Journal: Cell Death & Disease

Article Title: Targeting tumor intrinsic TAK1 engages TNF-α-driven cell death through distinct mechanisms and enhances cancer immunotherapy

doi: 10.1038/s41419-025-08013-0

Figure Lengend Snippet: A MC38 cells lacking Tak1 or B2m expression were pulsed with the MHC-I restricted chicken ovalbumin SIINFEKL peptide and subsequently cultured with increasing ratios of OT-I CD8 T cells, previously activated for 48 h by CD3 and CD28 stimulation. Tumor cell viability was assessed 24 h later by flow cytometry, using CD8 surface staining to discriminate T cells from tumor cells. B – D CD8 T cells isolated from naïve mouse spleens (Balb/c for CT-26 and EMT6, C57BL/6 for MC38) were activated by CD3 and CD28 stimulation in vitro and subsequently cultured with ( B ) MC38, ( C ) CT-26, or ( D ) EMT6 tumor cells lacking Tak1 expression. Viability was assessed using DRAQ7 uptake, imaging cells every 2 h for the indicated times. E – G Tak1- deficient MC38 ( E ) or CT-26 ( F ) cells were cultured in media derived from 48 h activated CD-8 T cells. Viability was assessed via DRAQ7 uptake. G Conditioned media from 48 h activated CD8 T cells was added to EMT6 cells deficient for Tak1 , Tnfr1 , or Tak1 and Tnfr1 and viability was assessed via DRAQ7 uptake. All panels represent the mean +/− SD from a single experiment, n = 2 independent experiments.

Article Snippet: For the CD8 depletion protocol, mice were injected with anti-mouse CD8 (BioXCell; BP0061, New Haven, CT) or IgG2a Isotype control (BioXCell; BP 00085) at 10 mg/kg i.p. at the dose schedule of Day -2, Day -1 prior to flank inoculation of cells, then again on Days 4, 8, and 11 post inoculation.

Techniques: Expressing, Cell Culture, Flow Cytometry, Staining, Isolation, In Vitro, Imaging, Derivative Assay

Journal: Cell Death & Disease

Article Title: Targeting tumor intrinsic TAK1 engages TNF-α-driven cell death through distinct mechanisms and enhances cancer immunotherapy

doi: 10.1038/s41419-025-08013-0

Figure Lengend Snippet: A Immunocompetent mice (C57BL/6, n = 15/group) were implanted with pooled clones from Tak1 -deficient or parental MC38 cells and tumor growth was monitored. B , C Immunocompetent mice (BALB/c, n = 8/group) were implanted with Tak1 -deficient CT-26 cells ( B ) and treated at the indicated days with an α-PD-1 antibody ( C , 10 mg/kg) and tumor growth was monitored. D Immunodeficient mice (NSG, n = 10/group) were implanted with Tak1 -deficient CT-26 cells and tumor growth was monitored. E Quantification of the percentage of complete tumor clearance from panels ( B – D ). F Balb/c mice implanted with Tak1 -deficient CT-26 tumors and exhibiting complete responses were rechallenged with CT-26 parental cells and tumor growth was monitored. Naïve mice challenged with parental CT-26 cells served as controls. G CT-26 parental and Tak1 -deficient tumor growth was monitored in Balb/c mice administered an α-CD8 depleting antibody for two sequential days prior to tumor engraftment and during tumor progression at the indicated days ( n = 10/group). H CT-26 parental and Tak1 -deficient tumor growth was assessed in Balb/c mice administered an α-TNF-α neutralizing antibody (15 mg/kg) at the indicated days ( n = 10/group). Tumor growth curves represent the mean tumor volume +/− SEM. Statistical differences between tumor volumes at the final measurement was determined using a two-way ANOVA with Tukey’s multiple correction where * p < 0.05; ** p < 0.01; and **** p < 0.0001 was considered significant.

Article Snippet: For the CD8 depletion protocol, mice were injected with anti-mouse CD8 (BioXCell; BP0061, New Haven, CT) or IgG2a Isotype control (BioXCell; BP 00085) at 10 mg/kg i.p. at the dose schedule of Day -2, Day -1 prior to flank inoculation of cells, then again on Days 4, 8, and 11 post inoculation.

Techniques: Clone Assay

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: (A–E) ATs from naïve C57BL/6 mice were analyzed by flow cytometry. (A) Representative plots of transcription factor expression or cytokine production by CD8+ or CD4+ T cells from the mAT after PMA/ionomycin restimulation with BFA. (B) Numbers of CD8+ T cells and non-Treg CD4+ T cells from the mAT producing the indicated cytokine. (C) Frequencies of IFN-γ- and TNF-α-producing CD8+ and CD4+ T cells after PMA/ionomycin restimulation with BFA isolated from gAT, scAT, and mAT. (D) CD8+ (top) and CD4+ (bottom) T cells isolated from the mAT. Left: representative plots of CD44 and CD62L expression, Right: representative histograms of CD69 expression on CD44+CD62L− (red) or CD44−CD62L+ (blue) T cells. (E) Numbers of central memory (Tcm: CD44+CD62L+), effector memory (Tem: CD44+CD62L−CD69−) and resident memory (Trm: CD44+CD62L−CD69+) CD8+ and CD4+ T cells per gram of gAT, scAT, and mAT. (F) Conjoined pairs of naïve CD45.1 and CD45.2 congenic C57BL/6 mice were analyzed 6 weeks after parabiosis surgery to quantify the origin of CD8+ (left) and CD4+ (right) memory T cell subsets (described in (E)) in the spleen (Spl), mLN, siLP, and mAT. The percentage of cells originating from host (black bars) or donor (white bars) animals is shown. nd not detected. (G–K) mAT isolated from healthy rhesus macaques was analyzed by flow cytometry. (G) Representative plot (gated on CD3+CD8+ T cells) indicating the gating strategy for naïve (CD95−CD28lo), central memory or stem cell memory (Tcm/scm) (CD95+CD28+), and Tem/rm (CD95+CD28−) T cells. (H) Representative plots showing CD8+ (left) and CD4+ (right) naïve, Tcm/scm, and Tem/rm T cells. (I) Representative histograms of CD69 expression on CD8+ (left) and CD4+ (right) naïve (shaded) and Tem/rm T cells (blue). (J) Frequencies of CD8+ (left) and CD4+ (right) memory T cell subsets from (HI). (K) Representative plots (gated on CD3+ T cells) from concatenation of all samples, showing cytokine production by T cell subsets. Numbers in representative plots indicate mean±SD. In all bar graphs, error bars represent standard deviation. Data are representative of at least 2 experiments with at least 4 mice, 3 pairs of parabiotic animals, or a total of 3 rhesus macaques. See Figure S1.

