Brain Advance Access originally published online on June 21, 2008
Brain 2008 131(7):1701-1711; doi:10.1093/brain/awn118
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Cytometric profiling in multiple sclerosis uncovers patient population structure and a reduction of CD8low cells
1Harvard Medical School/Partners Healthcare Center for Genetics and Genomics, 2Center for Neurologic Diseases, Department of Neurology, Brigham & Women's Hospital, Boston, MA, 3Broad Institute of Harvard University and Massachusetts Institute of Technology, 4Millenium Pharmaceuticals Inc., 5Amgen Inc. and 6Biogen IDEC Inc., Cambridge, MA, USA
Correspondence to: Philip L. De Jager, MD PhD, Harvard/Partners Center for Genomics and Genetics, Brigham & Women's Hospital, 77 Avenue Louis Pasteur, NRB 168C, Boston, MA 02115, USA E-mail: pdejager{at}rics.bwh.harvard.edu
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 CD8lowCD4– 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 CD8lowCD4– 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 CD8lowCD56+CD3–CD4– subset of CD8low 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 CD8lowCD4– 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.
Key Words: multiple sclerosis; flow cytometry; cluster analysis
Abbreviations: CIS, clinically isolated demyelination; syndrome;; CLIMB, Comprehensive Longitudinal Investigation of MS at the Brigham & Women's Hospital; CMS, comparative marker selection; FACS, fluorescence activated cell sorter; GMFI, geometric mean fluorescence intensity; MCV, mean channel value; MedFI, median fluorescence intensity; MFI, mean fluorescence intensity; MRI, magnetic resonance imaging; MS, multiple sclerosis; NK, natural killer; NMF, non-negative matrix factorization; PBMCs, peripheral blood mononuclear cells; QC, quality control; RRMS, relapsing–remitting multiple sclerosis; SVM, support vector machine
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Received September 24, 2007. Revised May 13, 2008. Accepted May 14, 2008.
*These authors contributed equally to this work.
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