human primary cd8 t cells  (Miltenyi Biotec)

Journal: Cancer Medicine

Article Title: SLC12A8 Drives Immune Evasion and Metastasis in Luminal B Breast Cancer by Inducing CD8 + T‐Cell Exhaustion via the TLR Signaling Pathway

doi: 10.1002/cam4.71712

Figure Lengend Snippet: SLC12A8 expression is negatively correlated with CD8 + T cell infiltration in the breast cancer immune microenvironment. (A) Circos plot from TIMER2 database analysis showing the correlation between SLC12A8 and CD8 + T cell infiltration. (B) ImmuCellAI database analysis shows a negative correlation between SLC12A8 expression and CD8 + T cell infiltration score (*** p < 0.001, Pearson correlation; n = 1087 samples).

Article Snippet: Human Luminal A breast cancer cell line MCF‐7, Luminal B breast cancer cell lines BT474 and MDA‐MB‐361, HER2‐overexpressing breast cancer cell line SKBR3, basal‐like breast cancer cell line BT549, normal breast epithelial cell line MCF‐10A, and human primary CD8 + T cells isolated from healthy donor peripheral blood using CD8 MicroBeads (Miltenyi Biotec, 130–045‐201) were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

Techniques: Expressing

Journal: Cancer Medicine

Article Title: SLC12A8 Drives Immune Evasion and Metastasis in Luminal B Breast Cancer by Inducing CD8 + T‐Cell Exhaustion via the TLR Signaling Pathway

doi: 10.1002/cam4.71712

Figure Lengend Snippet: Correlation between SLC12A8 expression and CD8 + T cell infiltration in different breast cancer subtypes. Heatmap shows the strongest negative correlation between SLC12A8 and CD8 + T cells in Luminal B breast cancer. Pearson correlation coefficients are shown; n for each subtype: Luminal A = 568, Luminal B = 219, HER2 = 82, Basal = 191.

Article Snippet: Human Luminal A breast cancer cell line MCF‐7, Luminal B breast cancer cell lines BT474 and MDA‐MB‐361, HER2‐overexpressing breast cancer cell line SKBR3, basal‐like breast cancer cell line BT549, normal breast epithelial cell line MCF‐10A, and human primary CD8 + T cells isolated from healthy donor peripheral blood using CD8 MicroBeads (Miltenyi Biotec, 130–045‐201) were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

Techniques: Expressing

Journal: Cancer Medicine

Article Title: SLC12A8 Drives Immune Evasion and Metastasis in Luminal B Breast Cancer by Inducing CD8 + T‐Cell Exhaustion via the TLR Signaling Pathway

doi: 10.1002/cam4.71712

Figure Lengend Snippet: Infiltration of CD8 + T cells in different types of breast cancer tissues. Immunofluorescence staining shows relatively less CD8 + T cell (green) infiltration in Luminal B breast cancer. DAPI (blue) marks nuclei. Scale bar: 50 μm. **( n = 10 samples per group; p < 0.01, one‐way ANOVA with Tukey's post hoc test).

Article Snippet: Human Luminal A breast cancer cell line MCF‐7, Luminal B breast cancer cell lines BT474 and MDA‐MB‐361, HER2‐overexpressing breast cancer cell line SKBR3, basal‐like breast cancer cell line BT549, normal breast epithelial cell line MCF‐10A, and human primary CD8 + T cells isolated from healthy donor peripheral blood using CD8 MicroBeads (Miltenyi Biotec, 130–045‐201) were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

Techniques: Immunofluorescence, Staining

Journal: Cancer Medicine

Article Title: SLC12A8 Drives Immune Evasion and Metastasis in Luminal B Breast Cancer by Inducing CD8 + T‐Cell Exhaustion via the TLR Signaling Pathway

doi: 10.1002/cam4.71712

Figure Lengend Snippet: Effect of SLC12A8 expression level on CD8 + T cell function. qPCR detection shows significantly increased mRNA expression levels of the effector molecules PRF1 and GZMB in CD8 + T cells in the SLC12A8 knockdown co‐culture group. ( n = 3 independent experiments; ns: Not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; one‐way ANOVA with Tukey's post hoc test). Data are mean ± SD.