Article Snippet: Anti-mouse CD8α (2.43) (depletion) , BioXcell , BE0061.

Techniques: Flow Cytometry, Expressing, Isolation, Standard Deviation

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: (A–D) C57BL/6 mice were orally infected with T. gondii. 6 weeks post-infection, T cell populations were evaluated by flow cytometry. (A) Number of IFN-γ+ CD8+ (left) and IFN-γ+ CD4+ (right) T cells from the mAT post PMA/ionomycin activation with BFA. (Student’s t test) (B) Representative plots show T. gondii-specific CD8+ (left) and CD4+ (right) T cells. (C) Representative plots of IFN-γ production by CD44+tgd057:Kb+ CD8+ T cells (left) and TGME49:I-Ab+ CD4+ T cells (right) after PMA/ionomycin stimulation with BFA in the indicated organ. (D) The mean fluorescence intensity (MFI) of IFN-γ+ tgd057:Kb+ CD8+ T cells (left) and IFN-γ+ TGME49:I-Ab+ CD4+ T cells (right) from (C). (E–G) C57Bl/6 mice were orally infected with Yptb WT. At the indicated time points post-infection, T cells were analyzed by flow cytometry. (E) Representative plots of YopE:Kb+ CD8+ T cells in the indicated organs at 15 days post-infection. (F) Time course of the numbers of CD44+YopE:Kb+CD8+ T cells in the mLN, siLP, and mAT. (G) Frequencies of Tcm, Tem, and Trm cells within YopE:Kb+CD8+ T cells in the mAT 31 days post-infection. (H–J) C57BL/6 mice were orally infected with Yptb WT or Yptb ΔyopM. At the indicated time points post-infection, T cell populations were analyzed by flow cytometry. (H) Representative plots of YopE:Kb+CD8+ T cells from the mLN or mAT 31 days post-infection. (I) Numbers of CD44+YopE:Kb+CD8+ T cells from the mAT 31 days post-infection. (J) Representative plots of YopE:Kb+ CD8+ T cells from the mAT and gAT >4 weeks post-infection with Yptb ΔyopM. (K) Representative plots of tgd057:Kb+ CD8+ T cells (left) and TGME49:I-Ab+ CD4+ T cells (right) from the gAT and mAT of mice >6 weeks post oral T. gondii infection. (L) Pairs consisting of one naïve and one previously infected (>4 weeks post Yptb ΔyopM infection) congenic C57BL/6 mouse were conjoined and analyzed 6 weeks after parabiosis surgery. The number of YopE:Kb+ CD8+ T cells isolated from the indicated organs of the naive (white bars) or previously infected (black bars) mouse in the pair was calculated. (One way Anova adjusted for multiple comparisons). Numbers in representative plots indicate mean±SD. Error bars in all bar graphs represent standard deviation. Data are representative of at least 2 experiments with at least 3 mice per group or 3 pairs of parabiotic animals. ns not significant, *p<0.05, ****p<0.0001. See Figure S2.

Article Snippet: Anti-mouse CD8α (2.43) (depletion) , BioXcell , BE0061.

Techniques: Infection, Flow Cytometry, Activation Assay, Fluorescence, Isolation, Standard Deviation

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: (A–B) Mice were orally infected with Yptb ΔyopM. >4 weeks post-infection, Tem and Trm memory CD8+ T cells were sorted for gene expression analysis by RNA-Seq. (A) Numbers of genes with a ≥2 fold increase (red) or decrease (blue) in expression levels between the indicated populations. (B) Pathway analysis was performed and gene pathways were organized into clusters, represented here as clustergrams showing gene pathways differentially regulated between Trm cells in the siLP and mAT. (C–G) Mice infected with Yptb ΔyopM were analyzed at >4 weeks post-infection. (C) Ki67 expression by Tem and Trm YopE:Kb+CD8+ T cells was determined by flow cytometric analysis. Left hand plots show YopE:Kb+ expression on CD8+ T cells. Center plots show CD69 expression on YopE:Kb+CD8+ cells. Right hand plots show Ki67 expression on Tem and Trm cells. Bar graphs show frequencies of Ki67+YopE:Kb+CD8+ Tem (left) and Trm (right) cells. (D) Representative Vybrant DyeCycle Violet staining of YopE:Kb+CD44+CD8+ T cells from the siLP (blue), spleen (green), and mAT (red). Bar graphs show the percentage of YopE:Kb+CD44+CD8+ Tem and Trm cells in the indicated cell cycle stages in the siLP (left), spleen (center), and mAT (right). (E) >4 weeks post-infection with the Yptb ΔyopM, mice received BrdU for 12 days. The percentage of BrdU+YopE:Kb+CD44+ CD8+ T cells found in the intraepithelial lymphocytes (IEL), lungs, salivary gland (SG), siLP, liver, mAT, and gAT is quantified in the bar graph. (F) >4 weeks post-infection, cells from the siLP, spleen, and mAT were incubated with Bodipy FL C16. Representative histogram indicates Bodipy uptake in YopE:Kb+CD8+ Trm cells (red) or YopE:Kb+CD8+ Tem cells (blue) from the siLP (open) and mAT (shaded). Bar graphs show the quantification of the bodipy MFI. (Student’s t test). (G) >4 weeks post-infection, cells from the siLP, spleen, and mAT were incubated with Mitotracker Deep Red. Representative histograms indicate Mitotracker Deep Red staining in YopE:Kb+CD8+ Trm cells (red) or YopE:Kb+CD8+ Tem cells (blue) from the siLP (open histogram) and mAT (shaded histograms). Bar graphs show the quantification of the Mitotracker Deep Red MFI. Numbers in representative plots indicate mean±SD. Error bars in all graphs represent mean±SD. Data are representative of at least 2 experiments with ≥3 mice per group. One way Anova adjusted for multiple comparisons. **p<0.01, ****p<0.0001 See Figure S3.

Article Snippet: Anti-mouse CD8α (2.43) (depletion) , BioXcell , BE0061.

