Targeting senescent cells with NKG2D-CAR T cells

Abstract

This study investigates the efficacy of NKG2D chimeric antigen receptor (CAR) engineered T cells in targeting and eliminating stress-induced senescent cells in vitro. Cellular senescence contributes to age-related tissue decline and is characterized by permanent cell cycle arrest and the senescence-associated secretory phenotype (SASP). Immunotherapy, particularly CAR-T cell therapy, emerges as a promising approach to selectively eliminate senescent cells. Our focus is on the NKG2D receptor, which binds to ligands (NKG2DLs) upregulated in senescent cells, offering a target for CAR-T cells. Using 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. In conclusion, 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 in vivo.

Fig. 1. Construction of NKG2D‑based CAR‑T cells.

A Schematic representation of retroviral (RV) and lentiviral (LV) NKG2D-CAR constructs encoding full-length mouse NKG2D receptor and cytoplasmic CD3ζ. B Western blot analysis of protein expression of FLAG-tagged NKG2D-CAR using anti-FLAG antibody in LV NKG2D-CAR construct-transfected and untransfected (UT) HEK293T cells. Data are presented as the mean ± SEM of three biological replicates; normalized to GAPDH protein level. ****P < 0.0001, determined by unpaired two-tailed Student’s t-test analysis. C Immunofluorescence co-staining of LV NKG2D-CAR construct-transfected HEK293T cells. Cells were co-stained with mouse NKG2D (red) and CD3ζ (purple) antibodies. Cell nuclei were stained with DAPI (blue). Orange and white arrows point to examples of NKG2D-CAR+ and NKG2D-CAR- cells, respectively. Scale bar = 10 μm. Cell viabilities (D) and transduction efficiencies (F) of RV and LV NKG2D-CAR-transduced T cells labelled with cell viability dye, analyzed by FACS, three to 4 days post-second round of transduction. The transduction efficiency was calculated as % mCherry/GFP/ZsGreen+ cells among viable cells. Data are presented as the mean ± SEM of six biological replicates. ns not significant, determined by one-way ANOVA followed by Tukey’s post-hoc tests. E Fluorescence microscopy images of packaging Plat-E cells (upper row) and T cells transduced with RV NKG2D-CAR 24 h post-second round of transduction (lower row). Scale bar = 200 μm. G Surface expression of NKG2D assessed by FACS. Histograms show the percentage of positive cells stained with APC-conjugated anti-mouse NKG2D antibody (purple) or isotype antibody (green). MFI of APC are presented as the mean ± SEM of six biological replicates. ***P < 0.001; ****P < 0.0001, determined by one-way ANOVA followed by Tukey’s post-hoc tests. H CD8 and CD4 subtypes of CD3 + UN-T cells and NKG2D-CAR T cells analyzed by FACS. NKG2D-CAR T cells were sorted by viable GFP+ or mCherry+ cells prior to analysis. Data are presented as the mean ± SEM of two biological replicates and determined by two-way ANOVA followed by Tukey’s post-hoc tests.

Fig. 2. Functional validation of NKG2D-CAR T cells.

A Schematic representation of PiggyBac construct encoding individual mouse NKG2DLs. B The surface expression of each mouse NKG2DL determined by FACS. Flow plots (upper panel) and histograms (lower panel) show the percentage of positive cells and MFI of PE-labeled mouse NKG2DL in UT-B16F10 cells (control) and NKG2DLs stably transfected B16F10 cells. The results are based on four independent technical experiments for each transfected cell colony. FSC-A, Forward-scatter area. C Schematic of Calcein-AM-based cytotoxicity assay. D Representative whole-well images of NKG2DL-transfected and UT-B16F10 target cells stained with Calcein-AM before (0 h) and after 4 h of co-culture with NKG2D-CAR T cells or UN-T cells at an E: T ratio of 20:1. Live cells labeled by Calcein-AM are shown in green. Scale bar = 800 μm. E Quantification of cytotoxicity of NKG2D-CAR T cells and UN-T cells against NKG2DLs over-expressing B16F10 cells and B16F10 control target cells at E: T ratios of 1:1, 10:1, and 20:1 after 4 h of co-culture. Data are presented as the mean ± SD of three biological replicates. **P < 0.01; ***P < 0.001; ****P < 0.0001, determined by two-way ANOVA followed by Tukey’s post-hoc tests. BF, B16F10.