Article Snippet: Human Luminal A breast cancer cell line MCF‐7, Luminal B breast cancer cell lines BT474 and MDA‐MB‐361, HER2‐overexpressing breast cancer cell line SKBR3, basal‐like breast cancer cell line BT549, normal breast epithelial cell line MCF‐10A, and human primary CD8 + T cells isolated from healthy donor peripheral blood using CD8 MicroBeads (Miltenyi Biotec, 130–045‐201) were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

Techniques: Expressing, Cell Function Assay, Knockdown, Co-Culture Assay

Journal: Cancer Medicine

Article Title: SLC12A8 Drives Immune Evasion and Metastasis in Luminal B Breast Cancer by Inducing CD8 + T‐Cell Exhaustion via the TLR Signaling Pathway

doi: 10.1002/cam4.71712

Figure Lengend Snippet: SLC12A8 functional expression is associated with the TLR signaling pathway. (A) GSEA analysis shows significant enrichment of the TLR signaling pathway in samples with high SLC12A8 expression. (B) Western Blot and quantitative analysis verify the expression changes of key proteins in the TLR signaling pathway after different interventions (si‐SLC12A8, GIT27) in isolated CD8 + T cells. ( n = 3 independent experiments; ns: Not significant; ** p < 0.01; *** p < 0.001; one‐way ANOVA with Tukey's post hoc test). Data are mean ± SD.

Article Snippet: Human Luminal A breast cancer cell line MCF‐7, Luminal B breast cancer cell lines BT474 and MDA‐MB‐361, HER2‐overexpressing breast cancer cell line SKBR3, basal‐like breast cancer cell line BT549, normal breast epithelial cell line MCF‐10A, and human primary CD8 + T cells isolated from healthy donor peripheral blood using CD8 MicroBeads (Miltenyi Biotec, 130–045‐201) were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

Techniques: Functional Assay, Expressing, Western Blot, Isolation

Journal: Cancer Medicine

Article Title: SLC12A8 Drives Immune Evasion and Metastasis in Luminal B Breast Cancer by Inducing CD8 + T‐Cell Exhaustion via the TLR Signaling Pathway

doi: 10.1002/cam4.71712

Figure Lengend Snippet: Inhibiting the SLC12A8/TLR axis reverses T cell exhaustion and suppresses tumor invasion. (A) Immunofluorescence detection shows that knockdown of SLC12A8 or addition of GIT27 significantly reduces PD‐1 (red) expression on CD8 + T cells. DAPI (blue) marks nuclei. Scale bar: 100 μm. ( n = 3 independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; one‐way ANOVA with Tukey's post hoc test). (B) Transwell assay shows that knockdown of SLC12A8 or addition of GIT27 significantly inhibits the invasive ability of BT474 cells. Scale bar: 50 μm. ( n = 3 independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; one‐way ANOVA with Tukey's post hoc test). Data are mean ± SD.

Article Snippet: Human Luminal A breast cancer cell line MCF‐7, Luminal B breast cancer cell lines BT474 and MDA‐MB‐361, HER2‐overexpressing breast cancer cell line SKBR3, basal‐like breast cancer cell line BT549, normal breast epithelial cell line MCF‐10A, and human primary CD8 + T cells isolated from healthy donor peripheral blood using CD8 MicroBeads (Miltenyi Biotec, 130–045‐201) were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

Techniques: Immunofluorescence, Knockdown, Expressing, Transwell Assay

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: Bioinformatics Analysis Identifies Key Factors in Histone Lactylation Modification. Note: ( A ) Schematic workflow of bioinformatics analysis for identifying key factors; ( B ) Volcano plot of DEGs in tumor tissues and adjacent normal tissues from dataset GSE2685 (Normal = 8, Tumor = 22); ( C ) Heatmap showing the correlation of co-expression module genes with tumor and normal tissues, with each cell displaying the correlation coefficient and p -value; ( D ) Venn diagram illustrating the intersection of Blue module genes, DEGs, CD8 + T cell-related genes, and "Histone lactylation"-related genes; ( E ) Box plot of p300 differential expression; ( F ) The tSNE distribution map of EP300 in various cell types in the scRNA-seq data

Article Snippet: Human CD8+ T cells were isolated from peripheral blood mononuclear cells (hPBMCs, PCS-800–011, ATCC, USA) using the CD8 MicroBead Kit (130–045–201, Miltenyi Biotec, USA).