Techniques: Infection, Gene Expression, RNA Sequencing, Expressing, Staining, Incubation

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: (A–D) Mice were either naïve, 6 days post-infection with Yptb (Yptb WT 6 days), >4 weeks post-infection with Yptb ΔyopM (Yptb ΔyopM), or >4 weeks post-infection with Yptb ΔyopM followed by challenge with Yptb for 6 days (Yptb ΔyopM + Yptb WT) before imaging or isolation of cells for flow cytometry. (A) Representative images of the entire mAT from actin-DsRed reporter mice are shown. White arrows indicate FALCs. (B) Number of FALCs per mAT represented as fold change over the number of FALCs per naïve mAT. (C) mATs from CD11c–YFP reporter mice were stained for CD8 and LYVE-1 and imaged by confocal microscopy. CD8, CD11c, and LYVE-1 staining (top) or CD8+ T cells alone (bottom) in areas of the mAT with (left) or without (right) FALCs are shown. (D) Numbers of CD44+YopE:Kb+CD8+ T cells from mAT. (E) Pooled mAT, scAT and gAT isolated from either naïve donors or donors >4 weeks post-infection with Yptb ΔyopM were subcutaneously transplanted into Rag1−/− mice. Rag1−/− mice receiving ATs from previously infected mice were either left untreated or injected with anti-CD4 and anti-CD8 depleting antibodies. 2 weeks post surgery, animals were challenged i.v. with 200 CFU of Yptb WT. Data are representative of at least 2 experiments with ≥5 mice per group. Error bars in all bar graphs represent standard deviation. Statistical comparisons in (B) and (D) were performed using one way Anova adjusted for multiple comparisons. Statistical comparisons in (E) were performed using Log-rank (Mantel Cox) test. ns not significant, *p<0.05, **p<0.01, ****p<0.0001. See Figure S4.

Article Snippet: Anti-mouse CD8α (2.43) (depletion) , BioXcell , BE0061.

Techniques: Infection, Imaging, Isolation, Flow Cytometry, Staining, Confocal Microscopy, Injection, Standard Deviation

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: (A–B) >4 weeks post-infection with Yptb ΔyopM, mice were injected i.v. with YopE69–77 peptide or vehicle control (ctrl.) and evaluated by flow cytometry 1 hour post-injection. (A) Representative histogram shows expression of CD69 on YopE:Kb+CD8+ T cells after vehicle control (dotted line) or peptide (gray shading) injection (left) and MFI of CD69 in the indicated organs after the indicated treatment is quantified (right). (B) Representative plots show IFN-γ and TNF-α production by YopE:Kb+CD8+ T cells 1 hour after vehicle control (left) or peptide (right) injection in the indicated organs. Frequencies of IFN-γ+ TNF-α+ YopE:Kb+ CD8+ T cells in the indicated organs are shown in the bar graph. (C) Mice were infected orally with a fluorescent reporter strain of T. gondii. 6 weeks post-infection, mice were injected i.v. with MHC-I and MHC-II peptides or vehicle control and analyzed by flow cytometry 1 hour post injection. Representative plots show IFN-γ and TNF-α in CD44+CD8+ T cells in the indicated organ. Bar graphs show the frequencies of IFN-γ+TNF-α+ cells within the CD44+ CD8+ T cells. (D–E) >4 weeks post-infection with Yptb ΔyopM, mice were injected i.v. with YopE69-77 peptide or an equal volume of vehicle control and evaluated by flow cytometry at 1 and 4 hours post-injection. (D) Representative plots show neutrophils in the indicated organs (gated on live CD45+TCRβ−Siglec F−NK1.1−B220− cells) 4 hours after injection. Bar graph shows frequency of neutrophils at 1 and 4 hours post-injection in the siLP and mAT. (E) Representative plots show monocytes (gated on live CD45+TCR-β−Siglec F−NK1.1−B220−CD11b+Ly-6G−CD64+CCR2+ cells) 4 hours after injection in the indicated organs. Bar graph shows the frequency of monocytes at 1 and 4 hours post-injection in the siLP and mAT. Numbers in representative plots indicate mean±SD. Error bars in all bar graphs represent standard deviation and statistics are calculated using one way Anova adjusted for multiple comparisons. Data are representative of at least 2 experiments with 2–6 mice per group. ns not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See Figure S5.

Article Snippet: Anti-mouse CD8α (2.43) (depletion) , BioXcell , BE0061.

Techniques: Infection, Injection, Control, Flow Cytometry, Expressing, Standard Deviation

Journal: Immunity

Article Title: The white adipose tissue is a reservoir for memory T cells that promotes protective memory responses to infection

doi: 10.1016/j.immuni.2017.11.009

Figure Lengend Snippet: DATA AND SOFTWARE AVAILABILITY

Article Snippet: Anti-mouse CD8α (2.43) (depletion) , BioXcell , BE0061.

Techniques: Software, Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation, SYBR Green Assay, Staining, Microarray

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 1 Pharmacological inhibition of BCL9 induces tumor regression and increases antigen presentation. a The BCL9 expression between tumors and normal tissues in TCGA COAD datasets (Normal, n = 41; Tumor, n = 462). b The antigen processing and presentation signature (left) and HLA-I signature (right) between low and high BCL9 expression (median value) in TCGA COAD datasets (BCL9Low , n = 209; BCL9High, n = 236). c Tumor growth of 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (n = 6). d Tumor growth of MC38 tumor-bearing Bcl9f/fBcl9lf/f mice and Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation (n = 6). e Heatmap of the genes included in the GO:0019882 from 30 mg/kg hsBCL9z96-treated CT26 tumors (Vehicle, n = 4; hsBCL9z96, n = 5). f, g The relative expression of Tap1, Tap2, B2m and Psmb9 of tumors from hsBCL9z96-treated CT26 tumor-bearing mice (f) and MC38 tumor- bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (g) analyzed by qPCR (n = 4–7). h–k Representative plot (h, j) and quantitative analysis (i, k) of OVA257-264-specific CD8+ T cells in TILs of tumors from MC38-OVA tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (h, i) and hsBCL9z96-treated MC38- OVA tumor-bearing mice (j, k) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation and analyzed by flow cytometry (n = 3). l Tumor growth of C57BL/6 WT (n = 6) and Batf3−/−mice (n = 5) that had been injected subcutaneously with MC38 tumor cells and were treated i.p. with vehicle or 40 mg/kg hsBCL9z96 every day for 2 weeks. These data are representative values expressed as the mean ± SD of each group; n indicates biological replicate; **p < 0.01; ***p < 0.001; ****p < 0.0001; Unpaired Student’s t test (a, b, i, k); Two-way ANOVA followed by Bonferroni test (c, d, f, g)

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Inhibition, Immunopeptidomics, Expressing, Cytometry, Injection

