Journal: Cancer Immunology Research

Article Title: An IL6–Adenosine Positive Feedback Loop between CD73+ γδTregs and CAFs Promotes Tumor Progression in Human Breast Cancer

doi: 10.1158/2326-6066.cir-19-0923

Figure Lengend Snippet: Figure 3. CD73þ gdT cells are the predominant Tregs in breast cancer and exhibit direct immunosuppression via the adenosine-mediated pathway. A and B, Sorted CD73þ gdT cells, CD4þ Tregs (CD45þCD3þCD4þCD25þCD127low), CD4þCD73þ and CD8þCD73þ T cells, and CD73 gdT cells (1.0 104 cells, respectively) from tumor tissues and CD73þ gdT cells (1.0 104) from paired normal tissue were cocultured with CFSE-labeled allogeneic CD4þ T cells (5.0 104) in the presence of anti-CD3 and anti- CD28. n ¼ 5. A, CD4þ T-cell proliferation was evaluated on day 6 by flow cytometry. B, Bar diagram summarizes the percentages of proliferated CD4þ T cells (CFSElow). Data, mean SEM. n ¼ 5; , P < 0.01; , P < 0.001; paired Student t test for the analysis between CD73þ gdT cells from tumor and paired normal tissue and unpaired Student t test for the analysis among other cells from tumor tissue. C, Sorted CD73þ gdT, CD4þ Tregs, CD4þCD73þ and CD8þCD73þ T cells, and CD73 gdT cells (1.0 104 cells, respectively) from tumor tissues and CD73þ gdT cells (1.0 104) from paired normal tissue were cocultured with allogeneic CD8þ T cells (5.0 104) in the presence of anti-CD3 and anti-CD28. Concentrations of perforin in the supernatants were detected on day 6 by ELISA. Data, mean SEM. n ¼ 5; , P < 0.05; , P < 0.001; paired Student t test for the analysis between CD73þ gdT cells from tumor and paired normal tissue and unpaired Student t test for the analysis among other cells from tumor tissue. D and E, Sorted CD73þ gdT cells (1.0 104) from tumor tissues were cocultured with CFSE-labeled allogeneic CD4þ T cells (5.0 104) in the presence of anti-CD3 and anti-CD28 and pretreated with anti-CD73, an antibody cocktail, control antibody, or A2A (SCH58261) and A2B (PSB603) adenosine receptor antagonists. n ¼ 5. D, CD4þ T-cell proliferation was evaluated on day 6 by flow cytometry. n ¼ 5. E, Bar diagram summarizes the percentages of proliferated CD4þ T cells (CFSElow). Data, mean SEM. n ¼ 5; ns, no significance; , P <0.001;unpaired Student t test.mAb cocktail, the mixture of antibodies for LAG-3, CTLA-4, IL10, and TGFb; N, normal tissue; T, tumor tissue.

Article Snippet: For CD4þ and CD8þ T-cell isolation, peripheral blood mononuclear cells (PBMC) were derived from peripheral blood of healthy donors with Lymphocyte Separation Medium (Human, Ficoll–Paque, Tbdscience) and were labeled with the human CD4þ T (Miltenyi Biotec, #130-096-533) or CD8þ T-Cell Isolation Kit (Miltenyi Biotec, #130-096-495) and separated by magneticactivated cell sorting following the manufacturer's instructions.

Techniques: Labeling, Cytometry, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancer Immunology Research

Article Title: An IL6–Adenosine Positive Feedback Loop between CD73+ γδTregs and CAFs Promotes Tumor Progression in Human Breast Cancer

doi: 10.1158/2326-6066.cir-19-0923

Figure Lengend Snippet: Figure 7. Tumor-infiltrating CD73þ gdTregs predict worse clinical outcome and impede the prognostic impact of CD8þ T cells in patients with breast cancer. A, Breast cancer tissue sections of 516 samples were stained with antibodies for TCR gd (green), EpCAM (dark pink), and CD73 (red). Nuclei were stained with DAPI (blue). Representative image indicates that cells positive for both CD73 and TCRgd in EpCAM-negative areas were considered as stromal CD73þ gdTregs. White arrows indicate target cells (stromal CD73þ gdTregs). Scale bar, 50 mm. B, Breast cancer tissue sections of 516 samples were stained with antibodies for CD8 (green) and EpCAM (pink). Nuclei were stained with DAPI (blue). Representative image indicates that cells positive for CD8 in EpCAM-negative areas were considered as stromal CD8þ T cells. White arrows indicate target cells (stromal CD8þ T cells). Scale bar, 50 mm. C and D, The Kaplan–Meier survival curves/log-rank tests were used to compare OS (C) and DFS (D) in groups with high and low numbers of CD73þ gdTregs; median: 16.8 cells. E and F, Kaplan–Meier survival curves for the CD73þ

Article Snippet: For CD4þ and CD8þ T-cell isolation, peripheral blood mononuclear cells (PBMC) were derived from peripheral blood of healthy donors with Lymphocyte Separation Medium (Human, Ficoll–Paque, Tbdscience) and were labeled with the human CD4þ T (Miltenyi Biotec, #130-096-533) or CD8þ T-Cell Isolation Kit (Miltenyi Biotec, #130-096-495) and separated by magneticactivated cell sorting following the manufacturer's instructions.

Techniques: Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: Tumor-intrinsic RRBP1 inhibition triggers antitumor immunity. ( A ) Representative images of IHC staining for RRBP1 and CD8 + T cells in BC samples. ( B ) The correlation between RRBP1 expression and CD8 + T-cell infiltration was analyzed based on 96 patients from in-house BC cohort. Scale bar: 50 µm. ( C ) Representative images of IHC staining for RRBP1 expression in PD, SD, PR, and CR samples. Scale bar: 50 µm. ( D ) Bar plot showed the response rates of anti-PD-L1 therapy. Blue bars represent CR/PR, Red bars represent PD/SD. ( E ) Volcano plot of RNA-seq data for shNC or shRRBP1 tumors (n=3). Differentially expressed genes were identified with the threshold of |log2 (fold change) | >1 and FDR<0.05. ( F ) GSEA for DEGs showed the activation of immune-associated pathways in shRRBP1 tumors in the RNA-seq data. ( G ) Representative images of IHC and mIHC staining for RRBP1 and CD8 + T cells in shNC, shRRBP1, control or radezolid tumor tissues. Expression levels of the indicated proteins were displayed. Scale bar: 20 µm. ( H, I ) Flow cytometry showed the percentages of CD8 + T cells in CD3 + cells in shNC, shRRBP1, control or radezolid tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using Spearman correlation analysis ( B ), unpaired two-tailed t-test ( I ). ****p<0.0001. BC, bladder cancer; CR, complete response; FDR, false discovery rate; progressive disease; PR, partial response; PD-L1, programmed death-ligand 1; RNA-seq, RNA sequencing; RRB1, ribosomal-binding protein 1; SD, stable disease; IHC, immunohistochemistry; GSEA, gene set enrichment analysis; DEGs, differentially expressed genes; mIHC, multiplex immunohistochemistry.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Immunohistochemistry, Expressing, RNA Sequencing, Activation Assay, Staining, Control, Flow Cytometry, Two Tailed Test, Binding Assay, Multiplex Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: Single-cell RNA sequencing reveals the difference of CD8 + T-cell subgroup. The UMAP plot of CD8 + T cells subpopulation, color-coded by cell cluster and cell type. ( A ) The expression of markers in each CD8 + T cells subpopulation. ( B ) Bar plot showed the proportion of CD8 + T cells subpopulation in the shNC and shRRBP1 groups. ( C ) The percentage of each CD8 + T-cell clusters in shNC and shRRBP1 groups. ( D ) Heatmap showed the differentially activated pathway among all the CD8 + T-cell clusters. ( E ) The differentially expressed genes in CD8 + T cells between shNC and shRRBP1 groups. ( F ) KEGG analysis for differentially expressed genes showed the enrichment of immune-associated pathways. ( G, H ) mIHC and flow cytometric analysis displayed the tumor-infiltrating IFN-γ + or GZMB + CD8 + T cells in shNC or shRRBP1 tumor tissues. Scale bar: 20 µm. ( I–K ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Isotype control (IgG) or anti-mouse CD8 antibody administered on days –6, –3, and –1 before tumor challenge, with the same dose repeated on days 7, 9 and 11 after tumor challenge. Tumor sizes ( I ), volumes ( J ), and weight ( K ) were measured. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( H, K ) and two-way ANOVA with Tukey’s multiple comparison test ( J ). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; GZMB, Granzyme B; IFN, interferon; TEX, exhausted T cells; UMAP, Uniform Manifold Approximation and Projection; mIHC, multiplex immunohistochemistry; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Single Cell, RNA Sequencing, Expressing, Injection, Control, Two Tailed Test, Comparison, Multiplex Assay, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: RRBP1 inhibition promotes antitumor immunity via the CXCL10-CXCR3 axis in BC. ( A ) ScRNA-seq data showed the CXCR3 expression of CD8+T cells in shNC and shRRBP1 groups. ( B ) The correlation between CXCR3 expression and CXCL10 expression or activated CD8 + T cell based on 571 patients from TCGA-BLCA cohort and GSE13507 cohorts. ( C ) MB49 cells were co-cultured with CD8 + T cells, and tumor cells were stained with crystal violet. ( D ) Evaluation of the effect of genetic inhibition of RRBP1 on the cytotoxicity of CD8 + T cells in vitro conditioned culture model. ( E ) Schematic diagram of in vitro CD8 + T-cell migration assays. ( F ) The number of CD8 + T cells passing through the membrane of a Transwell system was analyzed by flow cytometry. ( G–I ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Tumor-bearing mice received intraperitoneal injection of either vehicle or anti-CXCL10 when the tumor volume reached a calculated average of 100 mm 3 . The tumor sizes ( G ), volumes ( H ), and weights ( I ) were measured. ( J ) Representative images of IHC and mIHC staining for CD8, CXCR3, CXCL10, IFN-γ, GZMB in different tumor tissues. ( K ) Flow cytometric analysis of tumor-infiltrating CD8 + T cells, CXCR3 + CD8 + T cells, IFN-γ + CD8 + T cells or GZMB + CD8 + T cells in distinct tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( D, F, I, K ) and two-way ANOVA with Tukey’s multiple comparison test ( H ). The data presented represent on one or three independent experiments. *p<0.01, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BC, bladder cancer; GZMB, Granzyme B; IFN, interferon; RRBP1, ribosomal-binding protein 1; scRNA-seq, single-cell RNA sequencing; IHC, immunohistochemistry; mIHC, multiplex immunohistochemistry; BLCA, bladder urothelial carcinoma.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Expressing, Cell Culture, Staining, In Vitro, Migration, Membrane, Flow Cytometry, Injection, Two Tailed Test, Comparison, Binding Assay, Single Cell, RNA Sequencing, Immunohistochemistry, Multiplex Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: RRBP1 inhibition enhances response to anti-PD-L1 therapy in BC. ( A–D ) The protein expression of surface PD-L1 was analyzed in BC cells or tumor tissues by flow cytometry after RRBP1 inhibition and was shown as the mean fluorescence intensity. ( E–G ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Tumor-bearing mice were received intraperitoneal injection of either vehicle or anti-PD-L1 antibody when the tumor volume reached a calculated average of 100 mm 3 . The tumor sizes ( E ), volumes ( F ), and weights ( G ) were measured. ( H ) Representative images of IHC and mIHC staining for CD8, CXCR3, CXCL10, IFN-γ, GZMB in different tumor tissues. ( I ) Flow cytometric analysis of tumor-infiltrating CD8 + T cells, CXCR3 + CD8 + T cells, IFN-γ + CD8 + T cells or GZMB + CD8 + T cells in distinct tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( B, D, G, I ) and two-way ANOVA with Tukey’s multiple comparison test ( F ). The data presented represent on one or three independent experiments. *p<0.01, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BC, bladder cancer; GZMB, Granzyme B; IFN, interferon; PD-L1, programmed death-ligand 1; RRBP1, ribosomal-binding protein 1; IHC, immunohistochemistry; mIHC, multiplex immunohistochemistry.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Expressing, Flow Cytometry, Fluorescence, Injection, Staining, Two Tailed Test, Comparison, Binding Assay, Immunohistochemistry, Multiplex Assay

