Varsha Pattu

Background: CD8+ T lymphocytes (CTLs) act as serial killers of infected or malignant cells by releasing large amounts of interferon-gamma (IFNγ) and granzymes. Although IFNγ is a pleiotropic cytokine with diverse immunomodulatory functions, its precise spatiotemporal regulation and role in CTL-mediated cytotoxicity remain incompletely characterized. Methods: Using wild-type (WT) and granzyme B-mTFP knock-in mice, we combined in vitro approaches, including T-cell isolation and culture, plate-bound anti-CD3e stimulation, degranulation assays, flow cytometry, immunofluorescence, and structured illumination microscopy, to investigate IFNγ dynamics in CTLs. Results: IFNγ expression in CTLs was rapid, transient, and strictly dependent on T-cell receptor (TCR) activation. We identified two functionally distinct IFNγ-producing subsets: IFNγ ^high (IFNγ ^hi ) and IFNγ ^low (IFNγ ^lo ) CTLs. IFNγ ^hi CTLs exhibited an effector/effector memory phenotype, significantly elevated CD107a surface expression (a marker of lytic granule exocytosis), and pronounced colocalization with cis-Golgi and granzyme B compared to IFNγ ^lo CTLs. Furthermore, CRTAM, an early activation marker, correlated with IFNγ expression in naive CTLs. Conclusion: Our findings establish a link between elevated IFNγ production and enhanced CTL cytotoxicity, implicating CRTAM as a potential regulator of early CTL activation and IFNγ induction. These insights provide a foundation for optimizing T cell-based immunotherapies against infections and cancers.

Cytotoxic CD8⁺ T cells form immunological synapses with target cells and release effector molecules, including IFNγ, to mediate antitumor immunity. However, the mechanisms by which IFNγ contributes to cytotoxicity remain incompletely understood. Here, we identify a subset of IFNγ stored within GzmB⁺ cytotoxic granules (CGs) in activated mouse and human CD8⁺ T cells, termed ’lytic IFNγ’. Lytic IFNγ is polarized to the synapse and co-secreted with GzmB in both soluble and supramolecular attack particle (SMAP)-associated forms. Mouse CD8⁺ T cells lacking the vesicle priming factor Munc13-4 exhibit impaired both CG and early IFNγ release at the immunological synapse, while prolonged synaptic engagement restores IFNγ secretion. Super-resolution imaging demonstrates that sustained synaptic interactions drive IFNγ secretion at distal membrane sites, suggesting the existence of distinct IFNγ populations with potentially diverse functions beyond lytic IFNγ. These findings uncover an unrecognized mechanism of IFNγ storage and release, underscoring its pivotal role in CD8⁺ T cell-mediated tumor elimination.

SYNTAXIN-11 (STX11) is a SNARE protein that mediates the fusion of cytotoxic granules with the plasma membrane at the immunological synapses of CD8 T or NK cells. Autosomal recessive inheritance of deleterious STX11 variants impairs cytotoxic granule exocytosis, causing familial hemophagocytic lymphohistiocytosis type 4 (FHL-4). In several FHL-4 patients, we also observed hypogammaglobulinemia, elevated frequencies of naive B cells, and increased double-negative DN2:DN1 B cell ratios, indicating a hitherto unrecognized role of STX11 in humoral immunity. Detailed analysis of Stx11-deficient mice revealed impaired CD4 T cell help for B cells, associated with disrupted germinal center formation, reduced isotype class switching, and low antibody avidity. Mechanistically, Stx11−/− CD4 T cells exhibit impaired membrane fusion leading to reduced CD107a and CD40L surface mobilization and diminished IL-2 and IL-10 secretion. Our findings highlight a critical role of STX11 in SNARE-mediated membrane trafficking and vesicle exocytosis in CD4 T cells, important for successful CD4 T cell–B cell interactions. Deficiency in STX11 impairs CD4 T cell–dependent B cell differentiation and humoral responses.