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 2 Inhibition of BCL9/BCL9L enhances cDC1 activation and facilitates cross-priming of CD8+ T cells. a, b CD40 (left) and CD86 (right) expression by CD103+ cDC1 of TdLNs (a) and tumors (b) from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice analyzed by flow cytometry (n = 3-4). c, d CD40 (left) and CD86 (right) expression by CD103+ cDC1 of TdLNs (c) and tumors (d) from MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation analyzed by flow cytometry (n = 3–4). e The representative plot of OT-I CD8+ T cells in TdLNs from hsBCL9z96-treated MC38-OVA tumor- bearing mice analyzed by flow cytometry. f and g Quantitative analysis of the percentage of OT-I CD8+ T cells (f) and CFSE dilution of OT-I CD8+ T cells (mean fluorescent intensity, MFI) (g) based on the result of (e) (n = 3). h The representative plot of OT-I CD8+ T cells in TdLNs from MC38-OVA tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, and +6 post inoculation analyzed by flow cytometry. i, j Quantitative analysis of the percentage of OT-I CD8+ T cells (i) and CFSE dilution of OT-I CD8+ T cells (j) based on the result of (h) (n = 3). These data are representative values expressed as the mean ± standard deviation (SD) for each group, derived from three independent experiments; “n” denotes the number of biological replicates. An unpaired Student’s t test was used for statistical analysis of the data in groups a–d, f, g, i, and j

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Inhibition, Activation Assay, Expressing, Cytometry, Standard Deviation, Derivative Assay

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 3 Single-cell transcriptional profiling of CD8+ T cells and cDC1 in tumors and TdLNs from B16-OVA tumor-bearing Bcl9/Bcl9l deficiency mice. a Illustration of experiment and analysis process of single-cell transcriptional analysis. b TSNE plots of clustering process and marker genes (Zbtb46 for DCs, Cd68 for myeloid cells, Mlana for B16-OVA tumor cells, Cd3e for T cells, Cd4 for CD4+ T cells and Cd8a for CD8+ T cells) in tumors from B16-OVA tumor-bearing Bcl9f/fBcl9lf/f mice and Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6 and +11 post inoculation. c–e TSNE plots of DC reclustering (c, e) and marker genes (Xcr1 for cDC1 and Clec10a for cDC2) (d) in tumors from B16-OVA tumor-bearing Bcl9f/fBcl9lf/f mice and Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Marker

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 4 Bcl9/Bcl9l deficient cDC1 are superior to WT cDC1 in activation, antigen presentation and cross-priming of CD8+ T cells. a Expression of genes related to cDC1 maturation and antigen presentation in tumors and TdLNs from B16-OVA tumor-bearing Bcl9f/fBcl9lf/f mice and Bcl9f/f

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Activation Assay, Immunopeptidomics, Expressing

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 6 Targeting of BCL9/BCL9L increases cDC1 accumulation in tumors through XCL1-XCR1 axis. a Gating strategy of XCR+ cDC1 (CD45+ CD11b−CD11c+ MHC-II+ CD103+ XCR1+) in TILs. b The XCR+ cDC1 in TILs of 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (left) and MC38 tumor-bearing Bcl9f/fBcl9lf/fCre-ERT2 mice (right) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation were analyzed by flow cytometry (n = 4). c iCD103+ DC migration toward XCL1 for 3 h by trans well assay (n = 3). d Heatmap of the genes included in GO:0070098 of 30 mg/kg hsBCL9z96-treated CT26 tumors (vehicle, n = 4; hsBCL9z96, n = 5). e, f Xcl1 mRNA (left) and XCL1 protein (right) levels in tumors from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (e) and MC38 tumor-bearing Bcl9f/fBcl9lf/fCre-ERT2 mice (f) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation were analyzed by qPCR and ELISA, respectively (n = 4-5). g, h Representative plot (left) and quantitative analysis (right) of XCL1 expression of CD8+ T cells and NK cells in TILs from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice analyzed by flow cytometry (n = 4). g Representative plot (left) and quantitative analysis (right) of XCL1 expression among CD8+ T cells and NK cells in TILs from MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation were analyzed by flow cytometry (n = 4). h Results are presented as the mean ± standard deviation (SD) for each group, derived from three independent experiments; “n” denotes the number of biological replicates; Unpaired Student’s t test (b, c, e, f); Two-way ANOVA followed by Bonferroni test (g, h)

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Cytometry, Migration, Enzyme-linked Immunosorbent Assay, Expressing, Standard Deviation, Derivative Assay

Journal: Signal transduction and targeted therapy

Article Title: Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

doi: 10.1038/s41392-024-01838-9

Figure Lengend Snippet: Fig. 7 Targeting BCL9/BCL9L results in CD8+ T cells accumulation in tumors through CXCL9-CXCR3 axis. a Significantly upregulated GO terms related to IFN-γ response of 30 mg/kg hsBCL9z96-treated CT26 tumors are depicted (vehicle, n = 4; hsBCL9z96, n = 5). b and c Relative Ifng mRNA (left) and IFN-γ protein (right) levels in tumors from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (b) and MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (c) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation analyzed by qPCR and ELISA, respectively (n = 4–7). d, e Relative Cxcl9 mRNA (left) and CXCL9 protein (right) expression of tumors from 30 mg/ kg hsBCL9z96-treated CT26 tumor-bearing mice (d) and MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (e) treated i.p. with tamoxifen (1 mg/ 100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation analyzed by qPCR and ELISA, respectively (n = 4-7). f Assessment of CD8+ T cell migration toward CXCL9 or with the indicated doses of antibodies or chemokine for 4 h by trans well assay (n = 3). g Representative plot (left) and quantitative analysis (right) of CXCL9 expression in cDC1 of tumors from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice analyzed by flow cytometry (n = 3–4). h Representative plot (left) and quantitative analysis (right) of CXCL9 expression in cDC1 of tumors from MC38 tumor-bearing Bcl9/Bcl9l deficiency mice analyzed by flow cytometry (n = 4). i The expression of CXCR3 in CD8+ T cells of tumors from 30 mg/kg hsBCL9z96-treated CT26 tumor-bearing mice (left) and MC38 tumor-bearing Bcl9f/fBcl9lf/f Cre-ERT2 mice (right) treated i.p. with tamoxifen (1 mg/100 μL) in olive oil on days −7, −6, −5, +1, +6, and +11 post inoculation analyzed by flow cytometry (n = 4). Results are presented as the mean ± standard deviation (SD) for each group, derived from three independent experiments; “n” denotes the number of biological replicates; Unpaired Student’s t test (b–e, g–i); One-way ANOVA followed by Bonferroni test (f)

