Profiling Immune Gene Expression in SLE

Gene expression profiling was performed by NanoString, using the pre-designed nCounter Human Immunology v2 Panel (NanoString Technologies). Total CD4⁺ T cells from a subset of (i) four SLE patients with previously identified high transcriptional IFN signature, (ii) two SLE patients with low IFN signature, and (iii) four healthy controls, were obtained by negative selection (StemCell Technologies) from cryopreserved PBMCs. A total of 25,000 cells were collected into RLT lysis buffer (Qiagen) following in vitro stimulation for 120 min with PMA and ionomycin cocktail without addition of protein transport inhibitors (eBiosciences). RNA was extracted using the RNAeasy Micro Plus kit (Qiagen), with gDNA cleanup, and hybridized to the NanoString CodeSets, following manufacturer's instructions. Expression levels were assessed using an nCounter Sprint instrument (NanoString Technologies). Data were processed using the nSolver Analysis Software following normalization of the raw read counts to the geometric mean of positive control spike-ins, and the gene expression of 15 selected housekeeping genes that were found to have low variability following in vitro stimulation, as previously described (28 (link)).

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Ferreira R.C., Castro Dopico X., Oliveira J.J., Rainbow D.B., Yang J.H., Trzupek D., Todd S.A., McNeill M., Steri M., Orrù V., Fiorillo E., Crouch D.J., Pekalski M.L., Cucca F., Tree T.I., Vyse T.J., Wicker L.S, & Todd J.A. (2019). Chronic Immune Activation in Systemic Lupus Erythematosus and the Autoimmune PTPN22 Trp620 Risk Allele Drive the Expansion of FOXP3+ Regulatory T Cells and PD-1 Expression. Frontiers in Immunology, 10, 2606.

Publication 2019

nCounter Human Immunology v2 Panel negative selection RLT lysis buffer ionomycin cocktail protein transport inhibitors RNAeasy Micro Plus kit

Buffer Cd4 t cells Cells Gene expression Housekeeping genes Human Inhibitors Ionomycin Patients Protein transport Stemcell Transcriptional

Corresponding Organization : University of Oxford

Other organizations : University of Cambridge, King's College London, Institute of Genetic and Biomedical Research, University of Sassari, Guy's Hospital

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Variable analysis

independent variables

SLE patients with previously identified high transcriptional IFN signature
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dependent variables

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control variables

Total CD4+ T cells obtained by negative selection from cryopreserved PBMCs
Cells stimulated in vitro for 120 min with PMA and ionomycin cocktail without addition of protein transport inhibitors
RNA extracted using RNAeasy Micro Plus kit with gDNA cleanup
Expression levels assessed using nCounter Sprint instrument
Data processed using nSolver Analysis Software with normalization to positive control spike-ins and 15 selected housekeeping genes

positive controls

Positive control spike-ins

negative controls

Healthy controls

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