction of NK cell numbers and function in MS patients and evidence from experimental autoimmune encephalomyelitis (EAE) suggest a protective role of this population (Zhang et al., 1997; Kastrukoff et al., 2003; De Jager et al., 2008). However, this subset can also exert a detrimental role in the inflammatory process within the CNS (Winkler-Pickett et al., 2008). "
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    ABSTRACT: Interferon-β (IFN-β) is known to expand regulatory CD56(bright) natural killer (NK) cells in multiple sclerosis (MS). In this cross-sectional study we show that MS patients treated with IFN-β alone or in combination with low-dose prednisolone displayed increased proportion of all NK cell subsets in the active phase of the cell cycle (Ki-67+). There was no difference in NK cell apoptosis markers. In vitro experiments showed that both IFN-β and IFN-β in combination with corticosteroids increased the proportion of Ki-67(+) NK cells. This study, although limited, shows that treatment with IFN-β affects NK cell cycle without altering NK cell apoptosis in MS patients.
    Journal of neuroimmunology 06/2011; 236(1-2):111-7. DOI:10.1016/j.jneuroim.2011.05.005 · 2.47 Impact Factor
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