IL-4, IL-15, and Type I Interferon Orchestrate the Shaping of the Heterogeneity of Virtual Memory CD8 T Cells

The development of virtual memory CD8 T cells is dependent on IL-4, type I interferon, and IL-15. However, it remains unclear whether these cytokines individually contribute to the generation of specific subsets of virtual memory CD8 T cells. In this study, virtual memory CD8 T cells were categorized into four subsets based on Ly6C and Sca-1 expression, and their development was examined using knock-out mice lacking IFNAR1, IL-4, or IL-15Rα. Notably, both Ly6C⁺ Sca-1⁺ and Ly6C^- Sca-1⁺ subsets were significantly reduced in the spleen of IFNAR1 knock-out mice, while the proportion of Ly6C⁺ Sca-1^- VM CD8 T cells was reduced in IL-4-deficient mice. In IL-15Rα knock-out mice, both the Ly6C⁺ Sca-1^- and Ly6C^- Sca-1^- subsets were significantly reduced. Bulk RNA sequencing analysis revealed distinct gene expression patterns in naïve cells, true memory cells, and the four virtual memory cell subsets. Specifically, Ly6C⁺ subsets were enriched with IL-15 signal-related genes, whereas Ly6C^- subsets and true memory cells were enriched for cell cycle-related genes. Functionally, the Ly6C⁺ and/or Sca-1⁺ subsets exhibited higher production of IFN-γ and TNF-α compared with the Ly6C^- Sca-1^- subsets. Overall, this study demonstrates the heterogeneity of virtual memory CD8 T cells and highlights the cytokine-dependent nature of their development.