CD96 Downregulation Promotes the Immune Response of CD4 T Cells and Associates with Ankylosing Spondylitis

Abstract

Inhibitory receptors (IRs) play an indispensable role in regulating T cell activation and expansion. This study is aimed at exploring the correlation between IRs and ankylosing spondylitis (AS). Bioinformatics analysis of two datasets (GSE25101 and GSE73754), including 68 AS cases and 36 healthy controls, demonstrated that "T cell receptor signaling pathway" was significantly enriched, and two IRs (CD112R and CD96) were downregulated in AS cases. Real-time Quantitative PCR Detecting System (qPCR) analysis confirmed the decreased expression of CD112R and CD96 in the peripheral blood of AS patients. Flow cytometry demonstrated that the frequency of CD96-positive cells among CD4 T cells in AS patients was significantly reduced and that expressed on the cells was also significantly lower than the healthy controls. In addition, the expression of CD96 was altered on human primary CD4 T cells extracted from 3 healthy volunteers and cocultured with allogeneic dendritic cells (DCs). Also, low expression of CD96 elevated the phosphorylation of ERK in CD4 T cells and increased the level of TNF-α, IL-23, IL-17A, IL-6, and IFN-γ in the cell culture supernatant. These results suggested that CD96 is crucial for the pathogenesis of AS and may be a potential target in the treatment of the disease.

Figure 1

KEGG analysis and qPCR showed that T cell receptor signaling pathway and CD112R and CD96 are associated with AS. (a) Top 20 significantly enriched pathways of 642 DEGs. (b) CD112R and CD96 were significantly downregulated in AS patients through bioinformatics analysis. (c) qPCR validation of the expression of CD112R and CD96 between patients with AS and the healthy controls (^∗∗P < 0.01,  ^∗∗∗P < 0.001).

Figure 2

Flow cytometry. (a) Circling gate scheme of flow cytometry analysis. Gating lymphocytes according to the size and granularity (FSC and SSC). Circle the live immune cells “CD45 live” according to CD45 and 7AAD after obtaining single cells. In the gate of “CD45 live,” NK cells (CD3⁻CD56⁺) and conventional CD3 T cells (CD3⁺ CD56⁻) were sorted out. CD3 T cell (CD3⁺ CD56⁻) gate was divided into CD4 T and CD8 T cell subsets according to the expression of CD4 and CD8. (b) Histogram of the expression of CD112R and CD96 between samples and isotype. Compared to the isotype, the expression of CD112R increased slightly, but that of CD96 increased significantly. (c) Frequencies of each lymphocyte subset expressing CD96. The frequencies of CD96-positive CD45 live cells, CD3 T cells, and CD4 T cells in AS patients were lower than those in healthy controls, but no significant differences were observed on CD8 T and NK⁻ cells. (d) Comparison of the fluorescence intensities of CD96 expressed on lymphocyte subsets. The fluorescence intensities of CD96 expressed on CD45 live cells, CD3 T cells, and CD4⁺T cells were lower in AS patients than in healthy controls, but no significant differences were observed on CD8 T and NK cells (^∗P < 0.05,  ^∗∗P < 0.01).

Figure 3

Purification and phenotype identification of CD4 T cells and mature DCs. (a) The purity of CD4 T cells was 46.9% before sorting and 98.1% after sorting. (b) Mature DCs expressed HLA-DR, CD40, CD80, CD83, and CD86 but did not express CD14.

Figure 4

CD96 knockdown promoted the inflammatory response of CD4 T cells (three independent replicates were performed). (a) qPCR showed that the expression of CD96 in the shCD96 group was significantly inhibited compared to shNC group. (b) Western blotting showed that the level of p-Erk/Erk was significantly increased in the shCD96 group compared to the shNC group, but there was no significant difference observed in p-Akt/Akt. (c) Compared to the shNC group, the levels of TNF-α, IL-6, IL-17A, IFN-γ, IL-23, and IFN-γ were significantly increased in the cell culture supernatants of the shCD96 group (^∗P < 0.05,  ^∗∗P < 0.01).

Figure 5

CD96 overexpression alleviated the inflammatory response of CD4 T cells (three independent replicates were performed). (a) qPCR showed that the expression of CD96 in the CD96 group was significantly increased compared to the vector group. (b) Western blotting showed that the level of p-Erk/Erk was significantly downregulated in the CD96 group compared to vector group, but no significant difference was detected in p-Akt/Akt. (c) Compared to the vector group, the levels of TNF-α, IL-6, IFN-γ, IL-17A, and IL-23 were significantly decreased in the cell culture supernatants of the CD96 group (^∗P < 0.05).