Journal: iScience

Article Title: Transient tumor exposure induces persistent functional defects in memory CD8 + T cells

doi: 10.1016/j.isci.2026.115556

Figure Lengend Snippet: Phenotypic and transcriptional differences of memory CD8 + T cells generated after a viral or a tumoral challenge Naive F5 x CD45.1 cells (2.10 5 ) were i.v. transferred in B6 mice 1-day prior immunization with VV-NP68 (i.n., 2.10 5 pfu) or EL4-NP68 cells (s.c., 2,5.10 6 cells). (A) Viral load was measured in the lung by qPCR, or tumor volume (mm 3 ) was assessed by measuring its length, width, and thickness over time. (B) The number of Vir-CD8 + and Tum-CD8 + cells was determined over time in the blood by flow cytometry. (C and D) The expression of Ki67 (C) and Bcl2 (D) by Vir-CD8 + and Tum-CD8 + cells was measured over time in the blood. (E) The phenotype of Vir-CD8 + and Tum-CD8 + cells was analyzed 31 days after infection in the spleen, and the percentages of cells expressing each marker are represented as a heatmap. The statistical significance of differences was determined using a two-way ANOVA (C–E) (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001). Data are represented as mean ± SD and are representative of 3 independent experiments ( n = 5 to 10 mice per group). (F–I) 60 days after immunization, naive F5 and Vir-CD8 + and Tum-CD8 + cells were single-cell sorted, and stimulated with NP68 peptide (10 nM) for 2 h or left untreated. The transcriptome was analyzed by scRNAseq ( n = 476 cells). (F) Clustering of cells projected on a UMAP colored by populations. (G) Proportion of sorted populations in each cluster. (H and I) Volcano plot of the differentially expressed genes between quiescent (H) or restimulated (I) Vir-CD8 + and Tum-CD8 + .

Article Snippet: After 7 days, NP68 peptide (20 nM) and CpG ODN 1826 (2 mg/mL, InvivoGen, tlrl-1826) were added to the BMDC and cultured overnight before being washed and used for CD8 + T cells activation.

Techniques: Generated, Flow Cytometry, Expressing, Infection, Marker, Single Cell

Journal: iScience

Article Title: Transient tumor exposure induces persistent functional defects in memory CD8 + T cells

doi: 10.1016/j.isci.2026.115556

Figure Lengend Snippet: Tum-CD8 + memory cells express molecules associated with T cell exhaustion Naive F5 x CD45.1 cells (2.10 5 ) were i.v. transferred in B6 mice 1-day prior immunization with VV-NP68 (i.n., 2.10 5 pfu) or EL4-NP68 cells (s.c., 2,5.10 6 cells). (A and B) The expression of PD-1, TIM-3, CD9, and Gal3 was measured at the surface of CD8 + memory cells at 30 dpi by flow cytometry. (C and D) The expression of Gal3 was measured intracellularly in memory CD8 + T cells at 30 dpi by flow cytometry. (E) At 30 dpi, splenocytes were stimulated with NP68 peptide (10 nM) for 4 h, and the expression of PD-1, TIM-3, or intracellular Gal3 by memory CD8 + T cells was measured by flow cytometry. The statistical significance of differences was determined using the Mann-Whitney test (B and D) or two-way ANOVA (E) (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001). Data are represented as mean ± SD ( n = 5 mice per group) and are representative of 3 independent experiments.

Article Snippet: After 7 days, NP68 peptide (20 nM) and CpG ODN 1826 (2 mg/mL, InvivoGen, tlrl-1826) were added to the BMDC and cultured overnight before being washed and used for CD8 + T cells activation.

Techniques: Expressing, Flow Cytometry, MANN-WHITNEY

Journal: iScience

Article Title: Transient tumor exposure induces persistent functional defects in memory CD8 + T cells

