CD226 identifies effector CD8+ T cells during tuberculosis and costimulates recognition of Mycobacterium tuberculosis-infected macrophages

CD8⁺ T cells defend against Mycobacterium tuberculosis (Mtb) infection but variably recognize Mtb-infected macrophages. To investigate how chronic infection affects the diversity of lung parenchymal CD8⁺ T cells, we perform single-cell RNA sequencing (scRNA-seq) on cells from C57BL/6J mice infected for 6 and 41 weeks. We identify an effector lineage, including a cluster that expresses high levels of cytotoxic effectors and cytokines, and a dysfunctional lineage that transcriptionally resembles exhausted T cells. The most significantly differentially expressed gene between two distinct CD8⁺ T cell lineages is Cd226. Mtb-infected interferon (IFN)γ-enhanced yellow fluorescent protein (EYFP) reporter mice reveal that IFNγ production is enriched in CD226⁺CD8⁺ T cells, confirming these as functional T cells in vivo. Purified CD226⁺ but not CD226^- CD8⁺ T cells recognize Mtb-infected macrophages, and CD226 blockade inhibits IFNγ and granzyme B production. Thus, efficient CD8⁺ T cell recognition of Mtb-infected macrophages requires CD226 costimulation, and CD226 expression identifies CD8⁺ T cells that recognize Mtb-infected macrophages.