human cd8 t cells  (Miltenyi Biotec)

Journal: Oncoimmunology

Article Title: Ex vivo-generated conventional dendritic cells type 1 and type 2 from blood progenitors induce potent antigen-specific T-cell immunity

doi: 10.1080/2162402X.2026.2695692

Figure Lengend Snippet: Ex vivo-enerated cDC1s and cDC2s mount potent T-cell responses. (A) Representative dot plots showing the frequency of proliferating allogeneic pan T-cells (indicated by the low CFSE expression) upon coculture with untreated or TLR-matured cDC1s for 6 d. (B) Scatter dot showing the mean ± SEM of the percentage of proliferating T-cells upon coculture with cDC2s (blue) and cDC1s (red). Each data point represents an individual DC donor ( n ≥ 4). (C) Scatter dot showing the mean ± SEM of the raw concentration of IFNγ detected by standard sandwich ELISA on day 6 after pan T-cell coculture with cDC1s and cDC2s. Each data point represents an individual DC donor ( n = 5). (D) Scatter dot plot displays the ratio of proliferating CD8/CD4 T-cells upon coculture with either cDC1s or cDC2s for 6 d (mean ± SEM). Each dot represents an individual DC donor ( n ≥ 4). (E) Representative dot plots showing the intracellular cytokine levels of IFNγ and TNF- α detected on expanded naive CD4 T-cells upon coculture with untreated or TLR-matured cDC2s. (F) Scatter dot plot displays the mean ± SEM of the frequency of CD4 T-cells positive for IFNγ and TNFα expression after coculture with cDC1s and cDC2s. (G) The scatter dot plot shows the mean ± SEM of the frequency of CD4 T-cells positive for IFNγ expression alone. (H) Scatter dot plot displays the frequency of Tregs (identified as CD4 + CD127 - CD25 + FOXP3 + cells) after coculture with cDC1s and cDC2s (mean ± SEM). Each dot represents an individual DC donor ( n = 5). (I) Representative dot plots showing the intracellular levels of perforin (PRF) and granzyme-B (GRZB) detected on expanded naive CD8 T-cells upon coculture with untreated or TLR-matured cDC1s. (J) Scatter dot plot displays the mean ± SEM of the frequency of CD8 T-cells positive for PRF and GRZB expression after coculture with cDC1s and cDC2s. (K) The scatter dot plot shows the mean ± SEM of the frequency of CD8 T-cells positive for IFNγ expression alone. Each dot represents an individual DC donor ( n = 5). Statistical significance was calculated for paired data sets with a paired t-test or a Wilcoxon test, whereas for unpaired data sets significancy was calculated using a Mann–Whitney test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Pan naive T-cells were isolated using the MACS Naive Pan T-cell isolation kit (130-097-095, Miltenyi Biotec) and the MACS Naive CD8 T-cell isolation kit (130-093-244, Miltenyi Biotec).

Techniques: Ex Vivo, Expressing, Concentration Assay, Sandwich ELISA, MANN-WHITNEY

Journal: Oncoimmunology

Article Title: Ex vivo-generated conventional dendritic cells type 1 and type 2 from blood progenitors induce potent antigen-specific T-cell immunity