Journal: Vaccines

Article Title: Evaluation of the Effect of CD70 Co-Expression on CD8 T Cell Response in Protein-Prime MVA-Boost Vaccination in Mice

doi: 10.3390/vaccines11020245

Figure Lengend Snippet: Vaccination of C57Bl/6 mice with rMVA-HBc±CD70. ( A ) C57Bl/6 mice were immunized subcutaneously with HBcAg adjuvanted with CpG and PCEP at day 0 (group wt received adjuvants only). At day 21, mice were boosted with MVAwt (group wt), rMVA-HBc (group HBc) or rMVA-HBc-CD70 (group HBc-CD70) by intraperitoneal injection. At day 35, splenocytes of mice were isolated and stained with PE-labelled C 93 multimers to determine the amount of HBc-specific CD8 T cells ( B ) showing representative dot plots and ( C ) showing the group analysis. The table ( C ) shows the proportion of all CD8+ T cells. At day 35, splenocytes of vaccinated mice were also stimulated ex vivo with C 93 or B8R peptides for intracellular cytokine staining (ICS) to evaluate C 93 - or B8R- specific IFNγ-, TNFα- and IL2 production of CD8 T cells ( D ) showing representative dot plots and ( E ) showing the group analysis. ( F ) Simultaneous detection of IFNγ, TNFα and IL2 production of CD8 cells upon HBc stimulation ex vivo: dot plots show one representative mouse of each group. The table shows the mean values (% of CD8+ cells) of 4 mice/group. ( G ) The gain of function of CD8 T cells after CD70 co-stimulation during vaccination: the proportion of IFNγ+ CD8 T cells upon ex vivo C 93 stimulation was set in relation to the proportion of C 93 multimer-positive CD8 T cells in the same animals. Proportions were calculated for each mouse separately. C 93 : HBcAg CD8 T cell epitope; B8R: MVA-derived CD8 T cell epitope. Crosses and triangles represent individual mice; horizontal lines indicate mean values; error bars indicate standard deviation. Statistics: one-way ANOVA and subsequent unpaired t-test, n.s.: not statistically significant.

Article Snippet: Surface markers were stained with PB-conjugated anti-CD8 T cell antibody (clone 56.6-7, BD Biosciences, Heidelberg, Germany) and anti-CD4-PE (eBioscience, San Diego, USA).

Techniques: Injection, Isolation, Staining, Ex Vivo, Derivative Assay, Standard Deviation

Journal: Vaccines

Article Title: Evaluation of the Effect of CD70 Co-Expression on CD8 T Cell Response in Protein-Prime MVA-Boost Vaccination in Mice

doi: 10.3390/vaccines11020245

Figure Lengend Snippet: Amount and functionality of CD8 T cells in the livers and spleens of vaccinated HBV1.3tg mice with and without CD70 co-expression. HBV1.3tg mice were immunized with protein-prime (day 0, HBc + CpG + PCEP, group wt only CpG and PCEP) and MVA boost (day 21, with MVAwt (group wt), rMVA-HBc (group HBc) or rMVA-HBc-CD70 (group HBc-CD70)). Mice were divided into groups with low to moderate HBeAg levels (1–8 S/CO, “low viremia”) and high antigen levels (8–23 S/CO, “high viremia”) before the experiment. At day 35, splenocytes and liver-associated lymphocytes of mice were isolated. ( A ) Representative dot blot analysis of liver-associated lymphocytes (LAL) for single mice of each vaccination group, utilizing C 93 multimer staining. ( B ) Representative dot blot analysis of intracellular cytokine staining (IFNγ) after ex vivo C 93 stimulation of LALs. ( C ) Percentages of C 93 multimer-positive CD8 T cells in vaccinated mice. ( D ) HBV core-specific CD8 T cell response for the groups of mice with low and high viremia represented by the portion of CD8 T cells expressing IFNγ after ex vivo stimulation with C 93 peptide. ( E ) MVA-specific CD8 T cell response represented by the portion of CD8 T cells expressing IFNγ after ex vivo stimulation with B8R peptide. Mice with low and high viremia are presented together. C 93 : HBV core-derived CD8 T cell epitope; B8R: MVA-derived CD8 T cell epitope; Crosses and triangles represent individual mice; Horizontal lines indicate mean values; Error bars indicate standard deviation. Differences between groups HBc and HBc-CD70 (if >2 mice/group) were not statistically significant (one-way ANOVA and unpaired t-test).

Article Snippet: Surface markers were stained with PB-conjugated anti-CD8 T cell antibody (clone 56.6-7, BD Biosciences, Heidelberg, Germany) and anti-CD4-PE (eBioscience, San Diego, USA).

Techniques: Expressing, Isolation, Dot Blot, Staining, Ex Vivo, Derivative Assay, Standard Deviation

Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

Article Title: Redirected Lysis of Human Melanoma Cells by a MCSP/CD3-bispecific BiTE Antibody that Engages Patient-derived T Cells

doi: 10.1097/CJI.0b013e3182307fd8

Figure Lengend Snippet: CD3 + -positive cells in PBMC from 13 patients with melanoma

Article Snippet: Stimulated CD8 + T cells were isolated from the stimulated PBMC using a negative isolation kit for human CD8 T cells (Dynal Biotech, Oslo, Norway), according to the manufacturer's instructions.

Techniques:

Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

Article Title: Redirected Lysis of Human Melanoma Cells by a MCSP/CD3-bispecific BiTE Antibody that Engages Patient-derived T Cells

doi: 10.1097/CJI.0b013e3182307fd8

Figure Lengend Snippet: Use of OKT-3 and IL-2 to stimulate PBMC or CD8+ T cells from healthy donors increased the efficacy of MCSP-BiTE redirected lysis in MCSP-positive melanoma cell (M11-HI). A: Percent change of cytotoxicity in M11-HI cell line co-cultured with stimulated PBMC (sPBMC) or CD8+ T cells (sCD8) from healthy donors at E:T ratio 1:1. Value of MCSP-BiTE 0 ng/mL was used as a baseline in stimulated PBMC and CD8+ T cells, respectively. B: Number of live melanoma cells after FACS-based cytotoxicity assay. The anti-tumor effect of MCSP-BiTE was calculated according to the following formula: live melanoma cell number = total melanoma cell number (PKH67 positive) × percentage of live melanoma cell (7-AAD negative and annexin V negative). All data from 4 sets of PBMC or CD8+ T cells co-cultured with M11-HI was averaged. Each bar shows the mean number of live melanoma cells and SE. C: Melanoma cells were pre-stained with PKH-67 and co-cultured with stimulated PBMC or CD8+ T cells for 18 hours in presence of MCSP-BiTE 0, 0.1, 1 and 10 ng/mL. 7-AAD and annexin V were used to detect dead melanoma cells and early apoptotic cells, respectively. sPBMC: stimulated PBMC, sCD8: stimulated CD8+ T cell.

Article Snippet: Stimulated CD8 + T cells were isolated from the stimulated PBMC using a negative isolation kit for human CD8 T cells (Dynal Biotech, Oslo, Norway), according to the manufacturer's instructions.

Techniques: Lysis, Cell Culture, Cytotoxicity Assay, Staining

Journal: Nature Communications

Article Title: Charged substrate treatment enhances T cell mediated cancer immunotherapy

