Immunotherapy-based targeting of senescent cells

Abstract

Cellular senescence is a state of permanent cell cycle arrest, characterized by substantial macromolecular alterations and senescence-associated secretory phenotype (SASP) in response to various stressors. The accumulation of senescent cells is thought to play a crucial role in aging-associated physiological decline and the pathogenesis of various age-related diseases. Clearance of senescent cells by transgenic or pharmaceutical strategies has been reported to attenuate various age-related pathologies and extend lifespan in mice. However, current senolytics still have limitations in terms of potency and specificity. Immunotherapy, particularly chimeric antigen receptor (CAR)-T cell therapy, emerges as a promising avenue to selectively eliminate senescent cells. Despite its potential, the effectiveness of this therapy hinges on the precise identification of senescence-associated surface antigens (senoantigens). Even though some surface markers for cellular senescence have been first identified in human fibroblasts, the translational potential of these findings is constrained by the limited senescent cell models previously established. The comprehensive identification of senescence-associated surface proteins remains to be achieved. <br /> First, to investigate the efficacy of CAR-T cells in targeting and eliminating senescent cells in vitro, we focused on the Natural killer group 2D (NKG2D) receptor, which binds to NKG2D ligands (NKG2DLs) expressed on the surface of senescent cells, providing a target for CAR-T cells. Employing mouse embryonic fibroblasts (MEFs) and astrocytes (AST) as senescence models, we demonstrate the elevated expression of NKG2DLs in response to genotoxic and oxidative stress. NKG2D-CAR T cells displayed potent cytotoxicity against these senescent cells, with minimal effects on non-senescent cells, suggesting their potential as targeted senolytics. Our research presents the first evidence of NKG2D-CAR T cells’ ability to target senescent brain cells, offering a novel approach to manage senescence-associated diseases. The findings pave the way for future investigations into the therapeutic applicability of NKG2D-targeting CAR-T cells in naturally aged organisms and models of aging-associated brain diseases <em>in vivo</em>. <br /> Furthermore, to identify novel cell surface markers for potential senotherapeutic targeting, we conducted an extensive analysis of the cell surface proteome, or “surfaceome”, in senescent cells, spanning various senescence induction regimes and encompassing both murine and human cell types. Utilizing quantitative mass spectrometry, we investigated enriched cell surface proteins across eight distinct models of senescence. Our results uncover significant changes in surfaceome expression profiles during senescence, highlighting extensive modifications in cell mechanics and extracellular matrix remodeling. Our research also reveals substantive heterogeneity of senescence, predominantly influenced by cell type and senescence inducer. We identified four cell surface proteins with extracellular epitopes, namely PLXNA1, PLXNA3, PTK7, and CYB5R1. These proteins are expressed in senescent cells, absent or present at low levels in their proliferating counterparts, and notably upregulated in tissues from aged mice and an Alzheimer's disease mouse model. These proteins emerge as promising candidates for senotherapeutic targeting, offering potential pathways for identifying and selectively targeting senescent cell populations in aging and age-related diseases.