cd8a microbeads  (Miltenyi Biotec)

Journal: iScience

Article Title: Single-cell gene expression and TCR profiling reveal age-related differences in recent thymic emigrants

doi: 10.1016/j.isci.2026.115582

Figure Lengend Snippet: Neonatal and adult RTEs are phenotypically distinct (A) Schematic of the timestamping system marking T cells made at the time of tamoxifen administration. RTEs were collected at 2 weeks post-marking. (B) Left: Representative contour plots displaying virtual memory (VM, CD44 hi CD122 hi ) and true naive (TN, CD44 lo CD122 lo ) CD8 + populations. Right: Statistical analysis of VM population within the marked T cell population. (C) Statistical analysis and representative histograms of the RTE markers Qa2 (left) and CD103 (right). (D) Statistical analysis and representative histograms of the phenotypic markers CD127 (left) and CD11a (right). n = 8-9 mice per group from 2 independent experiments. For statistical analysis, unpaired t tests were performed. ns, not significant; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Article Snippet: CD8a Microbeads, mouse , Miltenyi Biotec , Cat# 130-117-044.

Techniques:

Journal: iScience

Article Title: Single-cell gene expression and TCR profiling reveal age-related differences in recent thymic emigrants

doi: 10.1016/j.isci.2026.115582

Figure Lengend Snippet: Neonatal RTEs exhibit enhanced immune functionality (A) Schematic of in vitro stimulation. CD8 + T cells from neonatal and adult timestamped (ts) mice were coated with CTV and stimulated for 48 h via antibody-mediated crosslinking of CD3/CD28. (B) Representative histograms of CTV dilution. (C) Division index. Unpaired t test was performed for statistical analysis. n = 8 samples per group from 2 independent experiments. (D) Schematic of the in vivo infection experiment. Thymic lobes from newborn GFP timestamped mice were grafted under the kidney capsule of adult RFP timestamped mice. Tamoxifen was administered to thymic graft recipients, and after 2 weeks, recipients were infected with 5 × 10 3 CFUs of LM-gB. Spleens were collected at 5 dpi. (E) Percentage of timestamped tetramer-positive cells with a short-lived effector phenotype (KLRG1 hi CD127 lo ). (F) Percentage of timestamped tetramer-positive cells with a memory precursor effector phenotype (KLRG1 lo CD127 hi ). (G) Percentage of timestamped cells producing IFN-γ. N = 19. For statistical analysis, paired t tests were performed. ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Article Snippet: CD8a Microbeads, mouse , Miltenyi Biotec , Cat# 130-117-044.

Techniques: In Vitro, In Vivo, Infection

Journal: Signal Transduction and Targeted Therapy

Article Title: Low-dose radiotherapy synergizes with PD-1 blockade to achieve durable survival in advanced NSCLC through antitumor neutrophil programming

doi: 10.1038/s41392-026-02712-6

Figure Lengend Snippet: TRIDENT expands TNF-α⁺ neutrophils and effector CD8⁺ T cells in second tumors. a UMAP visualization of intratumoral neutrophils from scRNA-seq. b Bubble plot showing representative marker gene expression across neutrophil subsets. Dot size indicates the fraction of expressing cells and color intensity the mean expression. c UMAP plot highlighting neutrophils scored by response-associated (left) and non-response-associated (right) gene signatures. d Box-and-violin plots showing the proportion of N1 neutrophils in second tumors across treatment groups. Boxes denote the interquartile range (IQR), with the horizontal line indicating the median; whiskers extend to the minimum and maximum values within 1.5 × IQR. Violins depict kernel-density distribution with individual points overlaid. Flow-cytometric quantification of TNF-α⁺ ( e ), TNF-α⁺CD14⁺ ( f ), CD14⁺CD101 − ( g ), and CD14⁺CD62L⁺ ( h ) neutrophils per milligram tumor in second tumors in the LLC model ( n = 7 per group). Flow cytometric analysis of CD8⁺ T cell infiltration in second tumors in the LLC model: i representative plots gated from CD45⁺CD3⁺ T cells; j absolute CD8⁺ T-cell counts per milligram tumor ( n = 7 per group) k UMAP plot of tumor-infiltrating T/NK cells from scRNA-seq. l Proportion of seven T/NK cell subsets across treatment groups. m Violin plots of cytotoxicity scores across different T/NK cell subsets derived from gene expression profiles. Embedded box plots indicate IQR (box), median value (horizontal line within the box), and whiskers extending to 1.5× IQR; outliers are shown as individual points. n Heatmap showing enrichment of functionally relevant pathways in CD8_Effector T cells under different treatments. Flow-cytometric counts of perforin⁺ ( o ), IFN-γ⁺ ( p ), TNF-α⁺ ( q ), and granzyme B⁺ ( r ) CD8 + T cells per milligram tumor in second tumors in the LLC model ( n = 7 per group). s – u Tumor growth curves and survival of LLC-bearing mice treated with TRIDENT or TRIDENT plus CD8⁺ T-cell-depleting antibodies ( n = 7 per group). Data are mean ± SEM. Statistical significance was assessed by one-way ANOVA ( e – h , j and o – r ), two-way ANOVA ( s , t ) or log-rank test for survival ( u ). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Article Snippet: CD8 + T cells were isolated from C57BL/6 spleens using CD8a (Ly-2) MicroBeads (Miltenyi Biotec, Cat# 130-117-044) and LS columns (Miltenyi Biotec, Cat# 130-042-401).