Article Snippet: For CD4 and CD8 T cells depletion, individual mice were injected i.p. with anti-mouse CD4 monoclonal antibodies (Abs) (100 μg/100 μL; BE0003-1, BioXcell) or anti-mouse CD8 Abs (100 μg/100 μL; BE0004-1, BioXcell) on days +2, +4, +6, +8 and +10 after inoculation.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Migration, Cytometry, Standard Deviation, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: METTL3 promotes an immunosuppressive microenvironment in bladder cancer via m6A-dependent CXCL5/CCL5 regulation

doi: 10.1136/jitc-2024-011108

Figure Lengend Snippet: METTL3 is highly expressed in tumors and is associated with an immunosuppressive microenvironment. (A) Flowchart for screening key N6-methyladenosine (m6A) modification genes related to immunotherapy response in bladder cancer (BLCA). (B) Pearson correlation analysis bar chart of the 10 target genes with the percentage of complete response (CR) patients to immunotherapy in the IMvigor210 cohort, and a scatter plot of METTL3 expression level versus CR patient percentage. (C) Proportion of immunotherapy responses among different Lund subtypes in the IMvigor210 cohort. (D) Violin plot of METTL3 expression levels in bladder tissues of patients with different Lund subtypes. (E–F) Expression and statistical analysis of METTL3 in normal and tumor cells from single-cell sequencing of clinical bladder cancer samples. Histogram of METTL3 expression levels in cancer tissues versus adjacent normal tissues in (G) non-paired samples and (H) paired samples from the The Cancer Genome Atlas (TCGA) bladder cancer cohort. (I) Representative immunohistochemistry staining of METTL3 in clinical BLCA samples. (J–K) Scatter plots of METTL3 expression levels with CD8+T cell, cytotoxic cell, and myeloid-derived suppressor cell (MDSC) infiltration levels based on ssGSEA algorithm and TIMER V.2.0 database. (L) Statistical plot of METTL3 expression levels and immune scores in BLCA from the CAMOIP database. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Anti-mouse Programmed Cell Death Protein 1 (PD-1) antibody (Bioxcell, #BE0146), anti-mouse CD8α antibody (Bioxcell, #BE0061), and anti-mouse Gr-1 antibody (Bioxcell, #BE0075) were also dissolved in PBS and administered intraperitoneally.

Techniques: Modification, Expressing, Sequencing, Immunohistochemistry, Staining, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: METTL3 promotes an immunosuppressive microenvironment in bladder cancer via m6A-dependent CXCL5/CCL5 regulation

doi: 10.1136/jitc-2024-011108

Figure Lengend Snippet: METTL3 regulates bladder cancer progression by chemotactic CD8+T cell infiltration through the IGF2BP1-AHR-CCL5 axis. (A) Venn diagram illustrating the screening process for key transcription factors regulated by METTL3-mediated m6A modification and involved in CCL5 transcription. (B) Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis of AHR and CCL5 mRNA expression levels after AHR knockdown in MB49 cells. (C) Assessment of CCL5 mRNA expression levels after overexpression of METTL3 and/or knockdown of AHR in MB49 cells. (D) Schematic representation of AHR binding sites within the CCL5 promoter region as predicted by JASPAR. (E) CHIP-qPCR analysis of AHR enrichment at the CCL5 promoter region. (F) mRNA and (G) protein expression levels of AHR after METTL3 knockdown in MB49 cells. (H) Peak plot of m6A modification sites in AHR in MB49 cells. (I) MeRIP-qPCR analysis showing changes in AHR m6A modification levels following METTL3 knockdown in MB49 cells. (J) RIP-qPCR analysis of METTL3 enrichment in AHR mRNA in MB49 cells. (K) MeRIP-qPCR showing changes in AHR m6A modification levels after treatment with the METTL3 inhibitor STM2457 in MB49 cells. (L) RT-qPCR analysis of AHR mRNA levels after STM2457 treatment to inhibit METTL3 in MB49 cells. (M) RNA decay assay showing AHR mRNA stability after silencing METTL3. (N) RNA decay assay showing AHR mRNA stability after treatment with METTL3 inhibitor STM2457 (2 µg/mL, 72 hours) in MB49 cells. (O) RT-qPCR analysis of IGF2BP1 and AHR mRNA expression levels in MB49 cells after silencing IGF2BP1. (P) RT-qPCR analysis of IGF2BP2 and AHR mRNA expression levels in MB49 cells after silencing IGF2BP2. (Q) RT-qPCR analysis of METTL3, IGF2BP1, and AHR mRNA expression levels in MB49 cells after overexpression of METTL3 and/or silencing of IGF2BP1. (R) Images of tumors formed by MB49 stable cell lines (control, AHR overexpression, METTL3 knockdown, METTL3 knockdown with AHR overexpression) subcutaneously implanted into the backs of C57BL/6J mice. (S) Growth curves of mouse bladder cancer tumors. (T) Volume of mouse bladder cancer tumors. (U) Schematic of the animal experiment. (V) Images of bladder cancer tumors in mice. (W) Growth curves of bladder cancer tumors in mice. (X) Tumor weights of bladder cancer tumors in mice; ns, no significance. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Anti-mouse Programmed Cell Death Protein 1 (PD-1) antibody (Bioxcell, #BE0146), anti-mouse CD8α antibody (Bioxcell, #BE0061), and anti-mouse Gr-1 antibody (Bioxcell, #BE0075) were also dissolved in PBS and administered intraperitoneally.

Techniques: Modification, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Knockdown, Over Expression, Binding Assay, ChIP-qPCR, Stable Transfection, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: METTL3 promotes an immunosuppressive microenvironment in bladder cancer via m6A-dependent CXCL5/CCL5 regulation

doi: 10.1136/jitc-2024-011108

Figure Lengend Snippet: Targeting METTL3 enhances the efficacy of anti-Programmed Cell Death Protein 1 (PD-1) immunotherapy in bladder cancer. (A) Control and METTL3-knockdown MB49 stable cell lines were subcutaneously injected into mice. Anti-PD-1 antibody (200 µg/mouse, every 3 days) was administered intraperitoneally starting on day 6. Tumors were harvested on day 12 for flow cytometric analysis of the immune microenvironment (n=5). (B–D) Images, growth curves, and tumor weights of subcutaneous bladder cancer tumors in mice. (E–F) Flow cytometric analysis of MDSCs and CD8+T cell infiltration levels in the tumor tissues of mouse bladder cancer. (G) Wild-type MB49 cells were subcutaneously injected into mice, and on day 6, the mice were randomly divided into groups. Treatment included anti-PD-1 antibody (200 µg/mouse, every 3 days, intraperitoneally), IgG antibody (200 µg/mouse, every 3 days, intraperitoneally), the METTL3 inhibitor STM2457 (250 µg/tumor, once daily, intratumorally), and a combination of STM2457 and anti-PD-1 antibody. (H, J) Images, growth curves, and tumor weights of bladder cancer tumors in mice. (K) Control or METTL3 knockdown MB49 stable cell lines were orthotopically injected into the mouse bladder wall to establish an orthotopic bladder cancer model. Anti-PD-1 antibody (200 µg/mouse, every 3 days, intraperitoneally) or IgG antibody (200 µg/mouse, every 3 days, intraperitoneally) was administered starting on day 6 (n=5). (L) In vivo imaging system (IVIS) Living imaging of tumor growth in the orthotopic bladder cancer model. (M) Images of orthotopic bladder cancer tumors in mice. (N) Statistical analysis of fluorescence signal values from IVIS Living imaging on day 16. (O) Tumor volume in the orthotopic bladder cancer model. (P) Tumor weight in the orthotopic bladder cancer model. (Q) Schematic diagram of the study content. ns, no significance. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Anti-mouse Programmed Cell Death Protein 1 (PD-1) antibody (Bioxcell, #BE0146), anti-mouse CD8α antibody (Bioxcell, #BE0061), and anti-mouse Gr-1 antibody (Bioxcell, #BE0075) were also dissolved in PBS and administered intraperitoneally.

