Impact of the Multiple Sclerosis associated genetic variant CD226 Gly307Ser on human CD8 T cell functions

Background and Objectives: The rs763361 non-synonymous (Gly307Ser) variant in the CD226 gene has been identified as a risk factor for several immune-mediated diseases, including multiple sclerosis (MS). Compelling evidence suggests that this allele may be one of the genetic driving forces contributing to suceptibility to MS by decreasing the immune-regulatory capacity of Treg cells and increasing the pro-inflammatory potential of CD4 T cells. However, the impact of this CD gene variant on CD8 T cell functions, a population that also plays a key role in MS, remain T to be determined. Methods: In order to study whether the CD226 risk variant affects human CD8 T cells functions, we used CD8 T cells isolated from PBMC of 16-age matched healthy donors homozygous for either the protective or risk allele of CD226. We characterized these CD8 T cells upon TCR stimulation using high-parametric flow cytometry, bulk RNAseq, and through characterization of canonical signaling pathways and cytokine production. Results: Upon TCR engagment, the phenotype of ex vivo CD8 T cells bearing the protective (CD226-307Gly) or the risk (CD226-307Ser) allele of CD226 was largely overlapping. However, transcriptomic signature of CD8 T cells from the donor carrying the risk allele presented an enrichment in TCR, JAK/STAT and INFg signaling. Peripheral blood mononuclear leukocytes (PBMCs) were purified from buffy coat preparations obtained from consenting healthy blood donors. PBMCs were prepared by gradient centrifugation of buffy coats and kept in frozen in liquid nitrogen. Genotyping of the CD226 SNP rs763361 was performed using TaqMan SNP Genotyping assay. rs763361T expressing CD226-307Ser represented the risk allele, whereas rs763361C expressing CD226-307Gly represented the protective allele. Experiments were performed on cells form male donors homozygous for either allele. Frozen PBMCs were thawed, monocytes removed by plastic adherence and CD8 T cells were purified by negative selection. The percentage of residual CD4 T cells after depletion was always less than 0.5% and the remaining populations consisted of 95-98% CD8 T cells. FACS-sorted CD3+CD8+ T cells from healthy donors homozygous for either the protective (CD226-307Gly, N = 10) or risk allele (CD226-307Ser, N = 10) cultured in complete medium RPMI and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies for 0h, 4h and 24h were subjected to RNA extraction using RNeasy Micro Kit. Paired-end (2x150pb) sequencing libraries were prepared using Illumina’s Stranded Total RNA prep kit starting from 7,5ng of total RNA using 14 PCR cycles for library amplification. Sequencing libraries were indexed using IDT Illumina UDI A Lig 96 indexes 96 Sp1. RNA sequencing was performed on one S4 lane on an Illumina Novaseq 6000 sequencer using Novaseq 6000 S4 1v5 Reagent Kit (300 cycles) yielding between 20 and 40 millions raw paired-end reads per library.