doi: 10.1016/j.isci.2026.115556

Figure Lengend Snippet: Tum-CD8 + memory cells display altered cytokine production but not cytotoxic capacities compared to Vir-CD8 + memory cells Naive F5 x CD45.1 cells (2.10 5 ) were i.v. transferred in B6 mice 1-day prior immunization with VV-NP68 (i.n., 2.10 5 pfu) or EL4-NP68 cells (s.c., 2,5.10 6 cells). (A and B) At 30 dpi, F5 memory cells were restimulated with NP68 (10 nM) for 4h in the presence of GolgiStop. The production of IFNγ, TNF, and IL-2 was measured by flow cytometry and expressed in % of total F5 (A) or MFI within the cytokine+ cells (B). (C and D) At 30 dpi, F5 memory cells were restimulated with NP68 (10 nM) for 4h in the presence (cytokines) or absence (CD69) of GolgiStop. The production of IFNγ and TNF and the upregulation of CD69 were measured by flow cytometry over time and expressed in % (C) or MFI (D). (E) The production of IFNγ was measured in supernatant after 4 or 24h of stimulation. (F and G) At 30 dpi, F5 memory cells were restimulated with various doses of NP68 for 4h in the presence of GolgiStop, and the production of IFNγ and TNF was measured by flow cytometry (F). EC 50 was determined (G). (H and I) Splenocytes were incubated with NP68 (10 nM) or control medium for 2h and labeled with CTV or CFSE, respectively. A 1:1 ratio of NP68-loaded splenocytes: control splenocytes (2.10 6 cells) was injected i.v. in Tum-CD8 + or Vir-CD8 + challenged mice at the memory stage. Representative histograms depicting control and CTV-labeled NP68-loaded splenocytes are shown (H). The percentage of NP68-loaded splenocytes killed was evaluated at 6-, 16-, or 44-h post-transfer (I). (J)Total CD8 + enriched from Tum-CD8 + or Vir-CD8 + challenged mice were labeled with CTV and stimulated with NP68-loaded DCs (1:1 ratio) for 4 days in the presence of IL-2. The expansion index of F5 cells was determined after 4 days. The statistical significance of differences was determined using 2-way ANOVA (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗ p < 0.0001). Data are represented as mean ± SD ( n = 5 mice per group) and are representative of 3 independent (A–G) or 1 (H–J) experiment(s).

Article Snippet: After 7 days, NP68 peptide (20 nM) and CpG ODN 1826 (2 mg/mL, InvivoGen, tlrl-1826) were added to the BMDC and cultured overnight before being washed and used for CD8 + T cells activation.

Techniques: Flow Cytometry, Incubation, Control, Labeling, Injection

Journal: iScience

Article Title: Transient tumor exposure induces persistent functional defects in memory CD8 + T cells

doi: 10.1016/j.isci.2026.115556

Figure Lengend Snippet: A transient tumoral challenge is sufficient to alter the protection capacity of F5 memory cells Naive F5 x CD45.1 cells (2.10 5 ) were i.v. transferred in B6 mice 1-day prior immunization with VV-NP68 (i.n., 2.10 5 pfu) or EL4-NP68 cells (s.c., 2,5.10 6 cells). (A–D) At 30 dpi, Vir- or Tum-challenged mice were infected with VV-NP68 (2.10 5 pfu). Six days after infection, mice received an i.v. injection of anti-CD8 antibody, and the proportion of cells within the tissue and the vasculature of the lung was determined (A). The proportion of memory CD8 + T cells in the lung tissue among all memory CD8 + T cells was determined (B). The expression of CD49a (C) and CD49d (D) was measured on memory CD8 + T cells within the lung tissue and vasculature. (E) At 30 dpi, Vir- or Tum-challenged mice were infected with Flu-NP68 (5.10 4 TCID50), and the weight loss was followed for 6 days. (F) At 30 dpi, Vir-CD8 + and Tum-CD8 + memory cells were FACS-sorted and transferred into B6 host (1,2.10 5 cells, i.v. route). One day after transfer, mice received a lethal dose of Flu-NP68 (2.10 6 TCID 50), and survival was followed for 10 days. The statistical significance of differences was determined with 1-way (B) or 2-way (C–E) ANOVA test (∗ p > 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001), or log rank test (F). Data are represented as mean ± SD ( n = 5 mice per group) and are representative of 2 (A–D, and G) or 1 (E and F) experiment(s).

Article Snippet: After 7 days, NP68 peptide (20 nM) and CpG ODN 1826 (2 mg/mL, InvivoGen, tlrl-1826) were added to the BMDC and cultured overnight before being washed and used for CD8 + T cells activation.

Techniques: Infection, Injection, Expressing

Journal: iScience

Article Title: Transient tumor exposure induces persistent functional defects in memory CD8 + T cells

