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

Abstract

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.

Keywords: CD8+ T cells; OT-I; adaptive immunity; aging; apoptosis; cytotoxicity; necrosis; perforin.

FIGURE 1

Conserved expression of the V_α2/V_β5 transgenic T‐cell receptor in CD8⁺ T cells from adult and elderly OT‐I^+/− mice. (a) Representative flow cytometric dot plots of V_α2 and V_β5 TCR chain expression in isolated CD8⁺ T cells from OT‐I^+/+, OT‐I^+/−, and C57BL/6J mice (WT). (b) Quantification of V_α2⁺V_β5⁺ and V_α2⁻V_β5⁻ cells among the CD8⁺ T‐cell populations. Data are presented as mean ± SEM, n = 4–5.

FIGURE 2

Subtype distribution and proliferative capacity of CD8⁺ T cells from OT‐I^+/+ and OT‐I^+/− mice. Flow cytometry‐based analysis of subtype distribution in unstimulated (a) and stimulated (b) CD8⁺ T cells from male (n = 6–10) and female (n = 5–11) OT‐I^+/+ and OT‐I^+/− mice. T‐cell subsets were defined based on CD62L and CD44 surface expression: T_N: CD62L^highCD44l^ow, T_CM: CD62L^highCD44^high, and T_EM: CD62L^lowCD44^high. (c) Flow cytometry‐based proliferation assay with CD8⁺ T cells from male and female OT‐I^+/+ and OT‐I^+/− mice. CD8⁺ T cells were stimulated with irradiated E.G7‐OVA mouse lymphoma cells, and cell divisions were quantified through dilution of cytoplasmatic dye CFSE 48 h after stimulation. Data are presented as mean ± SEM, n = 3–5.

FIGURE 3

Faster cytotoxicity of CD8⁺ T cells from elderly OT‐I^+/− mice. Time‐resolved killing assays with CD8⁺ T cells from male (a) and female (d) OT‐I^+/+ and OT‐I^+/− mice 3 days after stimulation with irradiated E.G7‐OVA mouse lymphoma cell line. E.G7‐OVA cells were used as target cells in an effector‐to‐target ratio of 10:1. Box plots represent the average target cell lysis after 60, 120, and 240 min (b, e) and the maximum target lysis per 10 min (c, f) as a measure of the kinetics. Data are presented as mean ± SEM, n = 5–11.

FIGURE 4

Increased mRNA expression of perforin, granzyme B, and Fas ligand in CD8⁺ T cells from elderly OT‐I^+/− mice. Normalized mRNA expression of perforin (a), granzyme B (b) and Fas ligand (c) in CD8⁺ T cells from OT‐I^+/+ and OT‐I^+/− mice. Expression levels were normalized to the reference genes hypoxanthine‐phosphoribosyl transferase 1 (HPRT1) and TATA box binding protein (TBP). Data from adult OT‐I^+/+ and elderly OT‐I^+/− mice are presented as relative fold change to the mRNA levels from adult OT‐I^+/− mice, respectively. Data are presented as mean ± SEM, n = 6–8.

FIGURE 5

CD8⁺ T cells from elderly OT‐I^+/− mice induce rapid necrotic target cell death. Cytotoxicity assays with activated OVA‐specific CD8⁺ T cells from OT‐I^+/+ and OT‐I^+/− mice. EG.7‐OVA pCasper cells were used as target cells to distinguish between viable (orange), apoptotic (green) and necrotic (fluorescence loss) cells with an effector‐to‐target ratio of 2:1. (a) Representative overlays of brightfield, GFP (green), and FRET (red) fluorescence at the indicated time points after effector cell contact. Images were acquired every 2 min for 4 h. (b) The percentages of viable, apoptotic, and necrotic cells were determined for each time point and plotted as color‐coded graphs over time. (c) Statistical quantification was performed at 60, 120, and 240 min. Data are presented as mean ± SEM, n = 4–6, scale bar 50 μm.