Journal: Aging Cell

Article Title: Reduction of class I histone deacetylases ameliorates ER ‐mitochondria cross‐talk in Alzheimer's disease

doi: 10.1111/acel.13895

Figure Lengend Snippet: Tac effect on InsP3R1‐VDAC1 interaction and MERCS morphology in AβO‐treated cells. HT22 cells were pre‐treated for 1 h with Tac (10 μM) and then co‐incubated with 1 μM AβO for the remaining 23 h or treated solely with Tac for 24 h. Representative confocal images of in situ PLA signal (gray) indicating a physical interaction between InsP3R1 and VDAC1 (scale bar = 20 μm). The lower panels represent enlargements of the boxed areas in the upper panels. Quantification of number of PLA puncta in about 200–240 cells from 8 image stacks per condition (a). WT hippocampal neurons were pre‐treated for 1 h with Tac (0.5 μM) and then co‐incubated with 1 μM AβO for the remaining 23 h or treated solely with Tac for 24 h. NFAT transcriptional activity evaluated by luciferase reporter assay in hippocampal neurons (b) and HT22 cells (c). Data are the mean ± SEM of 4 independent experiments, run in triplicates to quadruplicates. Schematic representation of MERCS structural parameters: MERCS length and distance, mitochondrial perimeter and Feret's diameter (d). HT22 cells were pre‐treated for 1 h with Tac (10 μM) and then co‐incubated with 1 μM AβO for the remaining 23 h. Representative TEM images of mitochondria (in purple) in close contact with ER (in blue) (scale bar = 200 nm) in which distances ≤25 nm were considered as contacts (e). MERCS length (f) and distance (g), number of MERCS per mitochondria (h), mitochondrial area (i) and perimeter (j), Feret's diameter (k) and mitochondrial aspect ratio (l). Representative TEM images of mitochondrial cristae (in green) (scale bar = 200 nm) (m). Number of cristae per mitochondria (n), and cristae area (o). About 70–90 individual mitochondria from 10 randomly selected cells obtained from approximately 30 images were assessed in 26–38 independent TEM images. Statistical analysis: Kruskal–Wallis test followed by uncorrected Dunn's multiple comparisons test, one‐way ANOVA followed by uncorrected Fisher's LSD multiple comparison test; * p < 0.05; ** p < 0.01; *** p < 0.0001; **** p < 0.0001 when compared to control.

Article Snippet: NFAT luciferase reporter was obtained from Addgene.

Techniques: Incubation, In Situ, Activity Assay, Luciferase, Reporter Assay, Comparison, Control

Journal: Cells

Article Title: NeoPAIR-T: Functional Mapping of Neoantigen–TCR Pairs Using a CRISPR-Engineered Jurkat Reporter System

doi: 10.3390/cells14221789

Figure Lengend Snippet: Establishment of reporter T cells and CRISPR/Cas9-mediated knock-in of a candidate T cell receptor (TCR). ( A ) Surface marker expression of parental Jurkat E6-1 cells. Histograms show the expression levels of the indicated markers. ( B ) Surface marker expression of engineered reporter T cells. Histograms show the expression levels of the indicated markers. ( C ) Schematic overview of the genetic modifications performed to generate reporter T cells. Parental Jurkat E6-1 cells were subjected to CRISPR/Cas9-mediated knockout of CD4, FAS, and TCRα, and lentiviral transduction of CD8α, CD8β, Cas9, and the NFAT–Luc–P2A–eGFP reporter cassette. ( D ) Targeted reconstitution of the TCR in reporter T cells at the endogenous TCRβ locus. ( E ) Flow cytometric analysis of CD3 restoration in reporter T cells after targeted reconstitution with the NY-ESO-1–specific 1G4 TCR. CD3 + cells were sorted to generate a uniform population of TCR-expressing reporter T cells. Pseudocolor plots indicate event density (blue, lower; red, higher).

Article Snippet: Finally, these cells were transduced with a lentiviral NFAT-Luciferase-eGFP reporter construct ( , BPS Bioscience, San Diego, CA, USA) at MOI of 20 and selected with 2 mg/mL G418 (FujiFilm Wako Pure Chemical Corporation).

