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: Immunity

Article Title: PIP5 Kinases Regulate Membrane Phosphoinositide and Actin Composition for Targeted Granule Secretion by Cytotoxic Lymphocytes

doi: 10.1016/j.immuni.2018.08.017

Figure Lengend Snippet:

Article Snippet: ASNENMDAM (NP68 peptide 366-374) , Anaspec , AS-60623.

Techniques: Blocking Assay, Control, Virus, Recombinant, Electron Microscopy, Fractionation, Software

Journal:

Article Title: Effect of a Small Molecule BCL-2 Inhibitor on Immune Function and Use with a Recombinant Vaccine

doi: 10.1002/ijc.25177

Figure Lengend Snippet: CD8 T lymphocytes from TCR-Tg F5 mice were stimulated with NP68 peptide in the absence or presence of Treg cells. Tregs were purified from spleens of untreated or GX15-070-treated C57BL/6 mice. Proliferation of CD8 cells was measured by [3H]thymidine incorporation. CD4+/CD25+/FoxP3+ phenotype of Tregs was confirmed by flow cytometry.

Article Snippet: Cells were cultured for three days in complete CTL medium (RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin) enriched with 10 −4 µg/mL of F5 TCR 366 ASNENMDAM 374 ligand peptide 68 (NP68) (American Peptide Company Inc., Sunnyvale, CA).

Techniques: Purification, Flow Cytometry