RNAseq of TLR agonist and SLE Immune complex stimulated SLE patient PBMCs with and without depletion of pDCs with CSL362
Stimulation experiments were done with PBMC from SLE or healthy donors treated with CSL362 or isotype control before stimulation with various stimuli including the TLR9 agonist 0.25 µM CpGc; the TLR4 agonist 10 µg/ml LPS; and the TLR3 agonist 10 µg/ml POLY I:C. As well as the SLE specific stimuli SLE immunoglobulin (Ig) + necrotic cell lysates (NCL) to form immune complexes; control healthy donor Ig + NCL; and SLE sera + NCL; or healthy donor sera + NCL to understand the specific effects of pDC depletion on different inducible gene transcripts. Overall design: PBMC from SLE or healthy donors were cultured with 1 microgram/ml CSL362 or isotype control for 20 hours. The supernatant was then removed and cells were resuspended in various stimuli (0.25 micromolar 0.25 µM CpGc, 10 microgram/ml 10 µg/ml LPS, 10 microgram/ml 10 µg/ml POLY I:C, 0.1 mg/ml healthy donor immunoglobulin (Ig) + 0.1 mg/ml necrotic cell lysates (NCL), 0.05-0.1 mg/ml SLE Ig + 0.1 mg/ml NCL, 20% SLE sera + 0.1 mg/ml NCL, 20% HD sera + 0.1 mg/ml NCL) for 24 hours at 37 degrees celcius. Cell pellets were then stored in RNAprotect before the RNA was later extracted using a qiagen RNAeasy miniplus kit. Samples underwent an illumina stranded RNA polyA library prep and HiSeq HT chemistry 100 bp single reads.