Characterization of CD8+ virtual memory T cells in IL-4 knockout mice using single-cell RNA sequencing

Antigen-inexperienced memory-phenotype CD8⁺ T cells are categorized as innate memory cells in the thymus or virtual memory (VM) CD8⁺ T cells in peripheral tissues. The key distinction between these cell types is their differing responses to IL-4, but the minimal effect of IL-4 on VM CD8⁺ T cell expansion in the periphery is not well understood. To address this, we investigated the development of VM CD8⁺ T cells in the periphery of IL-4 knockout (KO) C57BL/6 mouse. CD8⁺ splenocytes were isolated from the spleen of wilt-type (WT) and IL-4 KO mice, followed by single-cell RNA sequencing and Seurat analysis on sorted CD8⁺ cells using the 10x Genomics platform. This study identified various CD8⁺ T cell subtypes, including naïve, effector, IFN-stimulated, true memory (TM), and VM T cells. VM CD8⁺ T cells were characterized by high expression of Cd44, Cxcr3, Il2rb, Eomes, Tbx21, Ly6c2, and low expression of Itga4. In IL-4-deficient mouse, macrophages were significantly reduced, while memory T cell populations showed a slight increase compared to WT mouse. Both Itga4⁺ TM and Itga4^- VM CD8⁺ T cells were more abundant in IL-4 KO mouse. Within the VM T cell group, Ly6a^- VM CD8⁺ T cells were reduced, while Ly6a ⁺ VM CD8⁺ T cells were increased relative to WT mouse. These Ly6a⁺ VM CD8⁺ cells exhibited high expression of genes linked to type I IFN signaling, such as Isg15, Ifit1, and Stat1. Our findings suggest that IFN-influenced Ly6a ⁺ VM CD8⁺ T cells play a role in maintaining the peripheral VM CD8⁺ T cell population in the absence of IL-4.