doi: 10.1080/2162402X.2026.2695692

Figure Lengend Snippet: CD34-derived cDC1s and cDC2s induce tumor-derived antigen-specific CD8 T-cell clones. To address the capacity of these DCs to prime the induction of a T-cell clone specific against the tumor antigen MART1, matured CD34-derived pan-DCs (combined cDC1s and cDC2s) loaded with the MART1-derived peptide were cultured with autologous naive CD8 T-cells. (A) Schematic representation of the experimental layout. (B) Representative dot plot showing the frequency of MART1-positive CD8 T-cells after the induction protocol with CD34-derived pan-DCs, as indicated by the abundance of dextramer-PE positive CD8 T-cell. (C) Before–after dot plots showing the frequency of MART1-specific dextramer-stained CD8 T-cells for each donor compared to a negative control dextramer. Each dot represents an individual donor ( n = 4). (D) Schematic representation of the rechallenge experimental layout. To address the responsiveness of induced MART1-specific T-cells to a secondary antigen rechallenge, MART1-loaded autologous moDCs were cocultured overnight with the earlier primed CD8 T-cell pool containing the MART1-specific CD8 T-cells. (E) Representative dot plots showing the expression levels of CD137 and CD25 on CD8 T-cells cocultured with either unloaded or MART1-loaded moDCs. (F) Before–after dot plots showing the frequency of positive CD8 T-cells for the simultaneous expression of CD25 and CD137. (G) Before–after dot plots showing the frequency of positive CD8 T-cells for the activation marker CD25, CD137, CD69, CD107a, and the intracellular cytokines IFNγ, IL-2, and TNFα after coculture with either unloaded or MART1-loaded moDCs. Each dot represents an individual donor ( n = 4). Statistical significance was calculated using either a paired t-test or a Wilcoxon test. P- values are numerically depicted in the graph.

Article Snippet: Pan naive T-cells were isolated using the MACS Naive Pan T-cell isolation kit (130-097-095, Miltenyi Biotec) and the MACS Naive CD8 T-cell isolation kit (130-093-244, Miltenyi Biotec).

Techniques: Derivative Assay, Clone Assay, Cell Culture, Staining, Negative Control, Expressing, Activation Assay, Marker

Journal: Advanced Science

Article Title: Targeting the ATX‐LPA Axis Overcomes TKI Resistance and Immunosuppression in Renal Cell Carcinoma via Dual Inhibition of AKT/mTOR and TBK1/IRF3 Pathways

doi: 10.1002/advs.76352

Figure Lengend Snippet: ENPP2 drives immune evasion via the TBK1/IRF3/PD‐L1 axis and suppresses CD8 + T‐cell effector functions in renal cancer cells. (A) GSEA plots showing negative enrichment of interferon‐α and interferon‐γ response signatures following ENPP2 knockdown. (B) Representative flow cytometry histograms and quantification of median fluorescence intensity (MFI) for surface PD‐L1 expression in 786‐O and ACHN cells transduced with control shRNA (shNC) or shENPP2. (C) Immunoblot analysis of ENPP2, PD‐L1, total TBK1, phospho‐TBK1 (Ser172), total IRF3, and phospho‐IRF3 (Ser396) in 786‐O and ACHN cells following ENPP2 knockdown. (D) Immunoblot analysis of the TBK1/IRF3/PD‐L1 axis in ENPP2‐overexpressing 786‐O and ACHN cells treated with 10 µM GLPG1690, 10 µM GLPG1690 plus 20 µM LPA, or DMSO control for 48 h. (E) Immunoblot analysis of the specified cascade proteins in ENPP2‐knockout (sgENPP2) cells reconstituted with either wild‐type ENPP2 (oeENPP2) or the catalytically inactive T210A mutant. (F) Progenitor and terminal exhaustion scores of CD8+ T cells stratified by LPAR5 expression level. (G) UMAP visualization of T‐cell subclusters identified by single‐cell RNA‐seq (Tex, exhausted T cells; Teff, effector T cells; Tstr, stress‐response T cells; Trm, tissue‐resident memory T cells; Tcm, central memory T cells; Tpex, progenitor‐exhausted T cells; CXCL13+ T, CD8+CXCL13+ T cells). (H) Proportions of the indicated CD8+ T‐cell subpopulations in tumors with low versus high ENPP2 expression. (I) Feature plots showing co‐localization of LPAR5 and PDCD1 (PD‐1) expression in tumor‐infiltrating T cells. (J, K) LDH‐release cytotoxicity assays of primary human T cells co‐cultured with control (NC) or ENPP2‐knockout (sgENPP2) 786‐O cells across varying effector‐to‐target (E:T) ratios (J), and the corresponding rescue effect of exogenous LPA supplementation (K). (L, M) LDH‐release cytotoxicity assay (L) and representative crystal violet staining (M) of oeENPP2 RCC cells co‐cultured with T cells, demonstrating the restorative effects of pharmacological anti‐PD‐1 antibody treatment or genetic PD‐L1 knockdown (siPD‐L1) on T‐cell‐mediated killing. (N, O) Representative flow cytometry plots and quantification of Granzyme B (GZMB) and TNF‐α (N), as well as Perforin and IFN‐γ (O) in CD8 + T cells after co‐culture with control (NC) or ENPP2‐knockout (SG) 786‐O cells. (P, Q) In vivo antibody‐mediated CD4 + and CD8 + T‐cell depletion in an immunocompetent syngeneic RCC model. Tumor growth kinetics (P) and final endpoint tumor weights (Q) of the indicated depletion cohorts (αCD4, αCD8, or αCD4+αCD8) compared to the IgG control. Quantitative in vitro data are presented as mean ± SD from n = 3 independent experiments. In vivo data are presented as mean ± SD with n = 5 mice per group. Statistical significance was determined using an unpaired two‐tailed Student's t ‐test (B, F, N, O), one‐way ANOVA (L, Q), or two‐way ANOVA (J, K, P), followed by Tukey's post hoc test. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary human CD8+ T cells were purified using a CD8+ T‐cell Isolation Kit (Miltenyi Biotec, 130‐096‐495) following the manufacturer's instructions.

