Heterozygous OT-I mice reveal that antigen-specific CD8⁺ T cells shift from apoptotic to necrotic killers in the...

Numerous alterations in CD8⁺ T cells contribute to impaired immune responses in elderly individuals. However, the discrimination between cell-intrinsic dysfunctions and microenvironmental changes is challenging. TCR transgenic OT-I mice are utilized to investigate CD8⁺ T-cell immunity, but their immunodeficient phenotype hampers their use especially in aging. Here, we demonstrate that using a heterozygous OT-I model minimizes the current limitations and provides a valuable tool to assess antigen-specific T-cell responses even at old age. We analyzed phenotypic and functional characteristics of CD8⁺ T cells from OT-I^+/+ and OT-I^+/- mice to prove the applicability of the heterozygous system. Our data reveal that OVA-activated CD8⁺ T cells from adult OT-I^+/- mice proliferate, differentiate, and exert cytolytic activity equally to their homozygous counterparts. Moreover, common age-related alterations in CD8⁺ T cells, including naive T-cell deterioration and decreased proliferative capacity, also occur in elderly OT-I^+/- mice, indicating the wide range of applications for in vivo and in vitro aging studies. We used the OT-I^+/- model to investigate cell-intrinsic alterations affecting the cytotoxic behavior of aged CD8⁺ T cells after antigen-specific in vitro activation. Time-resolved analysis of antigen-directed target cell lysis confirmed previous observations that the cytotoxic capacity of CD8⁺ T cells increases with age. Surprisingly, detailed single cell analysis revealed that transcriptional upregulation of perforin in aged CD8⁺ T cells shifts the mode of target cell death from granzyme-mediated apoptosis to rapid induction of necrosis. This unexpected capability might be beneficial or detrimental for the aging host and requires detailed evaluation.