CITE-seq reveals inhibition of NF-kB pathway in B cells from vitamin D-treated multiple sclerosis patients
Vitamin D deficiency is a risk factor for multiple sclerosis (MS) and is correlated with disease activity and progression. Vitamin D treatment has emerged as potentially protective, even though results from randomized controlled trials are conflicting. Here, we used single-cell RNA-seq (scRNA-seq) coupled with barcoded antibodies targeting surface markers (CITE-seq) to discover candidate genes and pathways regulated in PBMC subpopulations from MS patients treated with high-dose vitamin D (n=5) or placebo (n=5). Best candidates were combined with genes involved in immune function and vitamin D metabolism for validation in a new cohort (n=8 in each group) by high-throughput qPCR (HT-qPCR) in naive CD4, Th1, Th17, Treg, naive CD8, memory and naive B cells, and MAIT cells, sorted by FACS. CITE-seq showed no significant changes in the proportions of these subpopulations in vitamin D-treated patients. Of the 92 candidate genes revealed by CITE-seq, differential expression of five genes (UXT, SNRPN, SUB1, GNLY and KLF6) was validated by HT-qPCR. CITE-seq also revealed the regulation of several pathways by vitamin D in naive and memory B cells, including MAPK, TLR and interleukin pathways, that contribute to counteract EBV-induced resistance to apoptosis through inhibition of the NF-kB pathway. Overall design: Five MS patients treated with high dose vitamin D (100,000 IU every 2 weeks for 3 months) and 5-matched MS patients treated with placebo were selected from the D-lay MS cohort (NCT01817166). Blood samples of study participants of the D-lay MS study had been previously collected in EDTA-containing tubes at D0 and M3 of treatment (placebo or vitamin D). PBMCs had been isolated by Ficoll® gradient and cryopreserved in liquid nitrogen using 4% human albumin solution + 10% DMSO medium in concentration of 5x106 cells per ml.  Cells were labeled with CITE-seq antibodies and live cells were isolated by fluorescence-activated cell sorting (FACS).