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: EL4-NP68 , Dr. T.N.M. Schumacher , Modified from EL4 lymphoma cell line (ATCC “TIB-39”).

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: EL4-NP68 , Dr. T.N.M. Schumacher , Modified from EL4 lymphoma cell line (ATCC “TIB-39”).

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: EL4-NP68 , Dr. T.N.M. Schumacher , Modified from EL4 lymphoma cell line (ATCC “TIB-39”).

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: EL4-NP68 , Dr. T.N.M. Schumacher , Modified from EL4 lymphoma cell line (ATCC “TIB-39”).

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: EL4-NP68 , Dr. T.N.M. Schumacher , Modified from EL4 lymphoma cell line (ATCC “TIB-39”).

Techniques: Expressing, Flow Cytometry

Journal: Nature Communications

Article Title: Aiolos restricts the generation of antigen-inexperienced, virtual memory CD8 + T cells in mice

doi: 10.1038/s41467-025-67540-8

Figure Lengend Snippet: Analysis of Eomes expression in CD8 + T cells isolated from the spleens of uninfected WT and Ikzf3 -/- mice. a Representative flow contour plots depicting expression of Eomes in WT and Ikzf3 -/- CD8 + T cells; and b respective bar graphs showing percent (%) of Eomes + and numbers of Eomes-expressing CD8 + T cells (# counts, normalized to 6 × 10 5 events). c Representative histogram overlay for Eomes expression and associated data showing differences in median fluorescence intensity (MFI) fold change relative to WT control. For ( a – c ), data shown for 4 independent experiments, n = 14/group, mean ± SEM, two-sided, unpaired Student’s t-test, ****p ≤ 0.0001. d Representative flow plots and e associated bar graphs showing percent (%) of CD122 + and numbers of CD122-expressing CD8 + T cells (#: counts, normalized to 6 × 10 5 events), Data shown for 3 independent experiments, n = 11/group, mean ± SEM, two-sided, unpaired Student’s t-test, ****p ≤ 0.0001. f Analysis of publicly available Aiolos Chromatin Immunoprecipitation (ChIP)-Seq data (GSM5106065) and STAT5b ChIP-Seq data (GSM7887512) showing enrichment of Aiolos and STAT5b at the Eomes promoter region. Sequencing tracks were viewed using Integrative Genomics Viewer. The gene region cloned into a reporter vector for Eomes promoter activity is indicated. g , h EL4 T cells were transfected with an Eomes promoter-reporter construct in conjunction with vectors for Aiolos, Aiolos DNA binding mutant (Aiolos DBM) , constitutively active STAT5b (STAT5b CA ), or empty vector control. As a control for transfection efficiency, cells were concurrently transfected with SV40- Renilla , and luciferase activity was used as a readout for promoter activity. Luciferase promoter-reporter values were normalized to SV40- Renilla control and presented as relative to the empty vector. Immunoblot analysis of the indicated proteins confirming the overexpression of respective vectors. β-actin was used as a loading control. Data are representative of 4 independent experiments, n = 4/condition, mean ± SEM, one-way ANOVA with Tukey’s multiple comparisons test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. Source data are provided as a file.

Article Snippet: The EL4 murine T cell lymphoma line (TIB-39) for transfection studies and YAC-1 cells (TIB-160) for cytotoxicity were acquired from the American Type Culture Collection (ATCC) and maintained in complete RPMI (RPMI media [catalog # 61870-036, Thermo Fisher Scientific] containing 10% FBS [catalog # 26140-079, Life Technologies] and 1% penicillin/streptomycin [catalog # 15140-122, Life Technologies]).

Techniques: Expressing, Isolation, Fluorescence, Control, Chromatin Immunoprecipitation, ChIP-sequencing, Sequencing, Clone Assay, Plasmid Preparation, Activity Assay, Transfection, Construct, Binding Assay, Mutagenesis, Luciferase, Western Blot, Over Expression