Techniques: Modification, Expressing, Quantitative Proteomics

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: p300 Regulates Histone Lactylation in CD8 + T Cells. Note: ( A ) Schematic representation of the experimental design, showing the workflow for detecting CD8 + T cells treated with lactate, p300 inhibitors, or activators. ( B–C ) WB analysis of PKla levels in CD8 + T cells over time (B) and under varying lactate concentrations ( C ). ( D–E ) WB analysis of the time-dependent ( D ) and dose-dependent ( E ) changes in H3K18la and H3K9la expression in CD8 + T cells following lactate treatment. *** p < 0.001, ** p < 0.01, and * p < 0.05 compared to the 0-h or untreated lactate group. ( F ) WB analysis of H3K18la and H3K9la expression in CD8 + T cells following p300 knockdown, activation, or inhibition. ( G ) ELISA detection of IFN-γ levels in the supernatant of CD8 + T cells across different treatment groups. (H) FCM analysis of GZMB expression in CD8 + T cells. ( I ) FCM analysis of CD8 + T cell proliferation. ( J ) LDH release assay showing the cytotoxic effects of CD8 + T cells on MKN-45 and SNU1 cells. In panels ( F–J ), * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared to the Lactate group; & p < 0.01 compared to the Lactate + sh-NC group. All cell-based experiments were performed in triplicate

Article Snippet: Human CD8+ T cells were isolated from peripheral blood mononuclear cells (hPBMCs, PCS-800–011, ATCC, USA) using the CD8 MicroBead Kit (130–045–201, Miltenyi Biotec, USA).

Techniques: Expressing, Knockdown, Activation Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Control

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: In Vivo and In Vitro Biocompatibility and Targeting Validation of NVEs. Note: ( A ) Schematic illustration of in vitro targeting and biocompatibility experiments of NVEs; ( B ) Hemolysis analysis of NVEs, NVEs@C646, and CD8a-NVEs@C646 after incubation with red blood cells (PC: positive control); ( C ) CD8 + T cell viability assessed using the CCK-8 assay after 48-h incubation with varying concentrations of NVEs; ( D ) Confocal microscopy and quantitative analysis of NVEs@C646 and CD8a-NVEs@C646 uptake in CD8 + T cells (Scale bars = 25 μm); (E) FCM analysis of the uptake efficiency of NVEs@C646 and CD8a-NVEs@C646 in CD8 + T cells; (F) Schematic illustration of the subcutaneous tumor model in mice and in vivo injection of NVEs; ( G ) FCM analysis of the percentage of CM-Dil-labeled CD8 + T cells in blood, spleen, tumor tissue, and TdLNs at different time points post-injection (* p < 0.05 compared with the NVEs@C646 group); ( H ) FCM analysis of CD3 + CD8 − T cells binding with CM-Dil-labeled NVEs recovered from blood, spleen, tumor tissue, and TdLNs at different time points; ( I ) Quantification of CD8 + T cell numbers 48 h after NVEs injection. Cell-based experiments were performed in triplicate. Animal experiments included nine mice per group, with three mice per time point

Article Snippet: Human CD8+ T cells were isolated from peripheral blood mononuclear cells (hPBMCs, PCS-800–011, ATCC, USA) using the CD8 MicroBead Kit (130–045–201, Miltenyi Biotec, USA).

Techniques: In Vivo, In Vitro, Biomarker Discovery, Incubation, Positive Control, CCK-8 Assay, Confocal Microscopy, Injection, Labeling, Binding Assay

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: CD8a-NVEs@C646 Facilitates Histone Delactylation Modification in CD8 + T Cells. Note: ( A ) Schematic workflow of RNA-seq and ChIP-seq experiments for CD8 + T cells treated with CD8a-NVEs@C646; ( B–C ) ChIP-seq analysis showing signals at TSS regions in PBS-treated (n = 3) and CD8a-NVEs@C646-treated groups (n = 3); ( D ) RNA-seq volcano plot illustrating significantly upregulated and downregulated genes in the CD8a-NVEs@C646-treated group compared to PBS (n = 3); ( E ) Venn diagram of overlapping genes from ChIP-seq and RNA-seq analyses related to CD8 + T cells; ( F ) ChIP analysis of p300 and H3K18la enrichment at the PDCD1 promoter region; ( G ) The protein interaction between p300 and H3K18la was detected by co-IP assay; ( H ) RT-qPCR analysis of PDCD1 mRNA expression in CD8 + T cells following CD8a-NVEs@C646 treatment; ( I ) WB analysis of PDCD1 and H3K18la protein expression levels in CD8 + T cells treated with CD8a-NVEs@C646. * p < 0.05, *** p < 0.001 compared to the PBS or sh-NC group; experiments were conducted in triplicate

