IL-27 induces a cytotoxic state in CD4 ⁺ T cells distinct from the Th1 lineage

CD4 ⁺ cytotoxic T lymphocytes (CD4-CTLs) are understudied immune mediators with ambiguous origins. Despite expressing RUNX3, granzyme B (GZMB), and perforin (PRF1), CD4-CTLs are frequently classified as Th1 extensions due to shared interferon-γ (IFNγ) and T-BET expression. Here, we identify interleukin-27 (IL-27) as a independent inducer of a distinct CD4-CTL program. Proteomics reveals that while IL-27-polarized CD4+ T cells share protein signatures with conventional Th1s and CD8 ⁺ T cells, they possess a unique molecular landscape with re-wired cytokine signaling networks and a potent cytotoxic protein profile. Mechanistically, this program requires STAT1 and T-BET but operates independently of the autocrine IFNγ feedback that sustains Th1 cells. During acute murine cytomegalovirus infection, IL-27 receptor signaling contributes to CD4-CTL differentiation in vivo , as its loss leads to reduced GZMB expression and skews CD4 ⁺ T cells toward IFNγ ⁺ and FOXP3 ⁺ subsets. Together, these findings establish IL-27 as a potent and previously unappreciated inducer of CD4-CTLs.