Lytic IFNγ is stored in cytotoxic granules and coreleased with granzyme B to mediate cytotoxic T lymphocyte killing.

Cytotoxic T lymphocytes (CTLs) eliminate target cells by forming immunological synapses and releasing effector molecules, including interferon gamma (IFNγ). However, how IFNγ contributes to cytotoxicity remains unclear. Here, we identify a subset of IFNγ stored within granzyme B⁺ cytotoxic granules (CGs) in activated mouse and human CTLs, which we term lytic IFNγ. This CG-associated IFNγ represents the primary pool released in a polarized manner at the immunological synapse together with canonical lytic molecules. Lytic IFNγ is present in tumor-infiltrating CTLs and is cosecreted with granzyme B (GzmB) in both soluble form and as part of supramolecular attack particles (SMAPs). Functional assays indicate that IFNγ contributes to CTL-mediated tumor cell death by acting in concert with granzyme B and perforin to increase cytotoxicity and promote apoptosis via the IFNγ-STAT1-caspase-3 pathway. CTLs lacking the vesicle priming factor Munc13-4 exhibit impaired release of both CGs and early-phase IFNγ. However, prolonged synapse engagement restores IFNγ secretion at distal membrane sites, revealing a second, nonpolarized IFNγ pool. Consistently, endogenous IFNγ is detected in both CG-enriched and multivesicular body (MVB)-enriched fractions. We propose that while lytic IFNγ is released from CGs at the synapse to directly promote target cell killing, nonpolarized IFNγ secretion originates from MVBs or small vesicles during sustained activation. Together, these findings reveal a previously unrecognized mechanism of IFNγ storage and release, establishing lytic IFNγ as a critical effector component of CTL cytotoxicity and antitumor immunity.