Techniques: Marker, Gene Expression, Expressing, Derivative Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Low-dose radiotherapy synergizes with PD-1 blockade to achieve durable survival in advanced NSCLC through antitumor neutrophil programming

doi: 10.1038/s41392-026-02712-6

Figure Lengend Snippet: TNF-α⁺ neutrophils exhibit antitumor properties and activate CD8⁺ T cells. a Spearman correlation between TNF-α + neutrophils and CD8 + T-cell abundance, with significance assessed by least-squares linear regression ( n = 7/group, 4 groups). b Pathway enrichment analysis of N1_TNF-α + neutrophils across treatment groups. c Cell–cell communication analysis between N1_TNF-α + neutrophils and CD8 _Effector T cells in second tumors across treatment groups. Flow-cytometric counts of MHC-I + ( d ), MHC-II + ( e ), CD86 + ( f ), CCR7 + ( g ), and 4-1BBL + ( h ) neutrophils per milligram tumor in second tumors in the LLC model ( n = 7 per group). ( i ) Flow-cytometric counts of 4-1BB + CD8 + T cells per milligram tumor in second tumors in the LLC model ( n = 7 per group). ( j ) Percentage of H-2K b -SIINFEKL (OVA) MHC-I complexes on Con- or N1_TNF-α⁺ neutrophils ( n = 3 per group). k Schematic of the in vitro study evaluating co-culture of control or N1_TNF-α⁺ neutrophils with CD8⁺ T cells, treated with or without αPD-1. Percentages of T-cell activation markers 4-1BB ( l ), CD69 ( m ), and CD25 ( n ) under co-culture conditions as in ( k ) ( n = 6 per group). o CD8⁺ T-cell proliferation in co-culture with N1_TNF-α⁺ neutrophils or control (untreated) neutrophils, with or without αPD-1 ( n = 8 per group). p Tumor-cell death assessed by propidium iodide (PI) staining after co-culture with CD8⁺ T cells, and N1_TNF-α⁺ or control neutrophils in the presence of αPD-1 ( n = 3 per group). q Experimental schematic of in vivo study evaluating the therapeutic efficacy of adoptively transferred N1_TNF-α⁺ neutrophils in combination with αPD-1. Tumor growth curves ( r ) and survival ( s ) of LLC-bearing mice treated with adoptive neutrophil transfer with or without αPD-1 ( n = 6 per group). Data are mean ± SEM. Statistical significance was assessed by one-way ANOVA ( d – i, l – o , p ), Student’s t test ( j ), two-way ANOVA ( r ), or log-rank test for survival ( s ). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Article Snippet: CD8 + T cells were isolated from C57BL/6 spleens using CD8a (Ly-2) MicroBeads (Miltenyi Biotec, Cat# 130-117-044) and LS columns (Miltenyi Biotec, Cat# 130-042-401).