doi: 10.1016/j.isci.2026.115556

Figure Lengend Snippet: Phenotype and cytokine production capacity of F5 memory cells is conserved after homologous or heterologous recall (A) Naive F5 x CD45.1 cells (2.10 5 ) were i.v. transferred in B6 mice 1-day prior immunization with VV-NP68 (i.n., 2.10 5 pfu) or EL4-NP68 cells (s.c., 2,5.10 6 cells). At 26 dpi, mice received a second immunization with VV-NP68 or EL4-NP68. (B) Thirty-one days post-recall, the number of F5 cells was measured in the spleen. (C) The expression of CD9, CD43, CD49a, and CD49d was measured on F5 memory cells 31 days after recall by flow cytometry. (D and E) Splenocytes were stimulated with NP68 (10 nM) for 4h in the presence (D) or absence (E) of GolgiStop. (D) The production of IFNγ, TFNα, and IL-2 was measured by flow cytometry. (E) The expression of PD-1 and TIM3 on F5 memory cells was determined in the spleen. The statistical significance of differences was determined with 1-way (B and C) or 2-way (D and E) ANOVA test (∗ p > 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001). Data are represented as mean ± SD ( n = 5 mice per group) and are representative of 2 independent experiments.

Article Snippet: After 7 days, NP68 peptide (20 nM) and CpG ODN 1826 (2 mg/mL, InvivoGen, tlrl-1826) were added to the BMDC and cultured overnight before being washed and used for CD8 + T cells activation.

Techniques: Expressing, Flow Cytometry

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: SPD1 comprises two peptides [SPDa, Ac-K(PpIX)-FF-DHLASLWWGTEL; SPDb, Ac-K(PpIX)-FF-AEEA-C(MAL-PEG 4 -DBCO)], which are composed of four discrete functional domains: (i) DHLASLWWGTEL, a GPC3-targeted motif; (ii) FF, a β sheet motif; (iii) PpIX, a fluorescent dye; (iv) DBCO, a click chemistry group. SPD1 self-assembles into nanoparticles, and SPD1 and Gd-DOTA-N 3 are administered via two sequential injections. SPD1 nanoparticles (first intravenous administration) first accumulate and transform into fibrillar networks on the cell membrane of high GPC3-expressing orthotopic liver tumor through intermolecular π-π stacking between FF. The fibrillar networks, functionalized with DBCO, trapped Gd-DOTA-N 3 (following a second intravenous administration) in the extracellular space via a bioorthogonal reaction and immobilized it in an ordered arrangement, thereby enhancing the T 1 -weighted signal for the precise detection of micro-HCC. Schematic elements were created by eBioart.

Article Snippet: SPD1 and SPD2 nanoparticles (50 μM) incubated in the presence or absence of human GPC3 recombinant protein (NovoProtein, C414) at molar concentration ratio of 1000:1 for 24 hours at room temperature, and then CD spectra of peptide nanoparticle solutions were acquired using a CD spectrometer (Chirascan, Applied Photophysics, UK).

Techniques: Functional Assay, Membrane, Expressing

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A and B ) UV-vis absorption spectra (A) and fluorescence (FL) emission spectra (B) of PpIX (excitation: 405 nm) following the gradual addition of H 2 O (from 0 to 99.5%) to a DMSO solution of SPD1 nanoparticles. a.u., arbitrary units. ( C ) CAC of SPD1 nanoparticles was determined using pyrene as a fluorescent probe. ( D ) Representative TEM image of self-assembled SPD1 nanoparticles (50 μM) in aqueous solution. ( E ) TEM images showing the initial SPD1 nanoparticles and nanofibers transformed from SPD1 nanoparticles (50 μM) after incubation with human GPC3 protein [molecular weight (MW) ≈ 61.6 kDa] at varying molar ratios. h, hours. ( F ) TEM images showing the initial SPD1 nanoparticles and nanofibers transformed from SPD1 nanoparticles (50 μM) after incubation with human GPC3 protein at different time points. The molar ratio of human GPC3 protein/SPD1 was ~1:1000. ( G ) CD spectra of SPD1 nanoparticles (50 μM) before and after incubation with human GPC3 protein (1:1000 molar ratio) for various durations, showing the secondary structure transition. mdeg, millidegrees. ( H ) FTIR spectra of SPD1 nanoparticles (50 μM) before and after 24 hours of incubation with GPC3 (1:1000 molar ratio), highlighting a shift in the amide I band consistent with β sheet formation. ( I ) Molecular simulation of the transformation of SPD1 into complex (i.e., SPD1 nanoparticles) in a water box based on the hydrophobic core of PpIX molecules, along with a hydrophilic corona formed by GPC3-targeted ligands. ( J ) Molecular docking simulation for SPD1 and GPC3. Rectangle: the possible binding sites between SPD1 and GPC3. ( K ) MD simulation of fibrillar transformation of SPD1 generated at t = 250 ns after interaction with GPC3. Rectangle: the interaction forces of the hydrogen bond and π-π stacking. All experiments were independently repeated three times with consistent and reproducible results.