Techniques: Control, Knockdown, Stable Transfection, Injection, In Vivo Imaging, Imaging, Fluorescence

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: Ascorbic acid improves the cognitive level of aged mice and increases the number of CD8 + cells in aged mice. A Trajectory tracking map of mouse movement in the open field test, illustrating time spent in the central zone (seconds), average speed during movement episodes (cm/s), and total distance traveled within the central area. B Statistical chart of New Object Cognition Index. C Flow cytometry was utilized to analyze immune cell populations, including CD3 + T, CD4 + T, CD8 + T, CD11b + , and B cells, in the whole blood of middle-aged mice assigned to either the control group or the AA group ( D ). E Statistical analysis of the flow cytometry data was presented in a histogram format, with significance levels denoted as follows: ns ( P > 0.05), * ( P < 0.05), ** ( P < 0.01), and *** ( P < 0.001)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Flow Cytometry, Control

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: AA facilitates the early differentiation of T cells while concurrently suppressing myeloid differentiation. A A schematic depicting the early and long-term differentiation of T cells in an OP9-DL1 and lin-CD117 + HSC co-culture system is presented. B The diagram illustrates the progression of T cell maturation from CD44 and CD25 negative to positive selection. C Flow cytometry analysis and statistical bar graphs are utilized to demonstrate the AA-promoted changes in T cell DN stages, including the DN1 phase (CD44 + CD25 − ), DN2 phase (CD44 + CD25 + ), DN3 phase (CD44 − CD25 + ), and DN4 phase (CD44 − CD25 − ). D Flow cytometry analysis is utilized to examine the inhibition of myeloid differentiation, as well as to assess the long-term T cell differentiation promoted by AA in vitro ( E ). F and G Statistical bar graphs are generated to represent the flow cytometry results of CD8 + and CD4 + T cells, with significance levels denoted as ns: not significant ( P > 0.05), * ( P < 0.05), and ** ( P < 0.01), ( n = 3 for each group)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Co-Culture Assay, Selection, Flow Cytometry, Inhibition, Cell Differentiation, In Vitro, Generated

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: The Myh9 protein exhibits binding affinity towards AA and affects the proliferation of CD8 + T cells. A MetPro protein-metabolite interaction experimental workflow diagram. B Predicted binding differential protein KEGG pathway enrichment diagram. C Mass spectrometry predicted binding differential protein heatmap. D Protein interaction average degree analysis diagram. E Protein interaction network analysis diagram. F Concentration gradient binding curves of AA with Rhoa and Myh9 highlight the binding dynamics between these proteins. G Flow cytometry diagrams of CD8 + T cells for (Ga) control group, (Gb) AA group, (Gc) blebbistatin group, and (Gd) blebbistatin + AA group with statistical results ( H ) ( n = 3 for each group. ** p < 0.01, * p < 0.05). I Flow cytometry diagrams of CD11b + cells for (Ia) control group, (Ib) AA group, (Ic) blebbistatin group, and (Id) blebbistatin + AA group with statistical results ( J ) ( n = 3 for each group. **** p < 0.0001, *** p < 0.001, ** p < 0.01)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Binding Assay, Mass Spectrometry, Concentration Assay, Flow Cytometry, Control

Journal: Immunity & Ageing : I & A

Article Title: Ascorbic acid attenuates immunosenescence and cognitive decline via MYH9-Mediated CD8⁺ T cell differentiation

doi: 10.1186/s12979-025-00538-4

Figure Lengend Snippet: Anti-CD8α antibody injection reduced the cognitive level of young mice. A Flow cytometry analysis of CD8 + T cells versus CD4 + T cells in the peripheral blood of mice after injection of IgG2b antibody and Anti-CD8α antibody ( B ). C Statistical histogram of CD8 + T flow cytometry results. D CD4 + T flow cytometry results statistical histogram. E Flow cytometry analysis of NK cells in mouse peripheral blood after IgG2b and Anti-CD8α antibody injection. F Statistical histogram of flow cytometry results of FNK cells. G Tracking diagram of mouse movement trajectory in open field test after antibody injection. H Time (s), speed, and distance traveled by the mouse in the central region during the open field test. I Statistical chart of new object cognition index ( n = 5 for each group, ns P > 0.05, * p < 0.05, *** P < 0.001)

Article Snippet: The Rat IgG2b isotype and Anti-mouse CD8α-InVivo antibodies (Selleckchem) were each diluted to a final volume of 1 ml using sterile PBS and injected intraperitoneally at a dosage of 200 μg per mouse.

Techniques: Injection, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: SMARCA4 deficiency impairs CD8 + T cell function and confers resistance to PD-1 blockade in NSCLC (A) Progression-free survival (PFS) of eight patients with SMARCA4-deficient (BRG1-deficient) NSCLC treated with ICIs. (B) Representative axial CT images of a patient (P6) with SMARCA4-deficient NSCLC before and after ICIs treatment. (C) Change in the sum of target lesion diameters for six evaluable patients with SMARCA4-deficient NSCLC from baseline to first radiographic assessment. (D) Schematic of the experimental design for the orthotopic lung cancer model ( n = 8/group). (E) Representative in vivo bioluminescence images of mice from the indicated treatment groups at different time points. (F) Quantification of total bioluminescence flux from mice in each group over time. (G) Kaplan-Meier survival curves of mice from the four treatment groups. (H) Individual tumor growth curves, as measured by bioluminescence, for mice in each treatment group. (I) UMAP visualization of major immune cell populations within the TME, analyzed by flow cytometry. (J) Quantitative comparison of the frequencies of indicated immune cell lineages between SMARCA4-WT and -KD tumors. (K and L) Representative flow cytometry plots showing the production of IFN-γ and TNF-α by tumor-infiltrating CD8 + T cells. (M) Quantification of the frequencies of IFN-γ + and TNF-α + cells among tumor-infiltrating CD8 + T cells. Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by unpaired Student’s t test or one- or two-way ANOVA where appropriate.

