Poster U19 - Dynamic biomarkers of T-cell senescence

Introduction: Cellular senescence is a hallmark of T-cell aging characterized by permanent cell cycle arrest, decreased activation capacity, and hypersecretion of pro-inflammatory molecules. Chronic exposure and unrepaired DNA damage during aging drives an increase in stressed, senescent T cells [1]. Senescent T cells are known to drive premature aging in solid organs and the age-associated decline of the immune system [2]. We are interested in developing an in vitro platform to study T-cell senescence to gain new insights and evaluate dynamic biomarkers of aging. Here, we show the development of an in vitro platform to induce senescence in T cells and identify dynamic motility patterns capable of identifying senescent from non-senescent cells.

Materials and

Methods: We induced senescent-like phenotypes in human peripheral blood derived CD4+ T cells by exposing them to repeated antigen stimulation using anti-CD3/CD28 Dynabeads over the course of 3 week intervals, plus or minus doxorubicin (a cancer drug). Cells were stained for protein-based biomarkers of senescence and imaged via immunofluorescence. Cells across all conditions were embedded within type-I collagen hydrogels, and we performed live-cell imaging for 3-hours using brightfield images. We then utilized open source image processing and tracking software to track the movement of single cells, from which we calculated biophysical features of cells. Following feature extraction, we performed dimensionality reduction to identify distinct patterns of single cell motility using CaMI [3].

Results, Conclusions, and Discussions: Chronic antigen exposure of CD4+ T cells over the course of 21 days completely diminishes their proliferative capacity (Fig. 1A) and leads to increased expression of biomarkers of senescence, p16 and yH2Ax. Coupling chronic stimulation and doxorubicin exposure (DNA damage) further inhibits T cell proliferation over time (Fig. 1B) and features increased biomarker expression. Chronically perturbed cells are characterized as having reduced motility along stimulation cycles, characterized by decreased speed, progressivity and increased tortuousness (Fig. 1C-E). This work provides a robust framework to induce senescence in CD4+ T cells and study the behavioral effects of senescence. This work offers a path to generating novel biomarkers of senescence in T-cells [4]. Furthermore, this platform could inform age-dependent immune phenotypes to determine whether T cells from older people are more vulnerable to senescence induction. Here we reveal that chronic activation and exposure to Doxorubicin to induce DNA damage leads to decreased T cell motility and the magnitude of dysfunction is time dependent.

Acknowledgements (Optional):

References: [1] Zhang et al. eBioMedicine 2021; 68:103409. [2] Yousefzadeh et al. Nature 2021; 594, 100–105. [3] Maity et al. bioRxiv 2022. [4] González-Bermúdez et al. Expert Reviews in Molecular Medicine 2022; 24, e35.