doi: 10.1038/s41467-025-56858-y

Figure Lengend Snippet: a Flow cytometric analysis of T cell membrane potential. OT-I cells were isolated and activated with OVA 257–264 peptides (2 μg/mL) for 6 h followed by DiBAC4(3) staining ( n = 5 biological replicates, mean ± sem, **** P < 0.0001, two-tailed unpaired Student’s t test). b Graphic illustration of the study design for evaluating T cell activation. OT-I cells were isolated and cultured on the nanocomposite membranes with varying surface charges, and activated by OVA 257–264 peptides (2 μg/mL) for 6 h. NC, non-charged nanocomposite membranes; LC, low-charged nanocomposite membranes; MC, mid-charged nanocomposite membranes; HC, high-charged nanocomposite membranes. c Flow cytometric analysis of T cell membrane potential. OT-I cells were isolated and cultured on the nanocomposite membranes with varying surface charges and treated with OVA peptides (2 μg/mL) for 6 h, followed by DiBAC4(3) staining ( n = 6 biological replicates, mean ± sem, *, compared with NC; &, compared with LC; #: compared with MC; *** P = 0.0002, **** P < 0.0001, & & & & P < 0.0001, #### P < 0.0001, two-tailed unpaired Student’s t test). d Whole-cell patch-clamp recording of T cell membrane potential. OT-I cells were isolated and activated with OVA 257–264 peptides (2 μg/mL) for 6 h, followed by electrophysiological recording ( n = 4 biological replicates, mean ± sem, *** P = 0.0002, two-tailed unpaired Student’s t test). e In vitro cytotoxicity assay of OVA expressing LLC cells by OT-I transgenic T cells at decreased E:T ratios. E, effector cells; T, target cells ( n = 4 biological replicates, mean ± sem, *, compared with NC; &, compared with LC; #: compared with MC; * P = 0.0169, ** P = 0.0047 (E:T = 1:1), ** P = 0.0030 (E:T = 0.5:1), **** P < 0.0001, & & P = 0.0023, & & & & P < 0.0001, ### P = 0.0006, #### P < 0.0001, one-way ANOVA). f A schematic illustration of the adoptive T cell therapy. Briefly, NOD-SCID mice were inoculated subcutaneously with LLC-OVA cells (2 × 10 6 ) on day 0. OT-I naïve T cells, isolated from lymph nodes and spleen of OT-I mice, were cultured on the nanocomposite membranes with varying surface charges, activated with OVA peptides (2 μg/mL) for 2 days, and expanded with IL-2 (10 U/mL) for another 4 days. OT-I cells (3 × 10 6 ) were intravenously injected into tumor-bearing mice on day 7 post-tumor inoculation. g Macroscopic evaluation of tumors from mice intravenously injected with OT-I cells ( n = 6 mice). h Tumor volume of mice intravenously injected with OT-I cells was monitored over time ( n = 6 mice, mean ± sem, *, compared with NC; &, compared with LC; ns, not significant ( P > 0.05), **** P < 0.0001, && & P = 0.0002, & & & & P < 0.0001, one-way ANOVA). i Quantity of tumor-infiltrating immune cells in mice intravenously injected with OT-I cells 21 days after implantation ( n = 6 mice, mean ± sem, *, compared with NC; &, compared with LC; #, compared with MC; ns, not significant ( P > 0.05), * P = 0.0371, ** P = 0.0043, **** P < 0.0001, & & P = 0.0050, # P = 0.0423, one-way ANOVA). j Flow cytometric analysis of the frequency of CD8 + T cells in tumors from mice intravenously injected with OT-I cells 21 days after implantation ( n = 6 mice, mean ± sem, *, compared with NC; &, compared with LC; #, compared with MC; ns, not significant ( P > 0.05), * P = 0.0162, *** P = 0.0004, **** P < 0.0001, & & & & P < 0.0001, ## P = 0.0019, one-way ANOVA). k Flow cytometric analysis of the frequency of CD8 + T cells in draining lymph node (dLN) from mice intravenously injected with OT-I cells 21 days after implantation ( n = 6 mice, mean ± sem, *, compared with NC; &, compared with LC; #, compared with MC; ns, not significant ( P > 0.05), * P = 0.0447, *** P = 0.0001, **** P < 0.0001, & & & P = 0.0001, # P = 0.0462, one-way ANOVA). l Flow cytometric analysis of the expression of TNF in tumor-infiltrated OT-I cells ( n = 6 mice, mean ± sem, *, compared with NC; &, compared with LC; ns, not significant ( P > 0.05), * P = 0.0326, ** P = 0.0012, **** P < 0.0001, & P = 0.0126, one-way ANOVA). m Flow cytometric analysis of the expression of IFNγ in tumor-infiltrated OT-I cells ( n = 6 mice, mean ± sem, *, compared with NC; &, compared with LC; ns, not significant ( P > 0.05), * P = 0.0299, & P = 0.0365 (MC vs. LC), & P = 0.0156 (HC vs. LC), one-way ANOVA). n Flow cytometric analysis of the expression of GZMB in tumor-infiltrated OT-I cells ( n = 6 mice, mean ± sem, *, compared with NC; &, compared with LC; #, compared with MC; ns, not significant ( P > 0.05), **** P < 0.0001, & & & & P < 0.0001, ## P = 0.0090, one-way ANOVA). o Graphic illustration of the study design for investigating the status of tumor-infiltrated OT-I cells. Briefly, NOD-SCID mice were inoculated subcutaneously with LLC-OVA cells (2 × 10 6 ) on day 0. OT-I naïve T cells, isolated from lymph nodes and spleen of OT-I mice, were cultured on the nanocomposite membranes with varying surface charges, activated with OVA peptides (2 μg/mL) for 2 days, and expanded with IL-2 (10 U/mL) for another 4 days. OT-I cells (3 × 10 6 ) were intravenously injected into tumor-bearing mice on day 7 post-tumor inoculation. Tumor-infiltrated OT-I cells were isolated on day 21 and subjected to SMART-sequencing. p , q Tumor-infiltrated OT-I cells were isolated and subjected to SMART-seq. GSEA of genes expressed in the HC group and NC group. ES, enrichment score; NES, normalized enrichment score. Data are representative of two ( d , e ) independent experiments. Source data are provided as a Source Data file.

Article Snippet: CD8 + T cells were separated from PBMCs by magnetic cell sorting (MACS) using CD8 microbeads (Miltenyi Biotec, 130-098-194).

Techniques: Membrane, Isolation, Staining, Two Tailed Test, Activation Assay, Cell Culture, Patch Clamp, In Vitro, Cytotoxicity Assay, Expressing, Transgenic Assay, Injection, Sequencing

Journal: Nature Communications

Article Title: Charged substrate treatment enhances T cell mediated cancer immunotherapy

doi: 10.1038/s41467-025-56858-y

Figure Lengend Snippet: a A schematic illustration of the study designed to assess the OT-I cell-mediated secondary immune response. Briefly, NOD-SCID mice were inoculated subcutaneously with LLC-OVA cells (2 × 10 6 ) on day 0. OT-I naïve T cells, isolated from lymph nodes and spleen of OT-I mice, were cultured on the nanocomposite membranes with varying surface charges, activated with OVA peptides (2 μg/mL) for 2 days, and expanded with IL-2 (10 U/mL) for another 4 days. OT-I cells (3 × 10 6 ) were intravenously injected into tumor-bearing mice on day 7 post-tumor inoculation. 21 days later, the subcutaneous tumor was excised and tumor cells (5 × 10 6 ) were re-implanted in the opposite axillary region. b Macroscopic evaluation of second tumors ( n = 8 mice). c Tumor volume of the secondary inoculation was monitored over time ( n = 8 mice, mean ± sem, **** P < 0.0001, two-tailed unpaired Student’s t test). d Flow cytometric analysis of the frequency of CD8 + T cells in second tumors ( n = 8 mice, mean ± sem, *** P = 0.0002, two-tailed unpaired Student’s t test). e A schematic illustration of the study designed to assess the CAR-T cell-mediated secondary immune response. Briefly, NSG mice were intravenously injected with luciferase-expressing Raji cells (5 × 10 5 ) on day 0. T cells were cultured on the nanocomposite membranes with varying surface charges and activated by anti-CD3/anti-CD28 mAb-coated beads. After transduced with CD19 CAR, T cells were expanded with IL2 and intravenously injected into tumor-bearing mice on day 7 post-tumor inoculation. After 30 days, tumor-free mice were selected and Raji cells (2 × 10 6 ) were re-injected into the mice via the tail vein. Naive mice refer to mice that have not previously been inoculated with tumors. f Tumor cells were measured by bioluminescence imaging at indicated times ( n = 4 mice). Source data are provided as a Source Data file.

Article Snippet: CD8 + T cells were separated from PBMCs by magnetic cell sorting (MACS) using CD8 microbeads (Miltenyi Biotec, 130-098-194).

Techniques: Isolation, Cell Culture, Injection, Two Tailed Test, Luciferase, Expressing, Transduction, Imaging

Journal: Nature Communications

Article Title: Charged substrate treatment enhances T cell mediated cancer immunotherapy

doi: 10.1038/s41467-025-56858-y

Figure Lengend Snippet: a Graphic illustration of the study design for the investigation the role of proliferative T cells in antitumor immunity. OT-I cells were cultured on HC nanocomposite membranes. OVA peptides (2 μg/mL) and IL-2 (10 U/mL) were used to stimulate T cell activation and ensuing proliferation. CFSE lo and CFSE hi cells were isolated by BD FACS Aria II flow cytometer and subjected to RNA-sequencing. b Volcano plot analysis of gene expression in CFSE lo and CFSE hi T cells. Red, genes upregulated in CFSE lo T cells. Blue, genes downregulated in CFSE lo T cells. Statistical significance was assessed by the Wald test. c Genes that were significantly upregulated in CFSE lo T cells were analyzed using GO database. Statistical significance was assessed by oa ne-sided hypergeometric test. d Macroscopic evaluation of tumors from mice intravenously injected with CFSE lo and CFSE hi T cells ( n = 5 mice). e Tumor volume of mice intravenously injected with CFSE lo and CFSE hi T cells cultured on HC nanocomposite membrane was monitored over time ( n = 5 mice, mean ± sem, *** P = 0.0005 (Day 13), *** P = 0.0008 (Day 15), *** P = 0.0003 (Day 19), **** P < 0.0001, two-tailed unpaired Student’s t test). f Quantity of tumor-infiltrating immune cells in mice intravenously injected with CFSE lo and CFSE hi T cells ( n = 5 mice, mean ± sem, ** P = 0.0055, two-tailed unpaired Student’s t test). g Flow cytometric analysis of the frequency of CD8 + T cells in tumors from mice intravenously injected with CFSE lo and CFSE hi T cells ( n = 5 mice, mean ± sem, **** P < 0.0001, two-tailed unpaired Student’s t test). h Flow cytometric analysis of the expression of IFNγ in tumor-infiltrated OT-I cells ( n = 5 mice, mean ± sem, ** P = 0.0042, two-tailed unpaired Student’s t test). i Flow cytometric analysis of the expression of TNF in tumor-infiltrated OT-I cells ( n = 5 mice, mean ± sem, ** P = 0.0013, two-tailed unpaired Student’s t t est). j Flow cytometric analysis of the expression of GZMB in tumor-infiltrated OT-I cells (n = 5 mice, mean ± sem, *** P = 0.0001, two-tailed unpaired Student’s t test). k Flow cytometric analysis of the expression of KLRG1 in tumor-infiltrated OT-I cells (n = 5 mice, mean ± sem, **** P < 0.0001, two-tailed unpaired Student’s t-test). Source data are provided as a Source Data file.

Article Snippet: CD8 + T cells were separated from PBMCs by magnetic cell sorting (MACS) using CD8 microbeads (Miltenyi Biotec, 130-098-194).