Techniques: CRISPR, Knock-In, Marker, Expressing, Knock-Out, Transduction

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , Binding affinities to human HER2 and CD3 at 37 °C by surface plasmon resonance. Data are K D ( n = 12 technical replicates of a single experiment for HER2-XPAT protein and HER2(1x-N); n = 8 for HER2(1x-C) and uTCE). Surface plasmon resonance sensorgrams for these data are provided in Supplementary Figs. – . b – d , In vitro tumor cytotoxicity of HER2-XPAT protein and its metabolites following a 48-h incubation with co-cultures of huPBMCs and the high HER2-expressing human tumor cell lines (1:1 effector–target ratio) SKOV3 ( b ), BT-474 ( c ) or the medium-low HER2-expressing MCF7 cell line ( d ). e , In vitro cytotoxicity of HER2-XPAT protein and its metabolites against BT-474 cells co-cultured with huPBMCs (1:1 effector–target ratio). f , Impact of the protease-cleavable linker on in vitro cytotoxicity versus BT-474 cells co-cultured with huPBMCs. g , h , CD69-positive T cells ( g ) and IL-2 secretion ( h ) following 72-h incubation of huPBMC/SKOV3 co-cultures with HER2-XPAT protein or its unmasked form (uTCE). i , Target-dependent T-cell activation with HER2-XPAT protein and its metabolites. CD3-expressing Jurkat reporter T cells were incubated with BT-474 cells at a 5:1 effector–target ratio for 6 h, followed by quantification of NFAT -induced luciferase activity and measured in relative luminescence units (RLUs). Mean data for n = 2 technical replicates within one single experiment ( b – i ). Extended Data Table provides a summary of EC 50 values for the different forms of the HER2-XPAT proteins in the cytotoxicity and reporter T-cell activation assays.

Article Snippet: The activation of CD3 + Jurkat T cells (Promega, J1625, J1601) by HER2-XPAT protein and unmasked HER2-TCE was assessed.

Techniques: Binding Assay, SPR Assay, In Vitro, Incubation, Expressing, Cell Culture, Activation Assay, Luciferase, Activity Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: CD3 + Jurkat reporter T cells were incubated with or without BT-474 cells at a 5ː1 effector-target ratio for 6 hours, followed by quantification of NFAT -induced luciferase activity. The graph shows individual data points from a single representative experiment. There were n = 2 biological repeats at each concentration tested within the same experiment for each cell line. HER2, human epidermal growth factor receptor 2; RLU, relative luminescence unit; uTCE, unmasked T-cell engager; XPAT proteins, TCE fused to XTEN polypeptides.

Article Snippet: The activation of CD3 + Jurkat T cells (Promega, J1625, J1601) by HER2-XPAT protein and unmasked HER2-TCE was assessed.

Techniques: Incubation, Luciferase, Activity Assay, Concentration Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , An XPAT protein comprises a TCE core with two scFvs, one targeting CD3 and the other, a TAA. Each scFv is masked by a protease-releasable XTEN mask, unstructured, hydrophilic polypeptides that act as modular, tunable masks, in addition to extending the half-life of the TCE. Each XTEN mask connects to the TCE core via a protease-cleavable linker, designed to be cleavable by any of eight proteases from three different classes (matrix metalloproteinases, serine proteases and cysteine proteases) involved in cancer progression . b , Predicted structure of HER2-XPAT protein visualized using AlphaFold2 v.2.0, a machine-learning-based computational method for predicting protein structures with reasonable accuracy . Colors indicate anti-HER2 domain, pale green; anti-CD3 domain, light orange; XTEN masks, blue; protease-cleavable linker, red; linkers, gray. The model represents a static picture showing a plausible conformation of the unstructured XTEN masks and the length of unstructured XTEN relative to the folded antibody domains in an XPAT protein. c , XPAT proteins are expected to remain largely intact in healthy tissues, where protease activity is well controlled by protease inhibitors. XPAT protein unmasking occurs in two steps via one of two potential paths to the fully unmasked state. The two requisite cleavage events can occur in either order and each sequence (either the top or bottom paths shown) is equally likely. In aggregate, both 1x-N and 1x-C partially unmasked forms will exist, depending on the cleavage path. Removal of both XTEN masks liberates the unmasked HER2-TCE (uTCE). d , XPAT proteins are designed to exploit the dysregulated protease activity present in tumors versus healthy tissues and expand the therapeutic index of TCEs through preferential unmasking in the TME. The active uTCE promotes the formation of immunologic synapses between tumor and T cells, resulting in potent cytotoxicity. Notably, the uTCE has a short half-life and should be rapidly cleared, thereby sparing healthy tissues when the uTCE diffuses away from the TME. By design, the molecular weight of the uTCE (∼59 kDa) is sufficiently small to allow rapid kidney filtration .

Article Snippet: The activation of CD3 + Jurkat T cells (Promega, J1625, J1601) by HER2-XPAT protein and unmasked HER2-TCE was assessed.

Techniques: Activity Assay, Sequencing, Molecular Weight, Filtration