Impact of the Multiple Sclerosis-Associated Genetic Variant CD226 Gly307Ser on Human CD8 T-Cell Functions

Abstract

Background and objectives: The rs763361 nonsynonymous variant in the CD226 gene, which results in a glycine-to-serine substitution at position 307 of the CD226 protein, has been implicated as a risk factor of various immune-mediated diseases, including multiple sclerosis (MS). Compelling evidence suggests that this allele may play a significant role in predisposing individuals to MS by decreasing the immune-regulatory capacity of Treg cells and increasing the proinflammatory potential of effector CD4 T cells. However, the impact of this CD226 gene variant on CD8 T-cell functions, a population that also plays a key role in MS, remains to be determined.

Methods: To study whether the CD226 risk variant affects human CD8 T-cell functions, we used CD8 T cells isolated from peripheral blood mononuclear cell of 16 age-matched healthy donors homozygous for either the protective or the risk allele of CD226. We characterized these CD8 T cells on T-cell receptor (TCR) stimulation using high-parametric flow cytometry and bulk RNAseq and through characterization of canonical signaling pathways and cytokine production.

Results: On TCR engagement, 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, the transcriptomic signature of CD8 T cells from the donors carrying the risk allele presented an enrichment in TCR, JAK/STAT, and IFNγ signaling. We next found that the CD226-307Ser risk allele leads to a selective increase in the phosphorylation of the mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) associated with enhanced phosphorylation of STAT4 and increased production of IFNγ.

Discussion: Our data suggest that the CD226-307Ser risk variant imposes immune dysregulation by increasing the pathways related to IFNγ signaling in CD8 T cells, thereby contributing to the risk of developing chronic inflammation.

Figure 1. Phenotype of Ex Vivo CD8 T Cells Carrying the Protective or the Risk Variant of CD226

PBMCs from healthy donors homozygous for either the protective (CD226-307Gly, N = 16) or the risk (CD226-307Ser, N = 16) allele were analyzed ex vivo by flow cytometry. (A) Uniform Manifold Approximation and Projection (UMAP) and frequency of central memory (CM, CD45RA- CCR7+), effector memory (EM, CD45RA- CCR7-), memory re-expressing CD45RA (EMRA, CD45RA+ CCR7-), and naïve (CD45RA+ CCR7+) CD8 T cells are shown for each allele. (B) Frequency and (C) MFI of CD226 expression are plotted in CM, EM, EMRA, and naïve CD8 T cells. MFI = mean fluorescent intensity; PBMCs = peripheral blood mononuclear cells.

Figure 2. Transcriptomics of CD8 T Cells Carrying the Protective or the Risk Variant of CD226

RNA sequencing of purified CD8 T cells from healthy donors homozygous for either the protective (CD226-307Gly, N = 10) or risk (CD226-307Ser, N = 10) allele stimulated with anti-CD3 and anti-CD28 mAbs for 0 and 24 hours. (A) Volcano plot representation of differential gene expression analysis after 24 hours of activation. Colored points show the differentially expressed genes (DEGs) (fold change >2 and Padj <0.05). In red are depicted the genes upregulated in the risk allele and in blue the genes upregulated in the protective allele. (B) The heat map shows the relative expression of the most upregulated genes in the risk allele for each sample. Metascape analysis based on DEGs and showing the pathways upregulated in the risk or protective allele using the gene ontology (C) and the Kyoto Encyclopedia of Genes and Genomes (D) pathway databases.

Figure 3. TCR Signaling of CD8 T Cells Carrying the Protective or the Risk Variant of CD226

Expanded CD8 T cells from healthy people homozygous for either the protective (CD226-307Gly, N = 6) or risk allele (CD226-307Ser, N = 6) were stimulated with anti-CD3 and anti-CD28 mAbs for 0, 30, and 120 seconds. Phosphorylation of ERK, AKT, and p38 was detected through Jess Replex Simple Western (A) and quantified. The ratio between the phosphorylated and total protein is shown in the graphs (B, C, and D). 2-way ANOVA was used for statistical analysis.

Figure 4. Functions of CD8 T Cells Carrying the Protective or the Risk Variant of CD226

Functional analysis of CD8 T cells isolated from PBMCs of healthy donors carrying either the protective (CD226-307Gly, N = 16) or risk (CD226-307Ser, N = 16) allele stimulated with anti-CD3 mAb alone or in combination with anti-CD28 mAb for 3 days. (A) IFNγ levels in culture supernatants were measured using LEGENDplex. Percentage (B) and MFI (C) of IFNγ+ CD8 T cells detected by flow cytometry after stimulation with PMA and ionomycin. 2-way ANOVA was used for statistical analysis. MFI = mean fluorescent intensity; PBMCs = peripheral blood mononuclear cells.

Figure 5. IFNγ Pathway in CD8 T Cells Carrying the Protective or the Risk Variant of CD226

Phosphorylation of STAT4 was measured as % and MFI through Phosflow in CD8 T cells isolated from healthy donors carrying either the protective (CD226-307Gly, N = 16) or risk (CD226-307Ser, N = 16) allele that was stimulated with anti-CD3 and CD28 Abs alone (A) or with addition of IL-12 (B) or IFNα (C) for 15 minutes. The graph bar shows the ratio between IL-12-stimulated and unstimulated cells. The Mann-Whitney test was used for statistical analysis. MFI = mean fluorescent intensity.

Figure 6. Functionality of CD8 T Cells Carrying the Protective or the Risk Variant of CD226 in Th1-Polarized Condition

Isolated CD8 T cells from healthy donors carrying either the protective (CD226-307Gly, N = 16) or risk (CD226-307Ser, N = 16) allele were stimulated with anti-CD3 mAb alone or in combination with IL-12 for 3 days. Frequency of CD69 (A), CD226 (B), and T-BET (C) expression by CD8 T cells was analyzed with flow cytometry. (D) Production of IFNγ was measured in the culture supernatants using the LEGENDplex CBA.

Figure 7. Expression of IL-12R by CD8 T Cells Carrying the Protective or the Risk Variant of CD226 in Th1-Polarized Condition

Isolated CD8 T cells from healthy donors carrying either the protective (CD226-307Gly, N = 16) or risk (CD226-307Ser, N = 16) allele were stimulated with anti-CD3 mAb alone or in combination with IL-12. Frequency of IL-12Rβ2 (A) and IL-12Rβ1 (B) expression by CD8 T cells was detected by flow cytometry after 5 days of activation. (C) Dot plot showing the expression of CD226 and IL-12Rβ2 by CD8 T cells harboring the protective or risk allele of CD226 after 5 days of activation. 2-way ANOVA was used for statistical analysis. (D) Proposed model on the consequence of the TCR activation with CD226 carrying the protective (left panel) or risk (right panel) allele in CD8 T cells. The engagement of the TCR induces phosphorylation of CD226 that synergizes into the TCR signaling pathways, leads to phosphorylation of STAT4, and results in IFNγ production. In CD8 T cells from donors carrying the risk variant, these pathways are amplified contributing to the risk of developing chronic inflammation.