Techniques: Cell Culture, Activation Assay, Isolation, Flow Cytometry, RNA Sequencing, Gene Expression, Injection, Membrane, Two Tailed Test, Expressing

Journal: Nature Communications

Article Title: Charged substrate treatment enhances T cell mediated cancer immunotherapy

doi: 10.1038/s41467-025-56858-y

Figure Lengend Snippet: a OT-I cells were isolated and cultured on the nanocomposite membranes with varying surface charges and treated with OVA peptides (2 μg/mL) for 24 h, followed by ATAC-seq. Volcano plot analysis of pairwise comparison of ATAC-seq density between HC and NC OT-I cells was shown. Statistical significance was assessed by the Wald test. b Genes upregulated in the HC group were analyzed with GO terms. Statistical significance was assessed by a one-sided hypergeometric test. c A schematic illustration of the identification of transcription factor EGR1. The sequence motifs identified from the peaks were analyzed and predicted binding transcription factors. d Representative EGR1 binding sites in the promoter region of key genes were displayed. e Immunoblot analysis of protein expression level of EGR1 in T cells cultured on the nanocomposite membranes with varying surface charges in the presence or absence of OVA peptides. f Flow cytometric analysis of the frequencies of CD25 − CD69 − cells, CD25 − CD69 + cells, and CD25 + CD69 + cells in OT-I cells cultured on the nanocomposite membranes with varying surface charges in the presence or absence of EGR1. OVA peptides (2 μg/mL) were used to stimulate T cell activation ( n = 4 biological replicates, mean ± sem, * P = 0.0353, **** P < 0.0001, two-tailed unpaired Student’s t test). g Flow cytometric analysis of the expression of IFNγ in OT-I cells cultured on the nanocomposite membranes with varying surface charges in the presence or absence of EGR1. OVA peptides (2 μg/mL) were used to stimulate T cell activation ( n = 4 biological replicates, mean ± sem, ns, not significant ( P > 0.05), **** P < 0.0001, two-tailed unpaired Student’s t test). h Flow cytometric analysis of the expression of TNF in OT-I cells cultured on the nanocomposite membranes with varying surface charges in the presence or absence of EGR1. OVA peptides (2 μg/mL) were used to stimulate T cell activation ( n = 4 biological replicates, mean ± sem, * P = 0.0115, **** P < 0.0001, two-tailed unpaired Student’s t test). i Flow cytometric analysis of the expression of Ki-67 in OT-I cells cultured on the nanocomposite membranes with varying surface charges in the presence or absence of EGR1. OVA peptides (2 μg/mL) and IL2 were used to stimulate T cell activation and expansion ( n = 4 biological replicates, mean ± sem, ns, not significant ( P > 0.05), *** P = 0.0001, two-tailed unpaired Student’s t-test). j NOD-SCID mice were inoculated subcutaneously with LLC-OVA cells (2 × 10 6 ) on day 0. OT-I cells with or without EGR1 were cultured on the nanocomposite membranes with varying surface charges, activated with OVA peptides (2 μg/mL) for 2 days, and expanded with IL-2 (10 U/mL) for another 4 days. OT-I cells (3 × 10 6 ) were intravenously injected into tumor-bearing mice on day 7 post-tumor inoculation. Macroscopic evaluation of tumors from mice intravenously injected with WT or Egr1 ─/─ OT-I cells 21 days post-implantation ( n = 5 mice). k Tumor volume of mice intravenously injected with WT or Egr1 ─/─ OT-I cells was monitored over time ( n = 5 mice, mean ± sem, * P = 0.0350, **** P < 0.0001, two-tailed unpaired Student’s ttest). l Flow cytometric analysis of the frequency of CD8 + T cells in tumors from mice intravenously injected with WT or Egr1 ─/─ OT-I cells ( n = 5 mice, mean ± sem, ns, not significant ( P > 0.05), **** P < 0.0001, two-tailed unpaired Student’s t test). m Flow cytometric analysis of the frequency of CD8 + T cells in dLN from mice intravenously injected with WT or Egr1 ─/─ OT-I cells ( n = 5 mice, mean ± sem, * P = 0.0182, **** P < 0.0001, two-tailed unpaired Student’s t test). n Flow cytometric analysis of the expression of GZMB in tumor-infiltrated WT or Egr1 ─/─ OT-I cells ( n = 5 mice, mean ± sem, ns, not significant ( P > 0.05), ** P = 0.0082, two-tailed unpaired Student’s t test). Data are representative of two ( e – i ) independent experiments. Source data are provided as a Source Data file.

Article Snippet: CD8 + T cells were separated from PBMCs by magnetic cell sorting (MACS) using CD8 microbeads (Miltenyi Biotec, 130-098-194).

Techniques: Isolation, Cell Culture, Comparison, Sequencing, Binding Assay, Western Blot, Expressing, Activation Assay, Two Tailed Test, Injection

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Cohort and in vitro studies reveal the role of GPR34 in macrophages. a Volcano plot showing differentially expressed genes between responder group and non-responder group from the macrophage subclusters of scRNA sequencing data. The horizontal dashed line represents the P -value cutoff ( P < 10⁻⁵⁰), and the vertical dashed line represents the log 2 FC cutoff (-1 or 1). FC fold change, sig significance, R Responder, NR Non-responder. b , c UMAP plot showing the expression of GPR34 in all cells ( b ) and macrophages ( c ). Mac: Macrophage, cl cluster. d Representative mIF staining images (100x) of surgical specimens from responders and non-responders in the clinical trial ( n = 26). Navy: GPR34, Magenta: CD68, Red: CD8, Green: Tim-3, Yellow: CK19, Blue: DAPI. White arrowheads: Tim-3 + CD8 + exhausted T cells (Tex); White arrows: GPR34 + CD68 + macrophages. White scale bar = 100 μm. e Bar plot comparing the proportion of CD68 + macrophages (top) and the proportion of GPR34 + cells among CD68 + macrophages (bottom) between responders and non-responders. Two-tailed unpaired t-test. Data are presented by mean ± SD. * P < 0.05. f Bar plot comparing the proportion of CD8 + T cells (top) and the proportion of Tim-3 + cells among CD8 + T cells (bottom) between responders and non-responders. Two-tailed unpaired t-test. Data are presented by mean ± SD. g Representative mIF staining images (200x) of surgical specimens from clinical trial patients ( n = 26). Navy: GPR34, Magenta: CD68, Red: MPO, Green: CD3, Dark yellow: CD20, Yellow: CK19, Blue: DAPI. White scale bar = 50 μm. h Representative mIF staining images (200x) of clinical trial patient surgical specimens ( n = 26). Navy: GPR34, Magenta: CD68, Red: α-SMA, Green: CD31, Dark yellow: CD117, Yellow: CD56, Blue: DAPI. White scale bar = 50 μm. i Bar plots showing the ratio of GPR34 + cells in different cell types ( n = 26). One-way ANOVA with Dunnett’s test was used to compare other cell types versus macrophages. Data are presented by mean ± SD. j Bar plots showing the ratio of GPR34 + cells in different cell types ( n = 26) examined by flow cytometry. One-way ANOVA with Dunnett’s test was used to compare other cell types versus macrophages. Data are presented by mean ± SD. k , l Kaplan-Meier curves for overall survival (OS) and Recurrence-free survival (RFS) in the prospective cohort ( k ) and retrospective cohort ( l ), comparing prognosis between groups with high versus low infiltration of GPR34 + macrophages. The cutoff for the proportion of GPR34 + cells among CD68 + cells were dichotomized using a 20% . Log-rank test was used for comparison. m Flow cytometry analysis of GPR34 + cells in tumor tissue versus adjacent non-tumorous tissue from prospective cohort patients ( n = 42). Scatter plot shows the paired infiltration proportion of GPR34 + cells in tumor and normal tissue from the same patient . Two-tailed paired t -test was used. n Flow cytometry analysis of GPR34 + cells in tumor tissue from prospective cohort patients ( n = 42). Bar plots show the infiltration proportions of CD45 + cells, CD8 + T cells, Tim-3 + PD-1 + T cells, MRC1 + macrophages, and MHC-I + macrophages in the low GPR34 ( ≤ 20%) versus high GPR34 ( > 20%) groups. Two-tailed unpaired t-test. Data are presented by mean ± SD. o BMDMs from C57BL/6 mice were cultured until day 5, stimulated with KPC cell TCM for 12 h, followed by treatment with Surufatinib (4 nM) or CSF-1R inhibitor (PLX3397, 20 nM) for 24 h, then analyzed by flow cytometry ( n = 3). Bar plot compares the gMFI of GPR34 among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

Techniques: In Vitro, Sequencing, Expressing, Staining, Two Tailed Test, Flow Cytometry, Comparison, Cell Culture

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Macrophage-specific GPR34 knockout improves chemotherapy outcomes in mice. a Orthotopic pancreatic injection of KPC-GFP-LUC cells into Gpr34 flox/flox and Gpr34 Δ Lyz2 mice. After tumor formation, chemotherapy was administered to simulate tumor killing. Tumor bioluminescence intensity was dynamically monitored by bioluminescence imaging. Representative bioluminescence images show tumor growth in each group ( n = 5). b Time-course curve of bioluminescence intensity in orthotopic tumor-bearing mice (n = 5). Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SEM. c Bar plot of tumor weight on day 21 post-implantation in orthotopic tumor-bearing mice ( n = 5). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS: no significance, *: P < 0.05. d Flow cytometry analysis of the proportions of CTL, Tex, M1 macrophages, and M2 macrophages in tumor tissues of orthotopic tumor-bearing mice (n = 5). Bar plots show the infiltration differences of immune cells among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. e KPC mice were irradiated with a total dose of 10 Gy, followed by tail vein injection of bone marrow cells from Gpr34 +/+ or Gpr34 −/− mice. After successful transplantation confirmed by flow cytometry. Tumor growth was monitored by B-ultrasound during chemotherapy simulating injury signals ( n = 8–10). Representative B-ultrasound images compare tumor size among groups. Black dashed lines outline tumor boundaries. Tumor volume was calculated as 0.5 * long diameter * (short diameter) ². S: spleen; K: kidney. Black arrows point to the tumor. f Time-course plot of tumor volume monitored by B-ultrasound. Each solid line represents an individual mouse. Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. g Kaplan-Meier curve of KPC spontaneous tumor-bearing mice ( n = 8–10). Log-rank test was used. h mIF staining of paraffin sections from orthotopic tumors in the bone marrow transplantation KPC mice ( n = 6). Representative fluorescence images (top 100x, bottom 400x) show exhausted T cell infiltration among groups. Red: CD8α, Green: Tim-3, White: CK19, Blue: DAPI. White scale bar = 50 μm. i Bar plot showing the proportion of Tim-3 + CD8 + T cells from mIF staining in KPC spontaneous tumor-bearing mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. j Fluorescence imaging of macrophages sorted by flow cytometry from tumors of KPC-GFP-LUC orthotopic injection mice model. Representative images show GFP phagocytosis by macrophages in each group ( n = 6). Green: GFP, Blue: DAPI. White scale bar = 20 μm. k Bar plot showing the gMFI of GFP phagocytosis by flow-sorted macrophages from orthotopic tumors ( n = 5). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. l Bar plot of the gMFI of GFP in macrophages from KPC cell orthotopic tumor-bearing mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. m Bar plot showing the proportion of MerTK + or AXL + macrophage from flow cytometry analysis in KPC mice ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used . Data are presented by mean ± SD. n BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, cultured with TCM for 12 h, followed by co-culture with CD8 + T cells for 24 h. Flow cytometry detected the proportion of MerTK + or AXL + macrophage. Bar plots show differences between Gpr34 +/+ and Gpr34 −/− groups ( n = 6). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