Article Snippet: SPD1 and SPD2 nanoparticles (50 μM) incubated in the presence or absence of human GPC3 recombinant protein (NovoProtein, C414) at molar concentration ratio of 1000:1 for 24 hours at room temperature, and then CD spectra of peptide nanoparticle solutions were acquired using a CD spectrometer (Chirascan, Applied Photophysics, UK).

Techniques: Fluorescence, Transformation Assay, Incubation, Molecular Weight, Circular Dichroism, Binding Assay, Generated

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) SEM-EDX elemental mapping of the Gd-DOTA-N 3 +SPD1+GPC3 probe. False-color maps show the spatial distribution of carbon (C, red), oxygen (O, cyan), nitrogen (N, green), and Gd (magenta), confirming uniform Gd incorporation and colocalization with organic matrix elements. Scale bars, 25 μm. All experiments were independently repeated three times with consistent and reproducible results. ( B ) Schematic illustration of the covalent conjugation attachment of Gd-DOTA-N 3 to SPD1 nanofibers via SPAAC click chemistry, designed to enhance r 1 relaxivity. ( C ) TEM images showing GPC3-modified gold (Au) nanoparticles alone or after incubation with SPD1 or SPD2 nanoparticles (50 μM, 24 hours). Scale bars, 50 nm. ( D ) T 1 -weighted images and pseudocolor T 1 maps comparing Gd-DOTA, Gd-DOTA-N 3 , Gd-DOTA-N 3 +SPD2-GPC3 (noncovalent incubation, molar ratio of 1000:1, 24 hours) for 6 hours, and Gd-DOTA-N 3 +SPD1-GPC3 (bioorthogonal conjugation, molar ratio of 1000:1, 24 hours) for 6 hours. Experiments were independently repeated three times, with consistent results observed across replicates. ( E ) r 1 relaxivity of Gd-DOTA (purple), Gd-DOTA-N 3 (pink), Gd-DOTA-N 3 +SPD2+GPC3 (blue), and Gd-DOTA-N 3 +SPD1+GPC3 (light green). Data are presented as means ± SD ( n = 3 independent experiments). ( F ) Room-temperature VSM showing the enhanced paramagnetic signal from Gd-DOTA-N 3 +SPD1+GPC3 (light green), compared to Gd-DOTA-N 3 +SPD2+GPC3 (blue), Gd-DOTA-N 3 (pink), and Gd-DOTA (purple).

Article Snippet: SPD1 and SPD2 nanoparticles (50 μM) incubated in the presence or absence of human GPC3 recombinant protein (NovoProtein, C414) at molar concentration ratio of 1000:1 for 24 hours at room temperature, and then CD spectra of peptide nanoparticle solutions were acquired using a CD spectrometer (Chirascan, Applied Photophysics, UK).

Techniques: Conjugation Assay, Modification, Incubation

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) Representative Western blot analysis of GPC3 expression in HepG2, WRL-68, and Hepa1-6 cells ( n = 3 independent experiments). ( B ) Quantitation of relative GPC3 protein level from (A). Data are presented as means ± SD ( n = 3). Statistical analysis was performed using one-way ANOVA with a Tukey’s post hoc test. ( C ) Flow cytometry analysis of surface GPC3 expression in HepG2, WRL-68, and Hepa1-6 cells. ( D ) Representative IHC images of GPC3 expression (brown) in tumor tissues from orthotopic Hepa1-6 tumor-bearing mice. Scale bar, 50 μm. ( E ) CLSM images of HepG2 and Hepa1-6 cells treated with SPD1 or SPD2 nanoparticles (50 μM; red fluorescence) for 6 hours. Scale bars, 20 μm. ( F ) Time-dependent CLSM imaging of HepG2 cells treated with SPD1 nanoparticles (50 μM) showing membrane-localized fibrillar transformation. Scale bars, 20 μm. ( G ) CLSM analysis of HepG2 cells sequentially incubated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours; red) and FITC-labeled anti-GPC3 antibody (green; 1:200; Abcam, #ab207080). Colocalization (yellow) indicates specific binding of SPD1 to membrane-bound GPC3. Fluorescence intensity and colocalization were quantified using MATLAB. Data are presented as means ± SD ( n = 3); n.s., not significant (one-way ANOVA with Tukey’s post hoc test). Scale bars, 20 μm. ( H ) SEM images of untreated HepG2 and WRL-68 cells or incubated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours). Magnified insets highlight membrane-associated fibrillar structures. ( I ) TEM images of untreated HepG2 cells (top) and those treated with SPD1 nanoparticles (50 μM, 24 hours; bottom). Red arrows indicate membrane-associated nanofibers. Scale bars, 500 nm. ( J ) SEM images showing the persistence of SPD1-derived fibrillar networks on HepG2 cells at 6, 24, and 72 hours posttreatment (50 μM). Scale bars, 2 μm. All experiments were independently repeated three times with consistent and reproducible results.

