Pattu, V.; Krause, E.; Chang, H.-F.; Rettig, J.; Li, X.

CD8+ T lymphocytes (CTLs) function as serial killers of infected or tumorigenic cells by secreting large quantities of interferon-gamma (IFN{gamma}) and granzyme B (GzmB). IFN{gamma} is a pleiotropic cytokine, its expression dynamics and precise role in CTL-mediated cytotoxicity remain incompletely understood. To investigate the relationship between IFN{gamma} expression and CTL cytotoxicity, we analyzed IFN{gamma} production, subcellular localization, cellular subtypes, degranulation capacity, and early activation markers in IFN{gamma}-expressing CTLs. First, we found that IFN{gamma} expression was rapid, transient, and required secondary T-cell receptor (TCR) stimulation, whereas GzmB was pre-stored in resting CTLs. Notably, two distinct IFN{gamma}-expressing subsets, IFN{gamma}hi and IFN{gamma}lo, were identified, while only a single GzmB-expressing population was observed. Subsequently, we classified IFN{gamma}-expressing CTLs into effector/effector memory (TE/TEM) and central memory (TCM) subsets. IFN{gamma}hi cells exhibited a predominantly TE/TEM phenotype, whereas IFN{gamma}lo cells showed reduced effector characteristics and a higher proportion of TCM. Next, degranulation assays revealed that IFN{gamma}hi CTLs displayed significantly higher surface expression of the lytic granule marker CD107a (33.75%) compared to IFN{gamma}lo CTLs (3.76%), despite comparable intracellular GzmB levels. This suggests that elevated IFN{gamma} production correlates with CTL killing capacity. Further analysis demonstrated that IFN{gamma}hi CTLs exhibited extensive colocalization with cis-Golgi and GzmB, indicating robust IFN{gamma} synthesis and efficient sorting of mature IFN{gamma} into cytotoxic granules (CGs) relative to IFN{gamma}lo CTLs. Finally, we compared IFN{gamma} and CRTAM expression in naive versus activated CD8+ T cells upon TCR stimulation, demonstrating that transient CRTAM expression may critically regulate initial CTL activation and IFN{gamma} production.