Techniques: Knock-Out, Injection, Imaging, Flow Cytometry, Irradiation, Transplantation Assay, Staining, Fluorescence, Cell Culture, Co-Culture Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Validation of GPR34 function in macrophage and CD8 + T cell co-culture system. a Gpr34 flox/flox and Gpr34 Δ Lyz2 mice were treated with anti-CD8α or IgG, followed by orthotopic pancreatic injection of KPC-LUC cells. After tumor formation, chemotherapy was administered to simulate an injury signal. Tumor bioluminescence was dynamically monitored. Representative bioluminescence images show tumor growth in different groups ( n = 6). b Time-course curve of bioluminescence imaging for the KPC-LUC orthotopic model ( n = 6). Two-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance, *** P < 0.001. c Bar plot showing tumor weight on day 21 in the KPC-LUC orthotopic model ( n = 6). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. NS no significance. d , e BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, then co-cultured with TCM and KPC-GFP cells for 12 h. BMDMs were then isolated and co-cultured with CD8 + T cells for 24 hours . Flow cytometry analyzed the expression of functional molecules in BMDMs ( d ) and CD8 + T cells ( e ). Bar plots show levels in Gpr34 +/+ vs Gpr34 −/− groups ( n = 3). Two-tailed unpaired Student’s t test was used. Data are presented by mean ± SD. f , g BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, pre-stimulated with SIINFEKL, then cultured with TCM for 12 h, followed by co-culture with CD8 + T cells from OT-1 mice for 24 h. Flow cytometry detected T cell-specific killing function ( f ) and BMDM antigen presentation function ( g ). Bar plots show differences between Gpr34 +/+ and Gpr34 −/− groups ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. h Violin-box plots of cytokine transcript expression in macrophage clusters from scRNA sequencing data. White dot and solid lines in boxes represent medians and quartiles. Two-tailed Wilcoxon test. i BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, then stimulated with TCM and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. qPCR detected Cxcl16 transcript levels. Bar plot compares Cxcl16 transcripts between groups ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. j ELISA detection of cytokine secretion in supernatant from BMDMs stimulated with apoptotic KPC-GFP cells. Bar plot shows CXCL16 protein secretion levels from Gpr34 +/+ and Gpr34 −/− BMDMs ( n = 10). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. k , l BMDMs from C57BL/6 mice were cultured until day 5, transiently transfected with siRNA, then co-cultured with TCM, LysoPS and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. BMDMs were then isolated and co-cultured with CD8 + T cells. Flow cytometry detected T cell exhaustion ( k ) and cytotoxicity levels ( l ) ( n = 3). One-way ANOVA with Dunnett’s test compared siRNA groups versus control. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

Techniques: Biomarker Discovery, Co-Culture Assay, Injection, Imaging, Cell Culture, Isolation, Flow Cytometry, Expressing, Functional Assay, Two Tailed Test, Immunopeptidomics, Sequencing, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: LysoPS-GPR34 modulates macrophage efferocytosis and inflammatory cytokine secretion. a , b BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM, LysoPS and chemotherapy-induced apoptotic KPC-GFP cells for 12 h. These were then co-cultured with CD8 + T cells for 24 hours. Flow cytometry detected GFP efferocytosis ( a ) and p-AKT levels ( b ) in BMDMs. Bar plots show differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. c BMDMs from Gpr34 +/+ and Gpr34 −/− mice were treated with LysoPS and co-cultured with CD8 + T cells. Flow cytometry detected MRC1, MHC-I on macrophages, and Tim-3, GZMB on CD8 + T cells. Bar plot shows gMFI differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. d , e BMDMs from C57BL/6 mice were treated with LysoPS and AKT inhibitor, then co-cultured with chemotherapy-induced apoptotic KPC-GFP cells for 12 h, followed by co-culture with CD8 + T cells for 24 h. Flow cytometry detected GFP uptake by BMDMs ( d ) and exhaustion/cytotoxicity levels of CD8 + T cells ( e ). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. f UMA P plot showing relative expression of efferocytosis-related genes from scRNA sequencing analysis. g BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM and either chemotherapy-induced apoptotic KPC-GFP cells or normal KPC-GFP cells for 12 h. After removing cells, RNA was extracted for qPCR. Bar plot compares transcript levels among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. h BMDMs from C57BL/6 mice were cultured until day 5, co-cultured with apoptotic KPC cells of negative control for 12 hours. RNA was extracted for qPCR. Bar plot compares transcript differences among groups. One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. i BMDMs from C57BL/6 mice were cultured until day 5, stimulated with TCM and LysoPS for 12 h. ELISA detected CXCL16 secretion in supernatant. Bar plot shows levels in LysoPS vs. control groups. Two-tailed unpaired Student’s t test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

Techniques: Cell Culture, Flow Cytometry, Co-Culture Assay, Expressing, Sequencing, Negative Control, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Targeting GPR34 in damage-associated macrophages enhances anti-tumor immunity and the efficacy of Surufatinib in pancreatic cancer

doi: 10.1038/s41392-026-02641-4

Figure Lengend Snippet: Macrophage efferocytosis function influences antigen presentation ability through MHC-I. a BMDMs from C57BL/6 mice were cultured until day 5, co-cultured with TCM and chemotherapy-induced apoptotic KPC-GFP cells for 12 hours, then analyzed by flow cytometry for GFP uptake. Bar plot shows gMFI of GFP in BMDMs treated with MerTK inhibitor vs control ( n = 3). One-way ANOVA with Dunnett’s test compared MerTKi groups to control. Data are presented by mean ± SD. b BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM and chemotherapy-induced apoptotic KPC-OVA-GFP cells for 12 hours, treated with MerTK inhibitor, then co-cultured with CD8 + T cells from OT1 mice for 24 hours. Flow cytometry detected MHC-I, SIINFEKL loading, CD80, CD86 on BMDMs. Bar plot shows differences between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. c Flow cytometry detection of Tetramer + , PD-1 + , Tim-3 + , and GZMB + cells after co-culture of BMDMs with OT1 CD8 + T cells. Bar plot shows differences between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. d Apoptotic KPC cells induced by chemotherapy and labeled with Caspase3/7 green were co-cultured with BMDMs. Phagolysosome formation was detected using pHrodo red. Representative fluorescence microscopy images (1000x) show differences between MerTK inhibitor and control groups ( n = 6). Green: Caspase3/7, Red: pHrodo, Blue: DAPI. White scale bar= 20 μm. e Bar plots show total pHrodo fluorescence intensity (left) and the number of Caspase3/7 + pHrodo + vesicles per cell (right) in BMDMs after incubation with apoptotic cells ( n = 6). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. f Flow cytometry analysis of pHrodo gMFI in BMDMs after incubation with apoptotic cells. Bar plot shows pHrodo gMFI levels between MerTK inhibitor and control groups ( n = 3). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. g Violin-box plots of lysosome-associated gene transcript expression in macrophage subpopulations from scRNA sequencing data. Solid lines represent medians and quartiles. One-way ANOVA with Kruskal-Wallis H test compared groups (Mac_cl1 as reference). h , i BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, co-cultured with TCM and chemotherapy-induced apoptotic KPC cells for 12 hours. After removing apoptotic cells, RNA was extracted for qPCR. Bar plots show transcript differences between Gpr34 +/+ and Gpr34 −/− BMDMs ( h ) efferocytosis receptors, ( i ) lysosome-related/transcription factors, ( n = 3). Two-tailed unpaired t-test was used. Data are presented by mean ± SD. j BMDMs from C57BL/6 mice were cultured until day 5, transiently transfected with siRNA, then co-incubated with TCM and chemotherapy-induced apoptotic KPC cells for 12 hours and analyzed by flow cytometry. Bar plot shows differences in MHC-I protein levels between knockdown and control groups ( n = 3). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. k BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM and chemotherapy-induced apoptotic KPC-OVA-GFP for 12 hours, treated with lysosomal inhibitor, then co-cultured with OT1 CD8 + T cells for 24 hours. Bar plot shows pHrodo gMFI in macrophages from flow cytometry, comparing lysosomal inhibitor group vs control. One-way ANOVA with Dunnett’s test was used . Data are presented by mean ± SD. l , m Flow cytometry detection of macrophage antigen presentation function ( l ) and CD8 + T cell specific killing capacity ( m ) in the BMDM-OT1 CD8 + T cell co-culture system. Bar plots show differences between lysosomal inhibitor and control groups. One-way ANOVA with Dunnett ’ s test was used. Data are presented by mean ± SD. n BMDMs from C57BL/6 mice were cultured until day 5, co-incubated with TCM, MerTK inhibitor/Lysosome inhibitor and chemotherapy-induced apoptotic KPC cells for 12 hours. Bar plot shows differences in MHC-I protein levels between different groups detected by flow cytometry ( n = 3). One-way ANOVA with post-hoc Tukey’s test was used. Data are presented by mean ± SD. o –q BMDMs from Gpr34 +/+ and Gpr34 −/− mice were cultured until day 5, transiently transfected with Cxcl16 siRNA, co-incubated with TCM, LysoPS and chemotherapy-induced apoptotic KPC-OVA-GFP for 12 hours, treated with MerTK inhibitor, then co-cultured with OT1 CD8 + T cells for 24 hours. Flow cytometry detected CD8 + T cell specific killing function (o ), cytotoxic function ( p ), and exhaustion levels ( q ). One-way ANOVA with Dunnett’s test was used. Data are presented by mean ± SD. NS no significance, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: CD8+ T cells were isolated from spleens of OT-1 or C57BL/6 mice using the human CD8 + T Cell Isolation Kit (Negative Isolation) (Miltenyi, Cat. No. 130-104-075).

Techniques: Immunopeptidomics, Cell Culture, Flow Cytometry, Control, Incubation, Co-Culture Assay, Labeling, Fluorescence, Microscopy, Expressing, Sequencing, Two Tailed Test, Transfection, Knockdown

Journal:

Article Title: Interleukin-10 Increases Th1 Cytokine Production and Cytotoxic Potential in Human Papillomavirus-Specific CD8 + Cytotoxic T Lymphocytes

doi:

Figure Lengend Snippet: (A) Effect of various concentrations of IL-10 on IL-2-induced proliferation of E7-specific CD8+ T cells at 6 to 7 days after antigen stimulation with autologous tumor cells. Pure populations of CD8+ T cells were cultured in CM (open bar) or in CM supplemented with increasing concentrations of IL-10 (solid bars). Cells were assayed for [3H]thymidine incorporation during the final 16 h of a 96-h culture. Results represent the means of triplicate wells ± standard deviations (SD). Thymidine incorporation in the presence of 1 to 20 ng of IL-10/ml plus IL-2, compared to that for control CTL cultured in IL-2 alone, was significant at P values <0.01 by Student's t test. No significant differences were noted when thymidine incorporation in the presence of 5 ng of IL-10/ml plus IL-2 was compared to levels in the presence of 10 and 20 ng of IL-10/ml plus IL-2. (B) Effect of 5 ng of IL-10/ml on IL-2-induced proliferation of E7-specific CD8+ T cells at 4 to 6, 8 to 10, and 14 to 16 days after antigen stimulation with autologous tumor cells. Pure populations of CD8+ T cells were cultured in CM (open bars) or in CM supplemented with 5 ng of IL-10/ml (solid bars). Cells were assayed for [3H]thymidine incorporation during the final 16 h of a 96-h culture. Results represent the means of triplicate wells ± SD. Thymidine incorporation in the presence of IL-10 plus IL-2, compared to that in control CTL cultured in IL-2 alone, was significant at P values <0.01 at all time points tested.

Article Snippet: Demonstration of HLA class I restriction of tumor-specific CD8 + T-cell responses was achieved in standard cytotoxicity assays ( 40 ) in the presence of blocking MAb specific for a nonpolymorphic HLA class I determinant (W6/32) or isotype-matched control MAb (hybridomas were obtained from the American Type Culture Collection).

Techniques: Cell Culture, Control

Journal:

Article Title: Interleukin-10 Increases Th1 Cytokine Production and Cytotoxic Potential in Human Papillomavirus-Specific CD8 + Cytotoxic T Lymphocytes

doi:

Figure Lengend Snippet: Effect of IL-10 on the expression of CD8β (A) and CD8α (B) by HPV-specific CTL as analyzed by flow cytometry. CD8+ T cells at 6 to 7 days after antigen stimulation with autologous tumor cells were stained with different MAb after incubation in CM alone (light lines) or in the presence of 5 ng of IL-10/ml for 72 to 96 h (heavy lines). Dashed lines, histograms from cells stained with control MAb.

Article Snippet: Demonstration of HLA class I restriction of tumor-specific CD8 + T-cell responses was achieved in standard cytotoxicity assays ( 40 ) in the presence of blocking MAb specific for a nonpolymorphic HLA class I determinant (W6/32) or isotype-matched control MAb (hybridomas were obtained from the American Type Culture Collection).

Techniques: Expressing, Flow Cytometry, Staining, Incubation, Control

Journal:

Article Title: Interleukin-10 Increases Th1 Cytokine Production and Cytotoxic Potential in Human Papillomavirus-Specific CD8 + Cytotoxic T Lymphocytes

doi:

Figure Lengend Snippet: Effect of IL-10 exposure on the percentage of CD56+ CD8+ T cells, as assessed by two-color flow cytometric analysis. The results from one experiment are shown and are representative of five separate experiments.

Article Snippet: Demonstration of HLA class I restriction of tumor-specific CD8 + T-cell responses was achieved in standard cytotoxicity assays ( 40 ) in the presence of blocking MAb specific for a nonpolymorphic HLA class I determinant (W6/32) or isotype-matched control MAb (hybridomas were obtained from the American Type Culture Collection).

Techniques:

Journal:

Article Title: Interleukin-10 Increases Th1 Cytokine Production and Cytotoxic Potential in Human Papillomavirus-Specific CD8 + Cytotoxic T Lymphocytes

doi:

Figure Lengend Snippet: (A) Effect of different cytokines in combination with IL-2 on the cytotoxic activity of E7-specific CTL. CD8+ T cells at 6 to 7 days from the last antigen stimulation with autologous tumor cells were cultured in CM (open bar; control) or in CM with different cytokines (solid bars), as described in Materials and Methods for 72 to 96 h before being tested in cytotoxicity assays against autologous tumor cells. Percent lysis (± standard deviation) at a 5:1 effector/target cell ratio is shown. The increase in cytotoxic activity, compared to that for control CTL cultured in IL-2 alone, was significant at P values <0.01 in the presence of IL-10 plus IL-2 and at P values <0.05 in the presence of IL-12 plus IL-2 by Student's t test. (B) Effect of various concentrations of IL-10 in combination with IL-2 on the cytotoxic activity of E7-specific CTL against autologous tumor targets. CD8+ T cells were cultured in CM (open bar; control) or in CM with the addition of various doses of IL-10 (solid bars) for 72 to 96 h before being tested in cytotoxicity assays against autologous tumor cells. Percent lysis (± standard deviation) at a 5:1 effector/target cell ratio is shown. The increase in cytotoxic activity, compared to that for control CTL cultured in IL-2 alone, was significant at P values <0.01 in the presence of 5 to 20 ng of IL-10/ml (C) Effect of 5 ng of IL-10/ml on the cytotoxic activity of E7-specific CD8+ T cells at 4 to 6, 8 to 10, and 14 to 16 days after antigen stimulation with autologous tumor cells. Pure populations of CD8+ T cells were cultured in CM (open bars) or in CM supplemented with 5 ng of IL-10/ml (solid bars) for 72 to 96 h before being tested in cytotoxicity assays against autologous tumor cells. Percent lysis (± standard deviation) at a 5:1 effector/target cell ratio is shown. Increased cytotoxic activity by CTL cultured in the presence of IL-10 plus IL-2, compared to that for control CTL cultured in IL-2 alone, was significant at P values <0.01 at all time points tested.

Article Snippet: Demonstration of HLA class I restriction of tumor-specific CD8 + T-cell responses was achieved in standard cytotoxicity assays ( 40 ) in the presence of blocking MAb specific for a nonpolymorphic HLA class I determinant (W6/32) or isotype-matched control MAb (hybridomas were obtained from the American Type Culture Collection).

Techniques: Activity Assay, Cell Culture, Control, Lysis, Standard Deviation

Journal:

Article Title: Interleukin-10 Increases Th1 Cytokine Production and Cytotoxic Potential in Human Papillomavirus-Specific CD8 + Cytotoxic T Lymphocytes

doi:

Figure Lengend Snippet: Effect of 5 ng of IL-10/ml in combination with IL-2 on the cytotoxic activity of E7-specific CTL measured in a 6-h 51Cr release assay against autologous tumor cells, autologous tumor cells plus anti-HLA class I blocking MAb (W6/32), autologous LCL, and K562. CD8+ T cells at 6 to 7 days after antigen stimulation with autologous tumor cells were cultured in CM (open bar; control) or in CM with the addition of 5 ng of IL-10/ml (solid bars) for 72 to 96 h before being tested in cytotoxicity assays. Percent lysis (± standard deviation) at a 5:1 effector/target cell ratio is shown. Inhibition of CTL-mediated killing by anti-HLA class I MAb (50 μg/ml) was significant at P values <0.01 for CD8+ T cells cultured in the presence of 5 ng of IL-10/ml plus IL-2 as well as control CTL cultured in IL-2 alone.

Article Snippet: Demonstration of HLA class I restriction of tumor-specific CD8 + T-cell responses was achieved in standard cytotoxicity assays ( 40 ) in the presence of blocking MAb specific for a nonpolymorphic HLA class I determinant (W6/32) or isotype-matched control MAb (hybridomas were obtained from the American Type Culture Collection).

Techniques: Activity Assay, Release Assay, Blocking Assay, Cell Culture, Control, Lysis, Standard Deviation, Inhibition

Journal:

Article Title: Interleukin-10 Increases Th1 Cytokine Production and Cytotoxic Potential in Human Papillomavirus-Specific CD8 + Cytotoxic T Lymphocytes

doi:

Figure Lengend Snippet: Effect of 72 to 96 h of exposure to IL-10 on the expression of intracellular perforin by HPV-specific CTL, as analyzed by flow cytometry. CD8+ T cells at 6 to 7 days (A) and 14 to 16 days (B) after antigen stimulation with autologous tumor cells were cultured in CM alone (light lines) or in the presence of 5 ng of IL-10/ml (heavy lines) before being stained with FITC-conjugated MAb against perforin, as described in Materials and Methods. Dashed lines, histograms from cells stained with isotype control MAb.

Article Snippet: Demonstration of HLA class I restriction of tumor-specific CD8 + T-cell responses was achieved in standard cytotoxicity assays ( 40 ) in the presence of blocking MAb specific for a nonpolymorphic HLA class I determinant (W6/32) or isotype-matched control MAb (hybridomas were obtained from the American Type Culture Collection).

Techniques: Expressing, Flow Cytometry, Cell Culture, Staining, Control

Journal:

Article Title: Interleukin-10 Increases Th1 Cytokine Production and Cytotoxic Potential in Human Papillomavirus-Specific CD8 + Cytotoxic T Lymphocytes

doi:

Figure Lengend Snippet: Two-color flow cytometric analysis of intracellular IFN-γ and IL-4 expression by tumor-specific CD8+ T cells. CD8+ T cells at 2 to 4 days (A and B), 8 to 10 days (C and D), and 14 to 16 days (E and F) after antigen stimulation with autologous tumor cells were cultured in CM alone (A, C, and E) or in the presence of 5 ng of IL-10/ml for 72 to 96 h (B, D, and F) before being activated overnight with solid-phase anti-CD3 in the presence of brefeldin A, as described in Materials and Methods. A representative experiment is shown.

Article Snippet: Demonstration of HLA class I restriction of tumor-specific CD8 + T-cell responses was achieved in standard cytotoxicity assays ( 40 ) in the presence of blocking MAb specific for a nonpolymorphic HLA class I determinant (W6/32) or isotype-matched control MAb (hybridomas were obtained from the American Type Culture Collection).

Techniques: Expressing, Cell Culture

Journal:

Article Title: Interleukin-10 Increases Th1 Cytokine Production and Cytotoxic Potential in Human Papillomavirus-Specific CD8 + Cytotoxic T Lymphocytes

doi:

Figure Lengend Snippet: Two-color flow cytometric analysis of intracellular IL-2 and IL-4 expression by tumor specific CD8+ T cells. CD8+ T cells at 2 to 4 days (A and B), 8 to 10 days (C and D), and 14 to 16 days (E and F) after antigen stimulation with autologous tumor cells were cultured in CM alone (A, C, and E) or in the presence of 5 ng of IL-10/ml for 72 to 96 h (B, D, and F) before being activated overnight with solid-phase anti-CD3 in the presence of brefeldin A, as described in Materials and Methods. A representative experiment is shown.

Article Snippet: Demonstration of HLA class I restriction of tumor-specific CD8 + T-cell responses was achieved in standard cytotoxicity assays ( 40 ) in the presence of blocking MAb specific for a nonpolymorphic HLA class I determinant (W6/32) or isotype-matched control MAb (hybridomas were obtained from the American Type Culture Collection).

Techniques: Expressing, Cell Culture

Journal: bioRxiv

Article Title: Blockade of β-adrenergic receptor signaling improves cancer vaccine efficacy through its effect on naive CD8 + T-cell priming

doi: 10.1101/497263

Figure Lengend Snippet: A , C57BL/6J mice were s.c. injected with E7 expressing-TCl tumor cells and vaccinated as mentioned in . Frequency of E7-specific CD8 + T cells from TDLN single-cell suspensions analyzed by flow cytometry at day 10 post-vaccination, with representative flow plots of Kb-E7-dextramer and CD44 staining in CD8 + T cells. Pool from three independent experiments ( n = 15). B , Confocal image of a TDLN slice stained for tyrosine hydroxylase (TH, red) and CD3 (green). 25x magnification. C-E, C57BL/6J mice were s.c. injected with 1 × 10 5 E7 expressing-TCl tumor cells. STxBE7 vaccine plus IFN-α were administrated ten days later (d0). The next day, vaccinated mice received an additional dose of IFN-α. Mice were daily treated with propranolol commencing 4 days after vaccination (d4). C, Experimental design. D , Tumor growth curves of non-vaccinated non-treated mice (dashed black line), vaccinated non-treated mice (black line) and vaccinated propranolol-treated mice (blue line). Pool from three independent experiments ( n = 15). E, Frequencies of CD45 + -cells among live cells and E7-specific CD8 + T cells among CD45 + -cells from tumor single-cell suspensions analyzed by flow cytometry at day 10 post-vaccination. Pool from two independent experiments ( n = 10). Statistical analysis by Mann-Whitney test: *, P <0.05, ****, P <0.0001. Mean ± SEM.

Article Snippet: For intracellular IFN-γ staining, cells were stimulated in vitro for 4 h at 37°C with either 5 μg/ml E7-peptide (kindly provided by Dr Eric Tartour) or Dynabeads Mouse T-activator CD3/CD28 (1 bead for 2 CD8 + T cells; Thermofisher) or with 0.

Techniques: Injection, Expressing, Flow Cytometry, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Blockade of β-adrenergic receptor signaling improves cancer vaccine efficacy through its effect on naive CD8 + T-cell priming

doi: 10.1101/497263

Figure Lengend Snippet: A and B , Naive CD8 + T cells were loaded with Indo-1, pre-treated with either adrenaline or noradrenaline at the indicated concentration during 10 min (A) or pre-treated with 10 μM propranolol during 30 min followed by treatment with either 10 μM adrenaline or noradrenaline during 10 min (B). iCa 2+ mobilization was assessed by flow cytometry before and after stimulation with hamster anti-CD3ε Abs (first arrow, above) cross-linked with antihamster IgG (second arrow) and is indicated as % response in treated cells relative to untreated cells, with representative normalized Indo-1 ratio histograms. A , Pool from four independent experiments ( n = 4). B , Pool from two independent experiments ( n = 5). C and D, Proliferation index of CVT-labeled naive CD8 + T cells stimulated with anti-CD3/CD28 Abs in the presence of adrenaline or noradrenaline at the indicated doses (C) or in the presence of 10 μM adrenaline or noradrenaline with or without IL-2 (D) during 3 days, indicated as % proliferation of treated cells relative to untreated cells, with representative histograms of CTV dilution. C , Pool from four independent experiments ( n = 20). D , Pool from two independent experiments ( n = 8). E and F , IL-2 (E) and IFN-γ (F) measured in the supernatants 24 h after stimulation of naive CD8 + T cells with anti-CD3/CD28 Abs in the presence of 10 μM adrenaline or noradrenaline. Pool from six independent experiments ( n = 8). Statistical analysis by one-way ANOVA test: *, P <0.05, **, P <0.01, ***, P <0.001, ****, p <0.0001. Mean ± SEM.

Article Snippet: For intracellular IFN-γ staining, cells were stimulated in vitro for 4 h at 37°C with either 5 μg/ml E7-peptide (kindly provided by Dr Eric Tartour) or Dynabeads Mouse T-activator CD3/CD28 (1 bead for 2 CD8 + T cells; Thermofisher) or with 0.

Techniques: Concentration Assay, Flow Cytometry, Labeling

Journal: bioRxiv

Article Title: Blockade of β-adrenergic receptor signaling improves cancer vaccine efficacy through its effect on naive CD8 + T-cell priming

doi: 10.1101/497263

Figure Lengend Snippet: A , Facs-sorted CD8 + TILs were loaded with Indo-1, pre-treated with either 10 μM adrenaline, noradrenaline or PGE 2 during 10 min. iCa 2+ mobilization was assessed by flow cytometry before and after stimulation with hamster anti-CD3ε Abs (first arrow, above) cross-linked with anti-hamster IgG (second arrow) and is indicated as % response in treated cells relative to untreated cells, with representative normalized Indo-1 ratio histograms. Pool from three independent experiments ( n = 5). B , Proliferation rate of CD8 + TILs cultured in the presence of 10 μM adrenaline or noradrenaline with or without IL-2 (20 U/ml) during 24 h, represented as ratio of absolute number at day 0 / absolute number at day 1. Pool from two independent experiments ( n = 9). C and D , IL-2 (C) and IFN-γ (D) measured in the supernatants 24 h after stimulation of CD8 + TILs cultured either alone or with IL-2 (20 Ul/ml) or restimulated with anti-CD3/CD28 Abs in the presence of 10 μM adrenaline or noradrenaline. Pool from two independent experiments ( n = 5). E, Percentage of IFN-γ + CD8 + TILs cells following in vitro restimulation with PMA/ionomycin or anti-CD3/CD28 Abs in the presence of 10 μM adrenaline or noradrenaline during 4 h, with representative flow cytometry histograms of intracellular IFN-γ staining in CD8 + T cells. Representative of two independent experiments [n = 5). Statistical analysis by one-way ANOVA test: ****, p <0.0001. Mean ± SEM.

Article Snippet: For intracellular IFN-γ staining, cells were stimulated in vitro for 4 h at 37°C with either 5 μg/ml E7-peptide (kindly provided by Dr Eric Tartour) or Dynabeads Mouse T-activator CD3/CD28 (1 bead for 2 CD8 + T cells; Thermofisher) or with 0.

Techniques: Flow Cytometry, Cell Culture, In Vitro, Staining

Journal: bioRxiv

Article Title: Blockade of β-adrenergic receptor signaling improves cancer vaccine efficacy through its effect on naive CD8 + T-cell priming

doi: 10.1101/497263

Figure Lengend Snippet: Activated CD8 + T cells were obtained by stimulation with anti-CD3/CD28 Abs during 3 days following by 4 days cultured with IL-2. A , Activated CD8 + T cells were loaded with Indo-1, pre-treated with either 10 μM adrenaline, noradrenaline or PGE 2 during 10 min. iCa 2+ mobilization was assessed by flow cytometry before and after stimulation with hamster anti-CD3ε Abs (first arrow, above) cross-linked with anti-hamster IgG (second arrow) and is indicated as % response in treated cells relative to untreated cells, with representative normalized Indo-1 ratio histograms. Pool from four independent experiments ( n = 4). B , Proliferation rate of activated CD8 + T cells cultured with or without IL-2 (20 UI/ml) in the presence of 10 μM adrenaline or noradrenaline during 24 h, represented as ratio of absolute number at day 0 / absolute number at day 1. Pool from two independent experiments ( n = 9). C and D , IL-2 (C) and IFN-γ (D) measured in the supernatants 24 h after stimulation of activated CD8 + T cells with anti-CD3/CD28 Abs in the presence of 10 μM adrenaline or noradrenaline. Pool from two independent experiments ( n = 4). (E) mRNA expression of β 2 -ARs in sorted-naive CD8 + T cells, CD8 + TILs and activated CD8 + T cells was evaluated by qPCR, ( n = 5 to 6 per group). Statistical analysis by one-way ANOVA test: ***, P <0.001, ****, p <0.0001. Mean ± SEM.

Article Snippet: For intracellular IFN-γ staining, cells were stimulated in vitro for 4 h at 37°C with either 5 μg/ml E7-peptide (kindly provided by Dr Eric Tartour) or Dynabeads Mouse T-activator CD3/CD28 (1 bead for 2 CD8 + T cells; Thermofisher) or with 0.

Techniques: Cell Culture, Flow Cytometry, Expressing

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Iron overload‐associated ZDHHC12 primarily participates in maintaining the activation of CD8+T cells in renal cell carcinoma. (a) UMAP plot showing the subpopulation classification of CD8+ T cells ( GSE121636 ). (b) UMAP visualization of ZDHHC12 expression across different CD8+ T cell subsets. (c) Violin plot depicting ZDHHC12 expression patterns in various CD8+ T cell subpopulations. (d) BALB/c mice fed either a high‐iron‐load diet or a normal diet were subcutaneously inoculated with Renca tumor cells on the dorsum, and starting from the day of inoculation, 200 µg of anti‐CD8 depletion antibody (αCD8) or an isotype control antibody was intraperitoneally administered every three days; tumor volume was measured from day 7 post‐inoculation. Data were presented as mean ± SD. e and f, Western blot (e) and RT‐qPCR (f) analyses were performed on CD8+ T cells sorted from renal cancer tissues of both the normal diet and high‐iron diet groups. (g–i), The figure illustrates schematic diagrams of different treatment groups (g). Specifically, tumor cells were isolated and sorted from the tumor tissues of the renal cancer model. Subsequently, the collected tumor cells were subcutaneously injected into the dorsum of either Zdhhc12 wild‑type or knockout mice. Tumor size was measured regularly, and growth curves were plotted (h). On day 19, the mice were sacrificed, and tumors were excised and weighed (i). (j–m), Portions of tumor tissues were processed for cell extraction, staining, and flow cytometry analysis to evaluate CD8+ T cell function in Zdhhc12 wild‐type and KO mice. Specifically, the proportions of CD3+/CD45+ (j), CD8+/CD3+ (k), GZMB+/CD8+ (l), and IFN‐γ+/CD8+ (m) T cells within tumor masses were quantified. (n,o) Portions of tumor tissues were embedded, sectioned, stained, and subjected to immunofluorescence analysis to assess CD8+ T cell function in Zdhhc12 wild‐type and KO mice. Specifically, multiplex staining was performed for DAPI/CD3/CD8/GZMB, and the proportions of positive cells were quantified (n); similarly, multiplex staining for DAPI/CD3/CD8/IFN‐γ was conducted with subsequent quantification of positive cell ratios (o).

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Activation Assay, Expressing, Control, Western Blot, Quantitative RT-PCR, Isolation, Injection, Knock-Out, Extraction, Staining, Flow Cytometry, Cell Function Assay, Immunofluorescence, Multiplex Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Iron overload promotes the ubiquitin‐proteasome pathway degradation of ZDHHC12 by enhancing its binding with TRIM28. (a) Cells were collected for Western blot analysis after treating sorted CD8+ T cells with FAC at the indicated concentrations for 48 h. (b) Cells were collected for Western blot analysis after treating sorted CD8+ T cells with FAC at the indicated concentration gradients for 48 h. (c) Cells were collected for Western blot analysis after treating 293T cells with FAC at the indicated concentrations for 48 h. (d) Cells were collected for Western blot analysis after treating 293T cells with FAC at the indicated concentration gradients for 48 h. (e) The degradation of ZDHHC12 in 293T cells was assessed by CHX chase assay after treatment with or without FAC (20 µg/mL) for 48 h. (f) HA‐ZDHHC12 was transfected into 293T cells, and co‐immunoprecipitation was performed using an anti‐HA‐ZDHHC12 antibody, followed by mass spectrometry to identify potential ZDHHC12‐interacting proteins. The peptide spectrum of TRIM28 is shown. (g) The degradation of ZDHHC12 in 293T cells was assessed by CHX chase assay with or without TRIM28 knockout. (h) Schematic diagrams depicting the protein domain architecture of TRIM28 and the TRIM28 △RING truncation mutant. (i) Sorted CD8+ T cells were stably transfected with the plasmids illustrated in the figure, followed by cell collection for Western blot analysis and quantitative assessment. (j) After transfection with the indicated plasmids, 293T cells were treated with FAC, followed by MG132 treatment, and then collected for Co‐IP and Western blot analysis. (k) After treatment with FAC (0, 20, 40 µg/mL), 293T cells were treated with MG132, collected, and subjected to Co‐IP and Western blot analysis. (l) After transfection with the indicated plasmids for 24 h, 293T cells were collected for Co‐IP and Western blot analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Ubiquitin Proteomics, Binding Assay, Western Blot, Concentration Assay, Transfection, Immunoprecipitation, Mass Spectrometry, Knock-Out, Mutagenesis, Stable Transfection, Co-Immunoprecipitation Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: ZDHHC12 inhibits cuproptosis in CD8+T cells through interaction with FDX1. (a) We reviewed the mass spectrometry results and found that FDX1 may interact with ZDHHC12, and the peptide spectrum of FDX1 is shown in the figure. (b) HA‐ZDHHC12 and Flag‐FDX1 were transfected into 293T cells, followed by cell lysis and co‐immunoprecipitation using Flag or HA antibodies, and then analyzed by western blot. (c) 293T cells were lysed and subjected to co‐immunoprecipitation with ZDHHC12 or FDX1 antibodies, followed by western blot detection. (d) Immunofluorescence staining was performed in 293T cells using ZDHHC12 and FDX1 antibodies, and images were acquired and analyzed by confocal microscopy. (e) GST pull‐down assay was performed using the recombinant protein of ZDHHC12. (f) GST pull‐down assay was performed using the recombinant protein of FDX1. (g) Protein structure prediction and molecular docking were conducted for ZDHHC12 and FDX1. (h,i) Western blot (h) and RT‐qPCR (i) were performed in 293T cells after transfection with the indicated plasmids. (j,k) Western blot (j) and RT‐qPCR (k) were performed in 293T cells after transfection with the indicated plasmids. (l) Western blot analysis was conducted in 293T cells 72 h after transfection with the indicated plasmids. (m) Western blot analysis was performed in 293T cells after transfection with the indicated plasmids. (n) CD8+ T cells were isolated from renal cancer tissues of Zdhhc12 WT and Zdhhc12 KO mice and subjected to western blot analysis. (o) After successful transfection with the indicated plasmids in 293T cells, cells were collected and treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu as shown in the figure for 48 h, followed by CCK‐8 assay to measure and quantify cell viability. (p,q) Following successful transfection with the indicated plasmids in 293T cells, cells were collected and immunostained with DLAT and Mito‐tracker, then imaged by confocal microscopy (p) to quantify (q) DLAT oligomerization. (r) After successful transfection with the indicated plasmids in 293T cells, cells were treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu for 48 h in the presence or absence of tetrathiomolybdate (TTM) (1 µ m ) as indicated, followed by CCK‐8 assay to measure and quantify cell viability. (s,t) Following successful transfection with the indicated plasmids in 293T cells, cells were collected after treatment with or without tetrathiomolybdate (TTM) (1 µ m ), then immunostained with DLAT and Mito‐tracker and imaged by confocal microscopy (s) to quantify (t) DLAT oligomerization. (u) After successful transfection with the indicated plasmids in CD8+ T cells, cells were treated with or without tetrathiomolybdate (TTM) (1 µ m ), then collected for western blot analysis. (v) Following successful transfection with the indicated plasmids in 293T cells, cells were collected and treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu as shown in the figure for 48 h, followed by CCK‐8 assay to measure and quantify cell viability. (w) After successful transfection with the indicated plasmids in sorted CD8+ T cells, cells were collected for western blot analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Mass Spectrometry, Transfection, Lysis, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Confocal Microscopy, Pull Down Assay, Recombinant, Quantitative RT-PCR, Isolation, CCK-8 Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: ZDHHC12‐mediated palmitoylation of FDX1 at Cys152/155 promotes its degradation. (a) In 293T and CD8+T cells, FDX1 was immunoprecipitated using an anti‐FDX1 antibody, followed by the acyl‐biotin exchange (ABE) assay performed either in the presence or absence of hydroxylamine (HAM) treatment, with subsequent streptavidin‐HRP pulldown of biotin‐conjugated proteins to specifically detect palmitoylated FDX1. (b) In 293T cells, FDX1 was immunoprecipitated using an anti‐ FDX1 antibody, followed by the acyl‐biotin exchange (ABE) assay performed with or without hydroxylamine (HAM) treatment, and subsequently enriched with streptavidin‐HRP to isolate biotin‐conjugated proteins, thereby enabling the detection of FDX1 palmitoylation levels in both 2‐BP (25 µ m , 24 h)‐treated and untreated conditions. (c) 293T cells treated with or without palmitic acid azide were collected for Click‐IT reaction and streptavidin pulldown. (d) 293T cells were treated with different concentrations of 2‐BP (+, 20 µ m ; ++, 40 µ m ) for 24 h, then collected for western blot analysis. (e) 293T cells treated with or without 2‐BP (25 µ m , 24 h) were exposed to CHX and collected at different time points for western blot analysis. (f) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (g) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (h) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (i) 293T cells transfected with the indicated plasmids were treated with or without 2‐BP (25 µ m , 24 h) after 24 h, then collected for western blot analysis. (j) The peptide spectrum for FDX1 palmitoylation site identification. (k) Conservation sequences of FDX1 C152 and FDX1 C155 sites across different species. (l) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to Click‐iT pull‐down assay and western blot analysis with or without palmitic acid azide treatment. m, AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (n) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to Click‐iT pull‐down assay and western blot analysis with or without palmitic acid azide treatment, followed by quantitative analysis of FDX1 palmitoylation levels. (o) CD8+ T cells were transfected with the indicated plasmids, then treated with PBS or FAC (20 µg/mL), collected, and stained with the indicated fluorescent dyes for flow cytometry analysis. (p) CD8+ T cells were transfected with the indicated plasmids, then collected and stained with the indicated fluorescent dyes for flow cytometry analysis. (q) CD8+ T cells were transfected with the indicated plasmids, then treated with PBS or FAC (20 µg/mL), collected, and stained with the indicated fluorescent dyes for flow cytometry analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Immunoprecipitation, Western Blot, Knock-Out, Transfection, Pull Down Assay, Staining, Flow Cytometry

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Development of lipid nanoparticles encapsulating Zdhhc12 mRNA (Zdhhc12‐LNP). (a) Schematic diagram of the LNP synthesis process; (b) The average zeta potential and the average particle size; (c) The average PDI and the average encapsulation efficiency; (d) Representative cryo‐EM image showing the morphology of synthesized LNPs. (e) Spectrum showing the purity of the sample. (f) Representative chromatogram showing mRNA capping efficiency. (g) Western blot analysis of Zdhhc12 expression in CD8+ T cells isolated from mice and treated with control, blank LNP, or Zdhhc12‐encapsulated LNP, following a 24 h pre‐stimulation with 2 µg/mL anti‐CD3/CD28 and subsequent 24 h incubation with LNPs. (h) ABE assay and Western blot analysis were performed on CD8+ T cells isolated from mice to assess the palmitoylation level of Fdx1, where cells were first cultured for 24 h post‐infection without anti‐CD3/CD28, restimulated with 2 µg/mL anti‐CD3/CD28 for 24 h, incubated with LNPs for 24 h, and then harvested for analysis. (i) Renca cells were subcutaneously injected into the backs of BALB/c mice, LNPs were administered via intratumoral injection, and when tumors reached an appropriate size, tumors were harvested for sorting of CD8+ T cells, followed by Western blot analysis to assess Zdhhc12 expression level in CD8+ T cells. (j–m) Renca cells were subcutaneously injected into the dorsal region of BALB/c mice. After tumor establishment, mice received intratumoral injection of LNPs or no injection, and serum levels of ALT (j), AST (k), CRE (l), and BUN (m) were measured at appropriate time points. Ns, not significant. (n,o) BALB/c mice were subcutaneously injected with Renca cells, intratumorally administered Zdhhc12‐LNP or control, and intraperitoneally injected with 200 µg anti‐CD8 (αCD8) or control; tumor growth was recorded starting from day 7 post‐tumor inoculation (n), and flow cytometry analysis of CD8+ T cell content in the peripheral blood of mice (n = 5) was performed at the end of the experiment (o), with data presented as mean ± SD.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Zeta Potential Analyzer, Encapsulation, Cryo-EM Sample Prep, Synthesized, Western Blot, Expressing, Isolation, Control, Incubation, Cell Culture, Infection, Injection, Flow Cytometry

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Under normal iron conditions, ZDHHC12‐mediated palmitoylation of FDX1 promotes FDX1 degradation by the mitochondrial matrix protease AFG3L2, ultimately suppressing cuproptosis in CD8+ T cells and maintaining their immune clearance function. Under iron overload conditions, increased binding between ZDHHC12 and TRIM28 leads to ZDHHC12 degradation, consequently inducing cuproptosis in CD8+ T cells and facilitating immune escape.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Binding Assay