Article Snippet: Human CD8+ T cells were isolated from peripheral blood mononuclear cells (hPBMCs, PCS-800–011, ATCC, USA) using the CD8 MicroBead Kit (130–045–201, Miltenyi Biotec, USA).

Techniques: Modification, RNA Sequencing, ChIP-sequencing, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Expressing

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: CD8a-NVEs@C646-Mediated Regulation of PDCD1 Enhances the Tumor-Killing Ability of CD8 + T Cells. Note: ( A ) Schematic diagram of the experimental design: CD8 + T cells were transduced with PDCD1 overexpression lentivirus and treated with CD8a-NVEs@C646, followed by co-culture with MKN-45/SNU1 cells; ( B ) RT-qPCR analysis of PDCD1 mRNA expression in CD8 + T cells across different groups; ( C ) WB analysis of PDCD1 and H3K18la protein expression in CD8 + T cells across different groups; ( D ) ELISA detection of IFN-γ levels in the supernatant of CD8 + T cells across different groups; ( E ) FCM analysis of GZMB expression in CD8 + T cells across different groups; ( F ) FCM analysis of CD8 + T cell proliferation activity; ( G ) LDH release assay to evaluate the cytotoxicity of CD8 + T cells on MKN-45 cells; ( H ) FCM analysis of apoptosis in MKN-45 cells across different groups. * indicates p < 0.05 compared to the PBS + vector group, *** indicates p < 0.001, # indicates p < 0.05 compared to the CD8a-NVEs@C646 + vector group, ### indicates p < 0.001. All cellular experiments were repeated three times

Article Snippet: Human CD8+ T cells were isolated from peripheral blood mononuclear cells (hPBMCs, PCS-800–011, ATCC, USA) using the CD8 MicroBead Kit (130–045–201, Miltenyi Biotec, USA).

Techniques: Transduction, Over Expression, Co-Culture Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, Lactate Dehydrogenase Assay, Plasmid Preparation

Journal: Journal of Nanobiotechnology

Article Title: CD8a antibody-functionalized biomimetic red blood cell membrane ectosomes delivering C646 reverse CD8⁺ T Cell exhaustion via H3K18la histone delactylation in gastric cardia adenocarcinoma

doi: 10.1186/s12951-025-03957-z

Figure Lengend Snippet: CD8a-NVEs@C646 Enhances CD8 + T Cell-Mediated Anti-Tumor Immunity. Note: ( A ) Schematic diagram of the experimental design, illustrating the establishment of the GCA orthotopic transplantation tumor model and treatment regimen; ( B ) In vivo fluorescence imaging of tumors in mice from different treatment groups and quantitative analysis of signal intensity over time; ( C ) Photographic images of tumor tissues from different treatment groups; ( D ) Tumor weight statistics for each group; ( E–F ) Immunohistochemistry and TUNEL staining showing cell proliferation and apoptosis in tumor tissues from each group (Scale bars = 50 μm); ( G ) Quantification of the proportion of infiltrating CD8 + T cells in tumor tissues from each group; ( H-J ) Expression levels of IFN-γ, TNFα, and GZMB in CD8 + T cells within tumor tissues from each group; ( K ) Proportion of PD-1 + CD8 + T cells in tumor tissues from each group. Six mice per group; * indicates p < 0.05 compared to the NS group, and # indicates p < 0.05 compared to the αPD-1 group

Article Snippet: Human CD8+ T cells were isolated from peripheral blood mononuclear cells (hPBMCs, PCS-800–011, ATCC, USA) using the CD8 MicroBead Kit (130–045–201, Miltenyi Biotec, USA).

Techniques: Transplantation Assay, In Vivo, Fluorescence, Imaging, Immunohistochemistry, TUNEL Assay, Staining, Expressing