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

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.

Faster cytotoxicity with age: Increased perforin and granzyme levels in cytotoxic CD8+ T cells boost cancer cell elimination.

A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8+ T cells responses in the elderly has come back into focus since the COVID-19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8+ T cells in aging. We focused on the different subpopulations and time-resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8+ T cells from elderly mice are much faster than those of CD8+ T cells from adult mice. This is true not only in the total CD8+ T cell population but also for their effector (TEM ) and central memory (TCM ) T cell subpopulations. TIRF experiments reveal that CD8+ T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8+ T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell-intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked.