Journal: Cell reports

Article Title: Defects in Antiviral T Cell Responses Inflicted by Aging-Associated miR-181a Deficiency

doi: 10.1016/j.celrep.2019.10.044

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cells were then stained with cell surface antigen-specific antibodies, permeabilized with Cytofix/Cytoperm kit (BD Biosciences) and stained with fluorescently labeled antibodies specific to the indicated cytokines.

Techniques: Infection, Recombinant, Cell Isolation, Sequencing, Transgenic Assay, Amplification, shRNA, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: ( A ) Pre-activated human and murine CD8 + T cells were treated with increasing doses of native IL-21 and 21h10 for 20 minutes and stained with AF488-phospho-STAT1 and AF647-phospho-STAT3 for flow cytometry analysis. ( B ) Genes related to cellular signaling and phenotype are compared for expression levels between murine CD8 + T cells treated with PBS, mIL-21, or 21h10 at 100 pM or 1 nM. * indicates memory-related genes, and ** indicates effector-related genes. Murine CD8 + T cells were pre-activated with TCR signals and treated with the cytokines at different concentrations for 24 hours, and cells were harvested for RNA-sequencing. ( C ) The percentage of IFN-𝛾-secreting cells within isolated CD8 + T cells upon treating PBS, mIL-21, or 21h10 at 1 nM, was quantified using flow cytometry after 5 hours of stimulation with PMA/Ionomycin in the presence of a protein transport inhibitor. ( D ) LCMV was inoculated into healthy mice along with daily injections of PBS, mIL-21 (2.5 µg per mouse), or 21h10 (2.5 µg per mouse). LCMV-specific CD8 + T cells from PBMC and spleen were analyzed for granzyme B expression. ( E ) Computational model of 21AT36, which is redesigned from 21h10 using ProteinMPNN, to bind IL-21R but not bind 𝛾 c . The mutated residues are highlighted in red, which abolish interactions with 𝛾 c . The 𝛾 c is included in the figure to show where the interface is positioned, not to imply that 21AT36 binds to 𝛾 c . ( F ) Western blot for STAT and pSTAT with vehicle (PBS), mIL-21, 21h10, and 21AT36 in TRP1 high CD8 + T cells. ( G ) MC38-bearing mice were treated with PBS, 21AT36, 21h10, or anti-PD-1 for 14 days. ( H ) Mice were inoculated with MC38 and treated with PBS, mIL-21, 10-fold dose mIL-21, and 21h10. ( I ) Western blot for STAT and pSTAT in the spleens of mice treated with vehicle (PBS), mIL-21, and 21h10 ( J ) Normalized pSTAT3/STAT3 in spleens of treated mice over 24 hours from ( I ). ( K ) MC38 rechallenges with MC38-survivor mice from previous 21h10 or anti-PD-1 treatment.

Article Snippet: Two days before and on the day of tumor inoculations, mice were treated with anti-CD4 (150 µg per mouse; Bioxcell Cat# BE0003-1) and/or anti-CD8 depleting antibodies (150 µg per mouse; Bioxcell (valid) # BE0061) or rat IgG2b isotype (150 µg per mouse; clone LTF-2, BioXCell Cat# BE0090) diluted in sterile endotoxin-free PBS.

Techniques: Staining, Flow Cytometry, Expressing, RNA Sequencing Assay, Isolation, Western Blot

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: 21h10 improves antitumor efficacy in in vivo B16F10 murine melanoma and ex vivo human PDOTs models, with non-neutralizing anti-drug antibodies, and toxicity alleviated with TNFα blockade. ( A ) Naïve CD8 + TRP1 high/low melanoma-specific T cells were adoptively transferred to mice prior to B16F10 inoculation. Cytokine therapy began on day 5 and continued every day until the stop of treatment (dashed line). ( B ) Mice received no prior T cell adoptive transfer before cytokine therapy. ( C ) Same as ( B ), but with adoptive transfer of naïve CD8 + TRP1 high/low T cells. ( D ) Scheme of PDOTS preparation. ( E ) Viability assessment of ex vivo human melanoma PDOTS following treatment of ICB (anti-PD-1, pembrolizumab, or anti-PD-1/anti-LAG-3), 21h10, or untreated control. ( F ) Representative images of PDOTS viability assessment shown in ( E ). PI-dead cells in red and Hoechst-nuclei in blue. ( G ) Mice died due to toxicity or after 40 days of treatment were bled and their serum was analyzed by ELISA for anti-drug antibodies against 21h10. Positivity (dotted line) was determined as two standard deviations above the mean of samples from mice treated only with PBS. Fractions indicate the number of mice classified as “positive” for anti-drug antibodies out of the total mice in each treatment group. ( H ) Mice with various titer levels of anti-21h10 antibodies were inoculated with MC38 tumors. Starting on day 6, mice were treated with 21h10 or PBS daily for 17 days. ( I ) Wild-type (WT), Rag2-/- , or Rag2-/- Il2rg-/- mice were treated with PBS or 21h10 daily and isotype or anti-NK1.1 depleting antibodies every three days. Mice were sacrificed when weight loss exceeded 20% of the initial starting weight. ( J ) Serum TNFα level was measured in C57BL/6 and Rag2-/- mice with PBS or 21h10 treatments. ( K ) Antitumor activity comparison on B16F10 melanoma between 21h10 and 21h10+aTNFα treatment groups.

Article Snippet: Two days before and on the day of tumor inoculations, mice were treated with anti-CD4 (150 µg per mouse; Bioxcell Cat# BE0003-1) and/or anti-CD8 depleting antibodies (150 µg per mouse; Bioxcell (valid) # BE0061) or rat IgG2b isotype (150 µg per mouse; clone LTF-2, BioXCell Cat# BE0090) diluted in sterile endotoxin-free PBS.

Techniques: In Vivo, Ex Vivo, Adoptive Transfer Assay, Control, Enzyme-linked Immunosorbent Assay, Activity Assay, Comparison

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: ( A ) Mice were treated as in , A and C, but on day 15 of tumor growth, tumors were processed for single-cell RNA-sequencing. UMAP of all samples are combined. ( B ) scRNAseq cluster composition average across treatment groups. ( C ) T cells in each scRNAseq sample from ( B ). Different colors for each bar indicate individual mice from each group. ( D ) Flow cytometry quantification of CD8 + T cell infiltration. ( E ) TRP1 high and TRP1 low melanoma antigen-specific cells captured by sequencing. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS groups. ( F ) T cells were sub-clustered from all samples from ( A ). The average T cell sub-cluster composition across treatment groups is shown on the right. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS samples. ( G ) IFN-𝛾-response score total UMAP from ( A ) based on scaled and summed Hallmark IFN-𝛾-response genes for each cell. ( H ) Heatmap from pseudo-bulk differential gene expression analysis across T cell sub-clusters. Significant differences compared to PBS samples. ( I ) Similar to ( H ), but for TRP1 cells only, values from individual mice are shown. P-values displayed vs. PBS samples. ( J ) Flow cytometry quantification of CD8 + IFN-𝛾/granzyme B levels in endogenous or transferred CD8 + TRP1 high/low T cells. ( K ) CD4 + T cell sub-clustering UMAP and cluster composition. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS samples. ( L ) Flow cytometry of IFN-𝛾 + CD4 + T cells. ( M ) Quantification and representative flow cytometry plots of Foxp3-GFP Treg cells. One-way ANOVA with Dunnett’s multiple comparisons for flow quantification and scRNA-seq total T cell comparison. For all dot plots with error bars: dots indicate individual mice, bars are S.E.M. *p ≤ 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001, or p-values displayed.

Article Snippet: Two days before and on the day of tumor inoculations, mice were treated with anti-CD4 (150 µg per mouse; Bioxcell Cat# BE0003-1) and/or anti-CD8 depleting antibodies (150 µg per mouse; Bioxcell (valid) # BE0061) or rat IgG2b isotype (150 µg per mouse; clone LTF-2, BioXCell Cat# BE0090) diluted in sterile endotoxin-free PBS.

Techniques: RNA Sequencing Assay, Flow Cytometry, Sequencing, Expressing, Comparison

Journal: Journal of Nanobiotechnology

Article Title: Targeting CD39 boosts PD-1 blockade antitumor therapeutic efficacy via strengthening CD8 + TILs function and recruiting B cells in cervical cancer

doi: 10.1186/s12951-025-03500-0

Figure Lengend Snippet: CD39 inhibitor combined with PD-1 blockade improved therapeutic efficacy by promoting CXCL13 release and B cell activation . ( A ) Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of CXCL13 in the serum samples of isotype, αPD-1, CD39i, and combined treatment groups of mice. ( B ) The content of CXCL13 in murine tumors was detected using ELISA. ( C ) Flow cytometry allowed detection of the proportion of CXCR5 + B cells in murine tumors. ( D - E ) Flow cytometry ( D ) and dot plots ( E ) showed the proportion of activated B cell infiltration in murine tumors. ( F ) The dot plot displayed the proportion of GCB cells in tumor B220 + B cells. ( G , H ) Dot plots showed the proportion of dark zone (DZ) ( G ) and light zone (LZ) ( H ) in GCB cells in mouse tumors. ( I , J ) Representative histograms ( I ) and summarized Mean Fluorescence Intensity (MFI) diagram ( J ) showed the expression of BCL6 in GCB cells. ( K ) The dot plot showed the proportion of plasma cells among CD19 + B cells of mouse tumors. ( L ) The dot plot displayed the proportion of memory B cells in mouse tumor B220 + B cells. The values were obtained using the Kruskal–Wallis test. * , P < 0.05; ** , P < 0.01; ***; P < 0.001; ns , not significant . Abbreviations: GCB, geminal center B cells; DZ, dark zone; LZ, light zone

Article Snippet: The sections were stained with a CD19 antibody (27949-1-AP; Proteintech; 1:500), Ki67 antibody(ab16667;Abcam;1:200), TUNEL antibody(G1507; Servicebio; 1:200) followed by a secondary antibody (GB23303; Servicebio; 1:200).

Techniques: Drug discovery, Activation Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Expressing, Clinical Proteomics

Journal: PLOS ONE

Article Title: Reactive oxygen species in endothelial signaling in COVID-19: Protective role of the novel peptide PIP-2

doi: 10.1371/journal.pone.0289854

Figure Lengend Snippet: A. Immunofluorescent labeling of Rac 1 (green) and membrane marker flotillin (red) in HPMVEC. HPMVEC were pre-treated with blank liposomes or PIP-2 carrying liposomes for 3 h followed by 1 h incubation with (media supplemented with) COVID-19 serum. Cells were fixed and immunostained with anti-Rac-GFP (1:100, green) and anti-flotillin (1:250). The secondary antibody was conjugated to Alexa 594 (1:200; red). DAPI (blue) was used to stain the nuclei. Rac1 is visualized as a green signal. Membranes were visualized using flotillin (red). The yellow colabel (white arrow) indicates the colocalization of Rac1 with the EC membrane. “COVID-19” denotes cells pre-treated with blank liposomes followed by COVID-19 serum; “COVID-19 + PIP-2” denotes cells pre-treated with PIP-2 carrying liposomes followed by COVID-19 serum. B. Rac translocation was analyzed by the ImageJ program. For each field, the perimeters of 3–4 cells were outlined (as shown by the dotted line). The green fluorescence intensity (Rac signal) along the cell border was measured and normalized to the cell perimeter. Three separate experiments were conducted for each sample. The average number from the three independent experiments was considered to represent the values for that sample. Data were obtained from N = 5 samples for each category (COVID-19 +PIP2 and COVID-19). Data are shown as a box & whiskers plot. The group average and median are indicated by a plus sign and horizontal bar, respectively. *p<0.05 as compared to COVID-19.

Article Snippet: The membranes were visualized using a polyclonal anti-flotillin antibody (1:200; Proteintech, Rosemont, IL, USA).

Techniques: Labeling, Membrane, Marker, Liposomes, Incubation, Staining, Translocation Assay, Fluorescence

Journal: Journal of translational medicine

Article Title: GPX2 inhibition enhances antitumor efficacy of lenvatinib via promoting immunogenic cell death in hepatocellular carcinoma.

doi: 10.1186/s12967-025-06468-5

Figure Lengend Snippet: Fig. 4 GPX2 downregulation augments lenvatinib’s efficacy against HCC by activating DC-mediated T-cell cytotoxicity. A and B Flow cytometry was utilized to quantify the expression of CD80 and CD86 biomarkers in mature DCs co-cultured with Huh7 cells treated with either si-GPX2 or lenvatinib. C The percent of CD3+CD8+ T cells was detected by flow cytometry following the co-culture of PBMCs with Huh7 cells treated with either si-GPX2 or lenvatinib. D and E ELISA assay detected IFN-γ and IL-2 levels in the cell supernatant of different treatment groups. Each experiment is conducted in triplicate, and the represented data are SD (±) mean for each experiment. *P < 0.05

Article Snippet: CD8 monoclonal antibody (66868-1) was obtained from Proteintech.

Techniques: Flow Cytometry, Expressing, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of translational medicine

Article Title: GPX2 inhibition enhances antitumor efficacy of lenvatinib via promoting immunogenic cell death in hepatocellular carcinoma.

doi: 10.1186/s12967-025-06468-5

Figure Lengend Snippet: Fig. 6 GPX2 downregulation enhances the lenvatinib’s efficiency against HCC via inducing ICD in vivo. A Representative images of GPX2 and HMGB1 stained by IHC of allograft subcutaneous tumors. Scale bar: 100 μm. B and C Quantitative analysis of GPX2 and HMGB1 expression in subcutaneous tumor tissue by IHC. D Representative images of CRT stained by IF of allograft subcutaneous tumors. Scale bar: 100 μm. E Representative images of CD8+GZMB+ T cell infiltration stained by IF of allograft subcutaneous tumors. Scale bar: 100 μm. Each experiment is conducted in triplicate, and the represented data are SD (±) mean for each experiment. *P < 0.05

Article Snippet: CD8 monoclonal antibody (66868-1) was obtained from Proteintech.

Techniques: In Vivo, Staining, Expressing

Journal: Molecular and Cellular Biology

Article Title: Nerve Growth Factor Stimulates the Concentration of TrkA within Lipid Rafts and Extracellular Signal-Regulated Kinase Activation through c-Cbl-Associated Protein

doi: 10.1128/mcb.01109-06

Figure Lengend Snippet: FIG. 2. TrkA, SOS, and CAP concentrate in the flotillin-containing membrane fraction following NGF treatment. PC12 cell lysates were fractionated using discontinuous sucrose gradients. Fraction 1 repre- sents the top of the gradient. PC12 cells were left untreated or stim- ulated with NGF for 5 min and lysed, and the lysates were resolved on sucrose gradients. Gradients were fractionated, and equal amounts of protein from corresponding fractions were separated by SDS-PAGE. Western blots were probed for flotillin, TrkA, phospho-TrkA (P- TrkA), CAP, SOS, FRS2, and TfR1. Flotillin, which is a marker for lipid raft membranes, was used as a marker for the lipid raft fraction, while TfR1 was used to mark the heavy membrane fractions. These results are representative of three independent experiments.

Article Snippet: Antibodies to CAP, ERK2, Trk, Cbl, SOS, Rap1, FRS2, and flotillin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Membrane, SDS Page, Western Blot, Marker

Journal: Molecular and Cellular Biology

Article Title: Nerve Growth Factor Stimulates the Concentration of TrkA within Lipid Rafts and Extracellular Signal-Regulated Kinase Activation through c-Cbl-Associated Protein

doi: 10.1128/mcb.01109-06

Figure Lengend Snippet: FIG. 3. Following NGF stimulation, CAP becomes associated with proteins important in lipid rafts and in MAPK activation. PC12 cells were stimulated with NGF for the indicated times, and cell lysates were prepared. (A) Flotillin was immunoprecipitated (IP) from cell lysates and the resulting Western blot (IB) was probed for CAP and flotillin. These data are representative of three indepen- dent experiments. (B) TrkA-CAP interactions were investigated by immunoprecipitation of TrkA followed by Western blotting with antibodies to CAP. The blots were stripped and reprobed for TrkA. (C) PC12 cells expressing Myc-tagged APS or GFP were stimulated with NGF. The tagged proteins were then immunoprecipitated from cell lysates with anti-Myc antibodies and then analyzed by Western blotting. The blots were probed with anti-CAP antibodies and then stripped and reprobed with anti-Myc antibodies to control for pro- tein expression levels and immunoprecipitation efficiency. (D) SOS was immunoprecipitated from cell lysates. Immunoprecipitates were resolved by SDS-PAGE. Western blots were probed for CAP. The blots were then stripped and reprobed with anti-SOS antibod- ies to control for immunoprecipitation efficiency. (E) PC12 cells were stimulated with NGF for the indicated times and then lysed. Cbl was immunoprecipitated from cell lysates. Immunoprecipitates were analyzed using Western blotting and probed for CAP and then reprobed with anti-Cbl antibodies.

Article Snippet: Antibodies to CAP, ERK2, Trk, Cbl, SOS, Rap1, FRS2, and flotillin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Expressing, Control, SDS Page

Journal: Molecular and Cellular Biology

Article Title: Nerve Growth Factor Stimulates the Concentration of TrkA within Lipid Rafts and Extracellular Signal-Regulated Kinase Activation through c-Cbl-Associated Protein

doi: 10.1128/mcb.01109-06

Figure Lengend Snippet: FIG. 4. Both TrkA and CAP colocalize with flotillin on the cell surface following NGF stimulation. (A) PC12 cells were treated with NGF for 0, 5, and 10 min. Cells were fixed and stained for CAP (red) and flotillin (green). Cells were then examined by confocal microscopy. (B) PC12 cells were treated with NGF for 0, 5, and 10 min. Cells were fixed and stained for TrkA (red) and flotillin (green). Cells were examined by confocal microscopy. Arrows indicate areas of colocal- ization. Scale bar, 5 m.

Article Snippet: Antibodies to CAP, ERK2, Trk, Cbl, SOS, Rap1, FRS2, and flotillin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Staining, Confocal Microscopy

Journal: Molecular and Cellular Biology

Article Title: Nerve Growth Factor Stimulates the Concentration of TrkA within Lipid Rafts and Extracellular Signal-Regulated Kinase Activation through c-Cbl-Associated Protein

doi: 10.1128/mcb.01109-06

Figure Lengend Snippet: FIG. 6. CAPSoHo inhibits colocalization of TrkA and flotillin following NGF stimulation. PC12 cells were transfected with FLAG- CAP or FLAG-CAPSoHo; 48 h later they were either left untreated or stimulated with NGF for 5 min. Cells were fixed and stained for TrkA (red), flotillin (green), or FLAG (blue) and examined by confo- cal microscopy. Arrows indicate points of colocalization. Open arrow- heads denote transfected cells. Scale bar, 5 m.

Article Snippet: Antibodies to CAP, ERK2, Trk, Cbl, SOS, Rap1, FRS2, and flotillin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Staining, Microscopy

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The APC function of B cells is closely associated with their FA metabolism in metastatic OvCa. A GSVA pathway enrichment analysis of APC function low and APC function high B cells in patients with metastatic OvCa ( GSE235951 , GSE147082 and GSE154600 dataset in the GEO database, n = 13). B Representative images of the IHC staining of TLS structure (composed of CD3 + T cells, CD19 + B cells, and CD21 + FDC), CD80, and CD86, respectively, in the area adjacent to or away from tumor or adipose tissues in clinical HGSOC specimens (n = 20 for TLS and n = 5 for CD80 or CD86). Red dashed line area: TLS structure. Magnification × 200. C Representative image of lymphoid aggregates (white dashed line area) by immunofluorescent staining in ascites of OvCa mice with 3 w and 6 w in tumor-bearing mice. D Mean fluorescence intensity of CD80, CD86, CD83, and MHC class II molecules in ascitic CD19 + B cells of OvCa mice were detected by flow cytometry. E Comparison of mRNA levels of FA metabolic genes in ascitic B cells in OvCa mice. The relative expression of each gene was calculated using β -actin as the internal reference. F Protein expression of β-actin and FA metabolic proteins in ascitic B cells in OvCa mice was assessed by WB. β -Actin was used as the internal control to calculate the relative expression level of FA metabolic proteins. G Mean fluorescence intensity of Bodipy C16 in ascitic B cells of OvCa mice detected by flow cytometry. H Expression of A-CoA in ascitic B cells of OvCa mice detected by ELISA. I Expression of ATP in ascitic B cells of OvCa mice detected by ELISA. J Mean fluorescence intensity of intracellular oxidized lipid in ascitic B cells of OvCa mice detected by flow cytometry. I Comparison of mRNA levels of FA metabolic genes in ascitic B cells in OvCa mice. The relative expression of each gene was calculated using β -actin as the internal reference. Data are presented as the mean ± SD of three independent experiments. FA, fatty acid; TLS, tertiary lymphoid structure. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: Immunohistochemistry, Staining, Fluorescence, Flow Cytometry, Comparison, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The APC function and resulting anticancer immunity of B cells can be enhanced by oleic acid (OA) via reprogramming FA metabolism in vitro. A Mean fluorescence intensity of CD80 and MHC Class II molecules in CD19 + B cells from peripheral blood of healthy volunteers (n = 3) treated with OA and PA (both 150 μM), respectively. B Mean fluorescence intensity of CD80, CD86, CD83, MHC Class II molecules, and Ki67 in splenic CD19 + B cells of WT mice treated with 150 μM OA. C Mean fluorescence intensity of CD80, CD86, CD83, and MHC Class II molecules in ascitic CD19 + B cells from 3 w OvCa-bearing mice when treated with 150 μM OA. D Analysis of FA metabolism-related signaling pathways based on RNA-seq results. GSEA was used to analysised the FA metabolic pathways. E Comparison of mRNA levels of main FA metabolic genes in ascitic B cells from 3 w tumor-bearing mice when treated with 150 μM OA. The relative expression of each gene was calculated using β -actin as the internal reference. F Experimental scheme to detect the influence of inhibiting OA uptake on ascitic B cells. G Comparison of protein expressions of main FA metabolic molecules in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS and treated with OA. β -Actin was used as the internal control to calculate the relative expression level of the main FA metabolic molecules. H Mean fluorescence intensity of Bodipy C16 in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS and treated with OA was detected by flow cytometry. I The expression of A-CoA, ATP and the FAO activity in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS treated with OA was detected by ELISA. J Mean fluorescence intensity of intracellular oxidized lipid in 3 w OvCa-bearing mouse ascitic B cells pretreated with BMS and treated with OA was detected by flow cytometry. K Mean fluorescence intensity of CD80, CD86, and CD83 in 3 w OvCa-bearing mouse ascitic CD19 + B cells pretreated with BMS and treated with OA was detected by flow cytometry. L Mean fluorescence intensity of Bodipy C16, CD80, CD86 and CD83 in FABP4-knockdown CD19 + B cells, which are from the ascites of 3 w OvCa-bearing mice, treated with OA, was detected by flow cytometry. PBMC, peripheral blood mononuclear cell; SP, spleen; AS, Ascites; OA, oleic acid; PA, palmitic acid; BMS, BMS309403. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: In Vitro, Fluorescence, Protein-Protein interactions, RNA Sequencing, Comparison, Expressing, Control, Flow Cytometry, Activity Assay, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: Reprogramming of the FA metabolism of B cells by OA can improve anticancer immunity in vitro. A Experimental scheme to detect the in vitro effects of ascitic B cells treated with OA on anticancer immunity. B Mean fluorescence intensity of CD80, CD86, and CD83 in 3 w OvCa-bearing mouse ascitic CD19 + B cells after pulsed with ID8-Luc-cell-prepared antigenic peptides and pretreated with BMS and treated with OA, T cells and ID8-Luc. C Levels of IFN- γ , GZMB, and TNF-α in the supernatant of the coculture system constructed by ID8-Luc cells and 3 w OvCa-bearing mouse ascitic B and splenic T cells were detected by ELISA. D Cytotoxicity of T cells in the coculture system mentioned above was detected by luciferase assay. AS, Ascites; Ag, Antigen; OA, oleic acid; BMS, BMS309403. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: In Vitro, Fluorescence, Construct, Enzyme-linked Immunosorbent Assay, Luciferase

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The enhanced APC function of B cells by OA in vitro is achieved through H3K27ac-mediated upregulation of PPAR γ expression. A Protein expression of β -actin and H3K27ac in 3 w OvCa-bearing mouse ascitic CD19 + B cells pretreated with BMS and treated with OA was assessed by WB. β -Actin was used as the internal control to calculate the relative expression level of H3K27ac. B The enrichment percentage of H3K27ac at the PPAR γ , CD80, CD86, and CD83 promoter regions was quantified using ChIP-seq analysis. C Correlation analysis of PPAR γ and APC function-related genes (CD80, CD86, CD83, MHC II) in B cells in OvCa patients in the TCGA database (n = 426). D Analysis of protein interaction among FABP4, PPAR γ , CD80, CD86, and CD83. E The enrichment percentage of PPAR γ at the CD80, CD86, and CD83 promoter regions was quantified using ChIP-seq analysis. F Mean fluorescence intensity of CD80, CD86, and CD83 in 3 w OvCa-bearing mouse ascitic CD19 + B cells pretreated with BMS, GW9662/Trog, and treated with OA was detected by flow cytometry. AS, Ascites; Ag, Antigen; OA, oleic acid; BMS, BMS309403. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: In Vitro, Expressing, Control, ChIP-sequencing, Fluorescence, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Promoting APC function of B cells via reprogramming the fatty acid metabolism enhances anticancer immunity in metastatic ovarian cancer

doi: 10.1007/s00262-026-04387-y

Figure Lengend Snippet: The combination of adoptive immunotherapy with APC function-enhanced B cells and LDC improved anticancer immunity in metastatic OvCa mice. A Experimental scheme to evaluate the effects of combining adoptive immunotherapy with APC function-enhanced B cells and LDC in metastatic OvCa mice. B Representative BLI images and comparison of OvCa progression in each group (n = 3). C Representative images ( a ) and the number of tumor nodules ( b ) in the abdominal wall of mice in each group (n = 3). Areas marked by yellow dashed lines: Representative tumor nodules in the abdominal wall. D (a) Representative images of lymphoid aggregates (composed of CD3 + T cells, CD19 + B cells, and CD21 + FDC) in the ascitic cells of mice in each group. (b) Comparison of the amount of CD3 + T, CD8 + T, Ki67 + CD3 + T, Ki67 + CD8 + T, CD19 + B, CD80 + B, CD86 + B, and lymphoid aggregates respectively in the ascitic cells of mice in each group (n = 3) in frozen sections detected by immunofluorescence. E Comparison of the proportion of CD3 + T, CD8 + T, CD19 + B, CD19 − CD138 + B cells, mean fluorescence intensity of Ki67 in CD3 + T and CD8 + T, and mean fluorescence intensity of CD80, CD86, CD83, MHC II in CD19 + B, respectively, in ascitic cells of each group of mice (n = 3) detected by flow cytometry. F Mean fluorescence intensity values of IL-2, IFN- γ , GZMB, and CTLA-4 in CD8 + T cells, respectively. G Kaplan Meier analysis of survival time of each group of mice (n = 7). LDC, low-dose chemotherapy; B OA , B cells that have been treated with OA for 24h; L-DDP, low-dose DDP (1 mg/kg); H-DDP, high-dose DDP (2 mg/kg); BLI, bioluminescence imaging. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant

Article Snippet: In the mechanistic study, mouse ascitic CD19 + B cells (1 × 10 6 /ml) were pretreated with fatty acid binding protein 4 gene (FABP4) inhibitor (BMS309403, MedChemExpress; Cat# HY-101903; 50 μM), PPARγ antagonist (GW9662, MedChemExpress; Cat# HY-16578; 25 μM), and PPAR γ agonist (Troglitazone, Trog, MedChemExpress; Cat# HY-50935; 10 μM) for 2h, respectively.

Techniques: Comparison, Immunofluorescence, Fluorescence, Flow Cytometry, Imaging