Techniques: Knockdown, Flow Cytometry, Fluorescence, Expressing, Transduction, Control, shRNA, Western Blot, Knock-Out, Mutagenesis, Single Cell, RNA Sequencing, Cell Culture, Cytotoxicity Assay, Staining, Co-Culture Assay, In Vivo, In Vitro, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting the ATX‐LPA Axis Overcomes TKI Resistance and Immunosuppression in Renal Cell Carcinoma via Dual Inhibition of AKT/mTOR and TBK1/IRF3 Pathways

doi: 10.1002/advs.76352

Figure Lengend Snippet: In vivo experiments demonstrate that targeting ENPP2 improves T cell function and enhances the efficacy of targeted‐immune combination therapy. (A–C) PDX subcutaneous tumor weights at the experimental endpoint (A), tumor volume changes recorded during the experiment (B), and representative tumor images (C) following treatment as outlined in the schematic. Immunotherapy refers to reinfusion of in vitro pre‐activated T cells and administration of αPD‐1 antibody. Combination therapy mimics clinical first‐line therapy, combining TKI with immune checkpoint inhibitor (ICI). (D, E) Representative flow cytometry histograms (D) and quantification of median fluorescence intensity (MFI) (E) for surface PD‐L1 expression on tumor cells dissociated from the excised xenografts. (F) Serum concentrations of TNF‐α and IFN‐γ in the indicated immune‐reconstituted murine cohorts, measured by ELISA. (G, H) Flow cytometric analysis and MFI quantification of the cytotoxic effector molecules Granzyme B (GZMB) (G) and Perforin (H) in tumor‐infiltrating CD8 + T cells. (I, J) Representative contour plots and quantitative analysis of the exhaustion markers PD‐1 (I) and LAG‐3 (J) on tumor‐infiltrating T cells. (K) Representative immunohistochemistry (IHC) images and quantification of the proliferation marker Ki‐67 in tumor sections from the specified treatment groups. Scale bars: 50 µm. All in vivo data are presented as mean ± SD with n = 5 independent biological replicates per group. Statistical significance was determined using one‐way ANOVA (A, E, F, G, H, I, J, K), or two‐way ANOVA (B), followed by Tukey's post hoc test. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary human CD8+ T cells were purified using a CD8+ T‐cell Isolation Kit (Miltenyi Biotec, 130‐096‐495) following the manufacturer's instructions.

Techniques: In Vivo, Cell Function Assay, In Vitro, Flow Cytometry, Fluorescence, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Marker