A CD8+ NK cell transcriptomic signature associated with clinical outcome in relapsing remitting multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) with the majority of cases characterised by relapsing/remitting (RRMS) attacks of neurologic dysfunction followed by variable resolution. Improving clinical outcomes in RRMS requires both a better understanding of the immunological mechanisms driving recurrent demyelination and better means of predicting future disease course to facilitate early targeted therapy. Here, we apply hypothesis-generating network transcriptomics to CD8+ cells isolated from patients in RRMS, identifying a signature reflecting expansion of a subset of CD8+ natural killer cells (NK8+) associated with favourable outcome. NK8+ are capable of regulating CD4+ T cell activation and proliferation in vitro, with reduced expression of HLA-G binding inhibitory receptors and consequent reduced sensitivity to HLA-G-mediated suppression. We identify surrogate markers of the NK8+ signature in peripheral blood leucocytes and validate their association with clinical outcome in an independent cohort, suggesting their measurement may facilitate early, targeted therapy in RRMS.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. The datasets generated during and/or analysed during the current study areavailable in the ArrayExpress repository at the European Bioinformatics Institute, accession number E-MTAB-9637. Clinical Trial data is available through the Immune Tolerance Network TrialShare Platform (https://www.itntrialshare.org). Cell and tissue-specific transcripts from the Human Gene Atlas (HGA) are accessible through GEO (accession number GSE1133) and the Immune Response In Silico (IRIS) dataset is publicly-available through GEO (accession number GSE22886) Sample size was determined as for the clinical study which served as the foundation for the current investigation and is described here: Neurology. 2012 Apr 10; 78(15): 1171-1178.
Samples for gene expression profiling were only excluded if they failed technical QC as described in the methods and outlined in Supplementary Material. Exclusion criteria were on the basis of established, pre-specificed QC metrics obtained using the ArrayQualityMetrics package in R.
An independent, publicly-available validation cohort was used to validate the study's principal findings as described in the manuscript. Validation was only attempted on this single cohort as no other appropriate cohort was available. For flow cytometry experiments and functional assays the number of independent replicates is as indicated in the figures with all data shown.
Subject randomization was performed as for the clinical study which served as the foundation for the current investigation and is described here: Neurology. 2012 Apr 10; 78(15): 1171-1178. For in vitro experiments subjects were selected in a blinded, random fashion by the collection of leucocyte cones from blood donors attending for donation.
Blinding to clinical treatment group was undertaken as for the clinical study which served as the foundation for the current investigation and is described here: Neurology. 2012 Apr 10; 78(15): 1171-1178. Processing of the transcriptomic data was undertaken by investigators who were blinded to the results of the clinical study until transcriptional networks were defined. For in vitro studies, investigators undertaking analysis were blinded to the source of samples and the treatments given until gates and expression values were determined.

Clinical data Policy information about clinical studies
All manuscripts should comply with the ICMJEguidelines for publication of clinical research and a completedCONSORT checklist must be included with all submissions.
Routine mycoplasma testing was undertaken for all established cell lines used and were repeatedly negative throughout.
No commonly misidentified cell lines were used in the study.
Population characteristics of human participants recruited into the clinical studies used are detailed in Supplementary Tables  1, 5, 6 and 7 Recruitment was undertaken as for the clinical study which served as the foundation for the current investigation and is described here: Neurology. 2012 Apr 10; 78(15): 1171-1178.
The trial was sponsored by NIAID in collaboration with the Immune Tolerance Network (clinicaltrials.gov NCT00094172). The study protocol and informed consent documents were approved by appropriate local ethics review committees and/or Institutional Review Boards. The study was approved by institutional review boards at 14 centers in the United States and Canada. Written informed consent was obtained from subjects prior to enrollment in the STAyCIS study (NCT00094172).
Data collection was undertaken as for the clinical study which served as the foundation for the current investigation and is described here: Neurology. 2012 Apr 10; 78(15): 1171-1178.