Article Snippet: SPD1 and SPD2 nanoparticles (50 μM) incubated in the presence or absence of human GPC3 recombinant protein (NovoProtein, C414) at molar concentration ratio of 1000:1 for 24 hours at room temperature, and then CD spectra of peptide nanoparticle solutions were acquired using a CD spectrometer (Chirascan, Applied Photophysics, UK).

Techniques: Western Blot, Expressing, Quantitation Assay, Flow Cytometry, Fluorescence, Imaging, Membrane, Transformation Assay, Incubation, Labeling, Binding Assay, Derivative Assay

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) Representative CLSM images of HepG2 cells incubated with SPD1 nanoparticles (red, 50 μM) for 6 hours, followed by treatment with FITC-N 3 (10 to 50 μM, green) for an additional 6 hours. Merged yellow fluorescence indicates successful copper-free click conjugation between DBCO and N 3 on the cell membrane. Cells treated with SPD2+FITC-N 3 (50 μM) served as the nontargeted controls, showing minimal colocalization. Scale bars, 20 μm. ( B ) Time-dependent kinetics of bioorthogonal conjugation quantified by BCA protein assay and ICP-MS. HepG2 cells were pretreated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours), followed by incubation with Gd-DOTA-N 3 (50 μM) for 0.5, 1, 6, or 12 hours. Cells treated with Gd-DOTA-N 3 alone served as baseline controls. ( C ) T 1 -weighted MR images of HepG2 cells treated with Gd-DOTA (50 μM), Gd-DOTA-N 3 (50 μM), or sequentially with SPD1 or SPD2 (50 μM, 6 hours) followed by Gd-DOTA-N 3 (50 μM, 6 hours). ( D ) Quantitative r 1 relaxivity under the corresponding treatment conditions in (C). ( E ) r 1 relaxivity of HepG2 cells preblocked with anti-GPC3 antibody (5 μg/ml, 12 hours; Abcam, #ab207080) before SPD1 treatment (50 μM, 6 hours) followed by Gd-DOTA-N 3 (50 μM, 6 hours). ( F to H ) Cell viability of WRL-68 (F), HepG2 (G), and Hepa1-6 (H) cells after sequential treatment with SPD1 or SPD2 for 6 hours followed by Gd-DOTA-N 3 (50 μM, 6 hours). Cell viability was quantified using the CCK-8 assay. Data are presented as means ± SD { n = 3 for [(A) to (E)]; n = 6 for [(F) to (H)]}. Statistical significance was performed using one-way ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant; n.s., not significant. All experiments were independently repeated three times with consistent results.

Article Snippet: SPD1 and SPD2 nanoparticles (50 μM) incubated in the presence or absence of human GPC3 recombinant protein (NovoProtein, C414) at molar concentration ratio of 1000:1 for 24 hours at room temperature, and then CD spectra of peptide nanoparticle solutions were acquired using a CD spectrometer (Chirascan, Applied Photophysics, UK).

Techniques: Incubation, Fluorescence, Conjugation Assay, Membrane, Bicinchoninic Acid Protein Assay, CCK-8 Assay

Journal: Physiological Reports

Article Title: Water intake regulates mucosal immunity in rat jejunal villi via IL ‐1β, IL ‐6, and IL ‐10

doi: 10.14814/phy2.70891

Figure Lengend Snippet: (a) Effects of water intake on changes in lymph volume collected over set intervals of 60 min in rat jejunum‐derived lymph vessels in the absence (white column) and presence of a MyD88 inhibitor (oblique line column). (b) Effects of water intake without (white column) and with a MyD88 inhibitor (oblique line column) on changes in the concentration of IL‐1β in the lymph. (c) Effects of water intake without (white column) and with a MyD88 inhibitor (oblique line column) on changes in the concentration of IL‐6 in the lymph. (d) Effects of water intake without (white column) and with a MyD88 inhibitor (oblique line column) on changes in the concentration of IL‐10 in the lymph. The error bars represent the SDs.

Article Snippet: Clodronate‐containing liposomes (catalog no 16001004, Sigma‐Aldrich, USA) and an inhibitor of MyD88 (catalog no 2‐29328 Novus Bio, USA) are purchased.

Techniques: Derivative Assay, Concentration Assay