Journal: Molecular cancer therapeutics

Article Title: STAT5 activation enhances adoptive therapy combined with peptide vaccination by preventing PD-1 inhibition

doi: 10.1158/1535-7163.MCT-24-0505

Figure Lengend Snippet: A ) CD45.1 mice were adoptively transferred with 1–2 × 10 5 gp100+ CD8 T cells co-transduced −CA-STAT5 or +CA-STAT5 and vaccinated with TriVax on the following day (n=3 mice/group). 7 days later, the mice were sacrificed and splenocytes were harvested for cytokine analysis. B ) Representative images demonstrating the percentage of splenocytes producing IFNγ, TNFα, and Granzyme B (GzmB) in response to gp100 peptide stimulation compared to irrelevant (ova) peptide. The number of cells from each group incubated with peptide were normalized based on tetramer staining so that each well had the same number of gp100 tetramer+ T cells. Summary of results depicted in bar graph. C ) Measurement of IFNγ production in an ELISpot assay. The number of CD8 T cells in each group per well were unsorted and normalized to the same number of total gp100 tetramer+ CD8 T cells so that each well had the same number of gp100+ CD8 T cells. D ) CD8 T cells isolated from the experiments described in A were co-cultured with EL4, EL4 pulsed with gp100 short peptide (positive control), or B16F10 tumor cells. Supernatant was harvested after overnight culture from triplicate wells and evaluated for IFNγ production using ELISA. E ) Cytotoxic killing potential was measured by co-culturing +CA-STAT5 or −CA-STAT5 CD8 T cells with CFSE-labeled EL4, EL4 pulsed with gp100 short peptide, or B16F10 at an effector-to-target ratio (gp100 tetramer+ CD8 T cells to B16 melanoma) of 10:1. Summary graphs of at least two independent experiments from each figure is shown.

Article Snippet: Mouse lymphoma cell line EL4 expressing mouse MHC-I (Db/Kb) was purchased from the American Type Culture Collection (2015) and maintained as recommended by the vendor.

Techniques: Incubation, Staining, Enzyme-linked Immunospot, Isolation, Cell Culture, Positive Control, Enzyme-linked Immunosorbent Assay, Labeling

Journal: Nature Communications

Article Title: H2A.Z primes an epigenetic landscape for memory CD8 + T cell recall response

doi: 10.1038/s41467-025-62976-4

Figure Lengend Snippet: a , b The log2 ratio of gene abundance between the memory and naïve OT-I cells, line plot with genes ranked according to increasing log2 ratios. Based on data from Fig. ( a ) and GEO dataset GSE15907 ( b ). c H2A.Z expression was analyzed in naïve (CD62L hi CD44 lo ) and memory (CD127 hi KLRG1 lo ) OT-I cells ( n = 5 mice per group). Values denote geometric mean fluorescence intensity (gMFI). d Experiment setup. CD45 congenically distinct naïve OT-I cells from wild-type (WT) and GzmB-Cre H2A.Z f/f (cKO) OT-I mice were mixed at a 1:1 ratio and co-transferred into recipients. On the next day, these mice were infected with LM-OVA. On day 30-45, WT and cKO memory OT-I cells were sorted and re-mixed at a 1:1 ratio (1.5 × 10 4 mixed cells/mice) and co-transferred into congenically marked WT recipients and followed by LM-OVA infection. e At day 7 post-infection, the frequencies of donor cells in the spleen of recipient mice were determined ( n = 10 mice). f Experiment setup. CD45 congenically distinct naïve OT-I cells from WT and Rosa26 cre-ERT2 H2A.Z f/f (icKO) OT-I mice were mixed at a 1:1 ratio and transferred into recipients. On the next day, these mice were infected with LM-OVA. After TAM injection, WT and icKO memory OT-I cells were sorted and re-transferred into congenically marked WT recipients and followed by LM-OVA infection. g , h On day 7 post-infection, the frequencies of donor cells in the blood ( g ) and spleen ( h ) of recipient mice were determined ( n = 10 mice). i , j Splenocytes isolated from recipient mice were stimulated with the OVA 257-264 peptide for 6 h, then cytokine production from OT-I cells was measured. Values denote gMFI ( n = 9 biologically independent samples). Data are representative of two independent experiments in ( c ), three independent experiments in ( e and g – j ). Data are mean ± SD. Statistical analysis was performed using two-tailed unpaired Student’s t test ( c ) or two-tailed paired Student’s t test ( e and g – j ). Source data are provided as a file.

Article Snippet: EL-4 mouse lymphoma cell line (ATCC) and Phoenix-ECO cells (ATCC) were maintained in DMEM containing 10% fetal bovine serum, 10 mM Hepes and 1% penicillin/streptomycin.

Techniques: Expressing, Fluorescence, Infection, Injection, Isolation, Two Tailed Test