Fig. 3. NKG2D ligands are expressed in senescent cells induced by DNA damage and oxidative stress.

A Representative images of SA-β-gal staining of MEFs (upper panel) and AST (lower panel) treated with or without ETO or H2O2 visualized under bright-field microscopy. Blue cells indicate SA-β-gal positive cells. Scale bar = 100 μm. Quantification of % SA-β-gal positive cells was performed in randomly selected view fields. Data are presented as the mean ± SEM of six biological replicates. B Western blot analysis (left panel) and quantification (right panel) of protein expression of senescence markers in MEFs and AST with or without ETO or H2O2 treatment. The band intensity of a given target protein was normalized to the corresponding actin signal for each sample. Data were normalized to the average of the corresponding control group and are presented as the mean ± SEM, based on n = 4 biological replicates. Statistical significance was assessed using one-way ANOVA followed by Tukey’s post-hoc tests, where appropriate, to compare each senescence marker within a specific cell type across various conditions (control and treatments). C qPCR analysis of mRNA expression of mouse NKG2DLs in senescent MEFs and AST induced by ETO or H2O2 treatment. Data are presented as the mean ± SEM of three or four biological replicates; normalized to β-actin mRNA level. D Cell surface expression of each mouse NKG2DL in senescent MEFs and AST (filled purple) compared to the corresponding proliferative control cells (green lines) determined by FACS. MFI of PE-labeled mouse NKG2DLs are presented as mean ± SEM of three biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, determined by one-way ANOVA followed by Tukey’s post-hoc tests (A–D).

Fig. 4. Cytotoxicity of NKG2D-CAR T cells against stress-induced senescent MEFs.

A Representative whole-well images of senescent MEFs induced by ETO or H2O2 treatment and untreated proliferative MEFs, stained with Calcein-AM, captured every 2 h during the 8 h of co-culture with NKG2D-CAR T cells or UN-T cells at an E: T ratio of 10:1. Live cells were labeled by Calcein-AM in green. Scale bar = 800 μm. B Quantification of cytotoxicity of NKG2D-CAR T cells and UN-T cells against senescent MEFs and proliferative MEFs at E: T ratios of 1:1, 5:1, and 10:1 after 8 h of co-culture. Data are presented as the mean ± SD of four biological replicates. ***P < 0.001; ****P < 0.0001, determined by two-way ANOVA followed by Tukey’s post-hoc tests.

Fig. 5. Cytotoxicity of NKG2D-CAR T cells against stress-induced senescent mouse AST.

A Representative whole-well images of senescent AST induced by ETO or H2O2 treatment and untreated proliferative AST, stained with Calcein-AM, captured every 2 h during the 8 h of co-culture with NKG2D-CAR T cells or UN-T cells at an E: T ratio of 10:1. Scale bar = 800 μm. B Quantification of cytotoxicity of NKG2D-CAR T cells and UN-T cells against senescent AST and proliferative AST at E: T ratios of 1:1, 5:1, and 10:1 after 8 h of co-culture. Data are presented as the mean ± SD of four biological replicates. ****P < 0.0001, determined by two-way ANOVA followed by Tukey’s post-hoc tests.

Fig. 6. Selective elimination of NKG2DLs-expressing senescent MEFs and mouse AST by NKG2D-CAR T cells.

NKG2D-CAR consists of an antigen recognition domain-NKG2D receptor fused to a CD3ζ cytoplasmic signaling domain. T cells are engineered with NKG2D-CAR-encoding retroviral or lentiviral vectors. Genotoxic insults and oxidative stress induce surface expression of NKG2DLs on MEFs and AST. Activated NKG2D-CAR T cells specifically recognize NKG2DLs expressed on the surface of senescent MEFs and AST, subsequently exerting cytotoxic effects on the target cells. SEN senescence.