Increased Frequency of T Peripheral Helper Cell and Plasmablast Correlates with Disease Activity in Childhood-Onset Systemic Lupus Erythematosus with Nephritis. Baxter et al.
Gene Expression and Module Score data from DxTerity analysis of whole blood from new-onset SLE patients as described in methods.
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See methods in associated publication. Method Details Blood processing and mass cytometry analysis Whole blood was collected into heparinized vacutainers. Freshly drawn whole blood was treated with protein transport inhibitor (ebioscience 00-4980-03) in the absence of immune-stimulatory agents, incubated for 6 hours, then lysed and fixed (BD lyse/fix buffer #558049) to remove RBCs. Fixed cells were stored in cell staining buffer (MaxPar Cell Staining Buffer, Fluidigm, # 201068) at -80°C. When specimen numbers for batch processing had been obtained, cells were thawed for downstream CyTOF barcoding and staining steps. Mass tag cell barcoding of fixed samples, followed by antibody staining and permeabilization was performed as previously described44,81. Antibody panel detailed in Supp Table 3. Targeted gene expression panel analysis Gene expression analyses of specified modules were measured by DxTerity Diagnostics, Rancho Dominguez, CA USA, using the DxTerity Modular Immune Profile (MIP) test, a chemical ligation-dependent probe amplification and gene expression test with relative quantitative analysis by capillary electrophoresis82. Modules analyzed were: DxTerity IFN Module (IFN-1), B Cell Module, Energy Module, IFN Beta Module, IFN Gamma Module, mRNA Translation Module, Neutrophil Module, pDC Module, Plasmablasts Module, T Cell Module. Sample testing and analysis was performed directly on PAXgene RNA Stabilized Blood as described by Kim et al. 82. The DxTerity MIP test measures the RNA expression levels of 51 immune response genes relative to the expression levels of 3 housekeeping normalizer genes (ACTB, GAPDH and TFRC). Normalized expression values of each respective response gene were calculated per the following function: Normalized ExpressionGene i = Log2(HeightGene i) – Mean (Log2(Normalized Gene Height). A 4-gene Type 1 Interferon (IFN-1) signature score was calculated by averaging the normalized expression values of HERC5, IFI27, IFIT1, and RSAD2 in the MIP panel. The IFN-1 signature score cutoff of -0.5 between IFN high and low was determined based on measurement of 281 healthy human blood samples and placing the cut-off at 2 standard deviations (95th percentile) above the mean healthy IFN-1 score (-0.5). This cut-off falls within the trough of the observed bimodal distribution of IFN-1 scores for this and other cohorts of SLE samples.
Childhood Arthritis and Rheumatology Research Alliance
National Institute of Arthritis and Musculoskeletal and Skin Diseases
National Institutes of Health
Anschutz Medical Campus, University of Colorado
Lupus Research Alliance