Techniques: In Vitro, Co-Culture Assay, Control, Activation Assay, Staining, In Vivo, Drug discovery

Journal: Signal Transduction and Targeted Therapy

Article Title: Low-dose radiotherapy synergizes with PD-1 blockade to achieve durable survival in advanced NSCLC through antitumor neutrophil programming

doi: 10.1038/s41392-026-02712-6

Figure Lengend Snippet: ICAM-1-LFA-1 interactions drive neutrophil-T cell engagement and cytotoxicity in TRIDENT-induced responses. a Comparison of ligand-receptor crosstalk between N1_TNF-α⁺ neutrophils and CD8_Effector T cells in second tumors across treatment groups, inferred from scRNA-seq. b Contribution scores of key ligand-receptor pairs within the ICAM signaling pathway between N1_TNF-α⁺ neutrophils and CD8_Effector T cells under TRIDENT. c Representative immunofluorescence (IF) images showing LFA-1α expression in second tumors in the LLC model across different groups. Scale bars, 50 µm. Flow-cytometric counts of ICAM1⁺ neutrophils ( d ) and LFA-1α⁺ CD8⁺ T cells ( e ) per milligram tumor in second tumors in the LLC model ( n = 7 per group). f Representative IF images showing ICAM-1 expression in control neutrophils versus N1_TNF-α⁺ neutrophils. g Percentage of ICAM-1 + neutrophils in control versus N1_TNF-α⁺ neutrophils, as determined by flow cytometry ( n = 10 per group). h Experimental schematic of in vitro study evaluating the potential active synapse formation between neutrophils and CD8 + T cells. i T-cell proliferation in co-culture with N1_TNF-α⁺ neutrophils and αPD-1, with or without anti-LFA-1α ( n = 9 per group). j – m T-cell activation under the co-culture conditions in ( h ): expression of 4-1BB, CD69, and CD25; representative flow-cytometry plots are shown in ( m , red, T + αPD-1 + N1_TNF-α⁺-Neu; gray, T + αPD-1 + N1_TNF-α⁺-Neu + anti-LFA-1α; n = 10 per group). Tumor growth curves ( n, o ) and survival ( p ) of LLC-bearing mice treated with TRIDENT with or without anti-LFA-1α antibody ( n = 8–9 per group). Data are mean ± SEM. Statistical significance was assessed by one-way ANOVA ( d, e, i – l ), Student’s t test ( g , h ), two-way ANOVA ( n , o ), or log-rank test for survival ( p ). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Article Snippet: CD8 + T cells were isolated from C57BL/6 spleens using CD8a (Ly-2) MicroBeads (Miltenyi Biotec, Cat# 130-117-044) and LS columns (Miltenyi Biotec, Cat# 130-042-401).

Techniques: Comparison, Immunofluorescence, Expressing, Control, Flow Cytometry, In Vitro, Co-Culture Assay, Activation Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Low-dose radiotherapy synergizes with PD-1 blockade to achieve durable survival in advanced NSCLC through antitumor neutrophil programming

doi: 10.1038/s41392-026-02712-6

Figure Lengend Snippet: High-resolution spatial mapping corroborates N1-mediated immune activation. a Study design and spatial profiling of NSCLC tumors before and after TRIDENT therapy. b Representative spatial transcriptomic maps showing spatial annotations deconvolved by RCTD. c Pathway enrichment analysis comparing post-treatment with baseline tumor tissues. d Dot plot showing log-fold changes (logFC) of major cell types across 11 niches defined by CellCharter. e Representative spatial niche map of a post-treatment tumor sample (patient #2). f Proportions of CD8⁺ T cells, neutrophils, and epithelial cells across the 11 identified niches. g Spatial diversity analysis. Left, heatmaps of the Shannon diversity index across tumor sections. Right, paired comparison of log2 fold changes (log2FC) in Shannon index between baseline and post-treatment samples. Center lines denote medians, boxes indicate interquartile ranges (IQR), and whiskers extend to 1.5×IQR. Paired comparisons were performed using paired t tests. ( h - j ) Pathway enrichment analyses of Niche 9 ( h ), Niche 8 ( i ), and Niche 5 ( j ). k Representative multiplex immunohistochemistry (mIHC) images showing MPO, CD14, ICAM-1, TNF-α, and CD8. Scale bars, 20 μm (left); 10 μm (right). l Schematic summarizing N1_TNF-α + neutrophil-driven immune remodeling after TRIDENT therapy

Article Snippet: CD8 + T cells were isolated from C57BL/6 spleens using CD8a (Ly-2) MicroBeads (Miltenyi Biotec, Cat# 130-117-044) and LS columns (Miltenyi Biotec, Cat# 130-042-401).

Techniques: Activation Assay, Comparison, Multiplex Assay, Immunohistochemistry