Article Snippet: InVivoMAb anti-human CD8α , BioXcell , Cat#BE0004-2; RRID: AB_1107673.

Techniques: Cell Function Assay, In Vivo, Flow Cytometry, Comparison

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: Attenuated IL-2/STAT5 signaling and an enhanced exhaustion phenotype in CD8 + T cells within SMARCA4-deficient tumors (A) Schematic of the workflow for transcriptomic profiling of CD8 + T cells. (B) Volcano plot displaying differentially expressed genes in CD8 + T cells from SMARCA4-KD versus WT tumors. (C–E) Pathway enrichment analyses of genes downregulated in CD8 + T cells from SMARCA4-KD tumors, including Gene Ontology (GO) terms, KEGG pathways, and Reactome pathways. (F) The gene set enrichment analysis (GSEA) plot. (G) Correlation matrix (pie chart) showing the association between IL-2 receptor subunits expression and key T cell exhaustion marker genes in tumor-infiltrating CD8 + T cells. (H) Radar plot comparing the normalized expression levels of genes encoding IL-2 receptor subunits and exhaustion markers in CD8 + T cells. (I) Quantification by flow cytometry of the expression frequencies of PD-1, TIGIT, and TIM-3 on tumor-infiltrating CD8 + T cells. (J and K) Representative multiplex immunofluorescence (mIF) images of SMARCA4-WT and -KD tumor sections stained for PanCK (cyan), CD8 (green), GZMB (white), PD-1 (red), TIGIT (orange), and TIM-3 (yellow). Scale bars, 20 μm. Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA where appropriate.

Article Snippet: InVivoMAb anti-human CD8α , BioXcell , Cat#BE0004-2; RRID: AB_1107673.

Techniques: Expressing, Marker, Flow Cytometry, Multiplex Assay, Immunofluorescence, Staining

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: SMARCA4 loss in tumor cells attenuates CD8 + T cell function via NF-κB-mediated suppression of ICAM1 (A) Flow cytometry analysis of surface expression of the exhaustion markers PD-1, TIGIT, and TIM-3 on human CD8 + T cells following co-culture. (B and C) Representative flow cytometry plots showing the production of IFN-γ and TNF-α and the surface expression of IL-2Rα (CD25) by CD8 + T cells following co-culture. (D) Quantification of the frequencies of IFN-γ + , TNF-α + , and IL-2Rα + cells among co-cultured CD8 + T cells. (E) Integrated single-nucleus RNA sequencing (snRNA-seq) analysis comparing IL2-STAT5 signaling activity. y axis: IL2-STAT5 signaling score. (F and G) Incoming and outgoing signaling patterns between major cell types in the TME, as inferred from snRNA-seq. (H) Specific cell-cell communication network illustrating the ICAM signaling pathway from tumor cells to CD8 + T cells in patients with SMARCA4-WT NSCLC. (I) Correlation analysis between SMARCA4 and ICAM1 mRNA expression in TCGA cohorts. (J) Immunohistochemistry staining and quantification of ICAM1 protein expression in tumor tissues from SMARCA4-WT ( n = 10) and -deficient ( n = 10) NSCLC patients. (K) Schematic illustrating the proposed link between SMARCA4 deficiency and impaired NF-κB activation. (L) Immunoblot analysis of ICAM1 and p65 protein levels in SMARCA4-WT H2122 cells treated with the NF-κB inhibitor PTDC or vehicle control. (M) ChIP-qPCR analysis showing NF-κB (p65) binding to a specific site within the ICAM1 promoter in SMARCA4-WT H2122 cells ( n = 3). (N) Dual-luciferase reporter assay in SMARCA4-WT H2122 cells ( n = 3). Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA where appropriate.

Article Snippet: InVivoMAb anti-human CD8α , BioXcell , Cat#BE0004-2; RRID: AB_1107673.

Techniques: Cell Function Assay, Flow Cytometry, Expressing, Co-Culture Assay, Cell Culture, RNA Sequencing, Activity Assay, Immunohistochemistry, Staining, Activation Assay, Western Blot, Control, ChIP-qPCR, Binding Assay, Luciferase, Reporter Assay

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: The PD-1/IL-2 bsAb exerts potent anti-tumor efficacy in SMARCA4-deficient models (A) Schematic of the therapeutic experiment in humanized mice ( n = 6/group). (B) Representative images of excised tumors from each treatment group at the endpoint. (C) Tumor growth curves of individual mice in the indicated treatment groups. (D) Kaplan-Meier survival curves of mice from the four treatment groups. (E) Quantification by flow cytometry of tumor-infiltrating CD3 + CD8 + T cells. (F) Quantification of the percentage of tumor-infiltrating CD8 + T cells expressing PD-1, TIGIT, and TIM-3. (G) Frequency of polyfunctional (TNF-α + IFN-γ + ) CD8 + T cells among tumor-infiltrating lymphocytes. (H) Schematic of the humanized patient-derived xenograft (PDX) model ( n = 5/group). (I) Representative images of excised PDX tumors from each treatment group. (J) Tumor growth curves of individual PDX-bearing mice. (K) Kaplan-Meier survival curves of PDX-bearing mice from the three treatment groups. (L) Quantification of tumor-infiltrating CD3 + CD8 + T cells in PDX tumors. (M) Quantification of the percentage of CD8 + T cells from PDX tumors expressing PD-1, TIGIT, and TIM-3. (N) Frequency of polyfunctional (TNF-α + IFN-γ + ) CD8 + T cells in PDX tumors. (O) Representative multiplex immunofluorescence (mIF) images of PDX tumor sections from different treatment groups, stained for PanCK (cyan), CD8 (green), GZMB (white), PD-1 (red), TIGIT (orange), and TIM-3 (yellow). Scale bars, 40 μm. Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA or two-way ANOVA where appropriate.

Article Snippet: InVivoMAb anti-human CD8α , BioXcell , Cat#BE0004-2; RRID: AB_1107673.

Techniques: Flow Cytometry, Expressing, Derivative Assay, Multiplex Assay, Immunofluorescence, Staining

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: STAT5 activation mediates the therapeutic effect of the PD-1/IL-2 bsAb in SMARCA4-deficient NSCLC (A) Representative flow cytometry plots showing the expression of the exhaustion markers PD-1, TIGIT, and TIM-3 on activated human CD8 + T cells. (B) Quantification of the mean fluorescence intensity (MFI) of the exhaustion markers PD-1, TIGIT, and TIM-3 on CD8 + T cells from the experiment in (A) ( n = 3). (C) Representative flow cytometry plots showing the production of IFN-γ and TNF-α by CD8 + T cells under the conditions described in (A). (D) Quantification of the frequencies of IFN-γ + and TNF-α + cells among CD8 + T cells ( n = 3). (E) Schematic of the adoptive T cell transfer experiment ( n = 8/group). (F) Representative in vivo bioluminescence images of mice from the indicated groups at different time points. (G) Tumor growth curves, as measured by bioluminescence, for mice in each treatment group. (H) Kaplan-Meier survival curves of mice from the four treatment groups. (I) Quantification of the frequency of donor-derived CD45.2 + CD8 + T cells among total tumor-infiltrating lymphocytes. (J) Flow analysis of donor-derived CD45.2 + CD8 + T cells expressing PD-1, TIGIT, and TIM-3. (K–M) Frequency of IFN-γ + (K–L) and TNF-α + (M) cells among donor-derived CD45.2 + CD8 + T cells. Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA or two-way ANOVA where appropriate.

Article Snippet: InVivoMAb anti-human CD8α , BioXcell , Cat#BE0004-2; RRID: AB_1107673.

Techniques: Activation Assay, Flow Cytometry, Expressing, Fluorescence, In Vivo, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: PD-1/IL-2 bsAb protects CD8 + T cells from macrophage phagocytosis via STAT5-mediated upregulation of CD47 (A) Schematic of the Cleavage Under Targets and Tagmentation (CUT&Tag) assays workflow. (B) Distribution of STAT5 binding signals relative to transcription start sites (TSS) in CD8 + T cells treated with or without the PD-1/IL-2 bsAb. (C) Genomic annotation of differentially enriched STAT5 binding peaks in the bsAb-treated group. (D) KEGG pathway enrichment analysis of genes associated with STAT5 binding peaks. (E and F) Strategy and Venn diagram for identifying potential STAT5 downstream genes. (G) ChIP-qPCR analysis of STAT5 binding to the promoter regions of selected candidate genes ( n = 3). (H) Genome browser tracks showing STAT5 binding signals at the CD47 locus in control and PD-1/IL-2 bsAb-treated CD8 + T cells. (I) Schematic of the macrophage phagocytosis assay. (J) Representative confocal microscopy images showing macrophages (red) engulfing CD8 + T cells (green). Scale bars, 20 μm. (K) Flow cytometry quantification of the percentage of macrophages that had phagocytosed CD8 + T cells under the indicated conditions ( n = 3). Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA where appropriate.

Article Snippet: InVivoMAb anti-human CD8α , BioXcell , Cat#BE0004-2; RRID: AB_1107673.

Techniques: Binding Assay, ChIP-qPCR, Control, Phagocytosis Assay, Confocal Microscopy, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: CD47 protects CD8 + T cells from macrophage clearance to boost antitumor immunity in SMARCA4-deficient NSCLC (A) Schematic of the adoptive T cell therapy experiment ( n = 8/group). (B) Representative in vivo bioluminescence images of mice from the indicated treatment groups at different time points. (C) Tumor growth curves, as measured by bioluminescence, for mice in each treatment group. (D) Individual tumor growth curves for mice in each treatment group. (E) Kaplan-Meier survival curves of mice from the four treatment groups. (F) Quantification by flow cytometry of donor-derived CD45.2 + CD8 + T cells among total tumor-infiltrating lymphocytes. (G) Representative flow cytometry plots for donor-derived CD45.2 + CD8 + T cells expressing the exhaustion markers PD-1, TIGIT, and TIM-3. (H) Quantification of the percentage of CD45.2 + CD8 + T cells expressing PD-1, TIGIT, and TIM-3. (I) The production of TNF-α by donor-derived CD45.2 + CD8 + T cells. (J) The production of IFN-γ by donor-derived CD45.2 + CD8 + T cells. (K) Representative immunofluorescence images of tumor sections. White: CD8, green: CD47, red: F4/80. Scale bars, 70 μm. (L) Schematic model depicting the proposed mechanism of action. Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by one-way ANOVA.

Article Snippet: InVivoMAb anti-human CD8α , BioXcell , Cat#BE0004-2; RRID: AB_1107673.

Techniques: In Vivo, Flow Cytometry, Derivative Assay, Expressing, Immunofluorescence

Journal: Nature immunology

Article Title: Inhibition of ENT1 relieves intracellular adenosine-mediated T cell suppression in cancer.

doi: 10.1038/s41590-025-02153-3

Figure Lengend Snippet: Fig. 6 | Adenosine suppresses TIL function, which is restored by EOS301984. a, Proliferation of CD8+ T cells in dissociated tumor cell suspensions in the presence of ATP as a source of high adenosine concentrations and EOS301984 and anti-PD-1 as indicated. Representative example from n = 9 donors. An example gating strategy is provided in Supplementary Fig. 5. b–i, Summary data of EOS301984-mediated rescue of CD8+ (b) and CD4+ (c) T cell proliferation, TNF (d), IFNγ (e), granzyme A (f), granzyme B (g), perforin (h) and sFASL (i) production from dissociated tumor cell suspension cultures. Rescue refers to the degree to which adenosine-mediated suppression has been reversed. j, FACS analysis of TIM-3, PD-1 and TOX expression by PD-1− CD8+ TILs isolated from NSCLC tumor samples, stimulated with anti-CD3/CD28 microbeads for 72 h in the

Article Snippet: The following antibodies were used for Western blot: Anti-human ENT1 (SP120, Abcam, Ab182023, 1:1000) HRP anti-rabbit IgG (7074S, Cell signaling Technology, 7074S, 1:1000) Anti-B-actin (D6A8, Cell signaling Technology, 8457S, 1:1000) The following antibodies were used for in vivo experiments: InVivoPlus anti-mouse CD8α (53-6.7, BioXcell, BP004-1) Anti-PD-1 (OPDIVO® (nivolumab)), (Bristol Meyers Squibb) Validation SAHENTA was validated for the binding to human ENT1 in this publication: Robins, M. J., Peng, Y., Damaraju, V. L., Mowles, D., Barron, G., Tackaberry, T., Young, J. D. & Cass, C. E. Improved Syntheses of 5ʹ- S -(2-Aminoethyl)-6- N -(4-nitrobenzyl)-5ʹthioadenosine (SAENTA), Analogues, and Fluorescent Probe Conjugates: Analysis of Cell-Surface Human Equilibrative Nucleoside Transporter 1 (hENT1) Levels for Prediction of the Antitumor Efficacy of Gemcitabine.

Techniques: Suspension, Expressing, Isolation