negative selection kit  (Miltenyi Biotec)

Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: mBH3@NPs induced effective antitumor immunity and remodeled the tumor microenvironment (A) Experimental timeline for mBH3@NPs administration in B16-F10-tumor-bearing mice. Mice were administered with mBH3@NPs three times, with a dosing interval of every 2 days (B) Average tumor volume curves for mice treated in the melanoma model ( n = 8). (C) Individual tumor volume curves for mice treated in the melanoma model ( n = 8). (D) Western blot analysis of Bax, Bak, Bcl-2, Bcl-x L , Mcl-1, Puma, Bim, caspase-3, and caspase-9 expression following different treatments. β-Actin was used as a loading control ( n = 3). (E) Representative images of tumor immunostaining and quantification for TUNEL (green)/DAPI (blue), CD8 (red)/DAPI (blue), and CRT (green)/DAPI (blue) at day 19 following the indicated treatments in the melanoma model. Scale bars, 200 μm. (F) HMGB1 expression in tumor tissues analyzed by ELISA following different treatments ( n = 5). (G) Heatmap of cytokine expression levels (GrB, IFN-γ, TNF-α, IL-12, and IL-6) in tumor tissues following different treatments ( n = 5). (H) Flow cytometric analysis of immune cell populations (T cells, CD8 + T cells, CD4 + T cells, Treg cells, NK cells, macrophages, M1-like TAMs, M2-like TAMs, and MDSCs) within tumors and CD11c + CD80 + CD86 + mature dendritic cells in lymph nodes ( n = 5). One-way ANOVA with Tukey’s multiple comparisons test was used for all statistical analyses. Data are presented as the mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant. See also and .

Article Snippet: Human CD8 + T cell Negative Selection , MedChemExpress , Cat#HY-KO351.

Techniques: Western Blot, Expressing, Control, Immunostaining, TUNEL Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: mBH3@NPs promoted the recruitment of adoptively transferred T cells (A) Schematic of transwell migration assay using B16-OVA cells and preactivated OT-1 T cells. (B) Transwell migration assay of OT-I T cells (CFDA-labeled). Representative flow cytometry graphs and quantification of T cell infiltration relative fold changes (calculated as the ratio of bottom well-located OT-I T cells to counting beads) ( n = 3). (C) Experimental timeline for mBH3@NPs administration combined with adoptively transferred T cells in B16-OVA tumor-bearing mice. (D) Serial IVIS images and quantification of infiltrating Cy5-T cell signals in B16-OVA tumor-bearing mice ( n = 5). (E) Quantification of tumor-infiltrating endogenous T cells and transferred CD8 + T cells per million cells ( n = 5). (F) Heatmap of RNA-seq data for key interleukin family member, chemokine family member, and granzyme family member from harvested tumor tissues, plotted as Z score of normalized gene expression for each gene ( n = 3). (G) Heatmap of the differences in pathway activities scored by GSVA from harvested tumor tissues, plotted as Z score of normalized gene expression for each gene ( n = 3). (H) Validation of key cytokines and chemokines expression by multiplex cytokine assay. Tumor tissue was homogenized, and the cell supernatant of each treatment group was collected ( n = 4). One-way ANOVA with Tukey’s multiple comparisons test was used for all statistical analyses. Data are presented as the mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant. See also .

Article Snippet: Human CD8 + T cell Negative Selection , MedChemExpress , Cat#HY-KO351.

Techniques: Transwell Migration Assay, Labeling, Flow Cytometry, RNA Sequencing, Gene Expression, Biomarker Discovery, Expressing, Multiplex Assay, Cytokine Assay

Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: mBH3@NPs combined with adoptive T cell therapy improved the therapeutic effect (A) Experimental timeline for mBH3@NPs administration combined with adoptively transferred T cells from PMEL mice in B16-F10-tumor-bearing mice. (B) The average tumor volume curves for mice treated in the B16-F10-tumor-bearing mice model ( n = 7). (C) Survival analysis for mice treated in B16-F10-tumor-bearing mice model ( n = 7). Mice were humanely euthanized when tumor volume reached the predefined ethical endpoint (15 mm × 15 mm). The x axis indicates days post-mBH3@NPs administration. (D) Experimental timeline for mBH3@NPs administration combined with adoptive transfered T cells from OT-1 mice in B16-OVA-tumor-bearing mice. (E) The average tumor volume curves for mice treated in B16-OVA-tumor-bearing mice model ( n = 7). (F) Survival analysis for mice treated in B16-OVA-tumor-bearing mice model ( n = 7). Mice were humanely euthanized when tumor volume reached the predefined ethical endpoint (15 mm × 15 mm). (G) Representative flow cytometry plot and quantification of CD44 and CD62L expression levels gated on total CD8 + T cells (upper panel) and the percentage of endogenous (CD90.2) CD8 + T cells and transferred PMEL (CD90.1) CD8 + T cells gated on total CD8 + T cells (lower panel) ( n = 5). (H) Representative flow cytometry plot and quantification of the expression level of granzyme B (GrB) and perforin in total CD8 + T cells from tumors and spleens ( n = 5). (I) Representative flow cytometry plot and quantification of the expression level of PD-1 and LAG-3 in both endogenous and transferred PMEL CD8 + T cells from tumors ( n = 5). (J) Immunofluorescence evaluation of CD8 + T cell exhaustion, effector, and memory function. Upper panel: representative immunofluorescence images of tumors from different groups for analyzing the exhaustion (PD-1 + , cyan) and effector function (IFN-γ + , green) of CD8 + T cells (CD8 + , pink). Scale bars, 100 μm. Lower panel: representative immunofluorescence images of tumor-infiltrating CD8 + T cells (CD8 + , green) along with markers for activation (CD69 + , red) and memory differentiation (CD44 + , pink; CCR7 + , cyan). Scale bars, 200 μm ( n = 3). One-way ANOVA with Tukey’s multiple comparisons test was used for all statistical analyses. Data are presented as the mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant. See also .

Article Snippet: Human CD8 + T cell Negative Selection , MedChemExpress , Cat#HY-KO351.

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Activation Assay

Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: Combination therapy drove CD8 + T cells toward memory-like states with expanded TCR diversity (A) Experimental design for scRNA-seq. Mice were treated with ACT alone or combined with mBim@NPs and mPuma@NPs as shown ( n = 3); CD3 + T cell isolated from mice for scRNA-seq. (B) UMAP representation and distribution of 68,176 cells of the CD8 + T cell atlas in each experimental condition, colored by 12 CD8 + T cell subtypes annotated in this study. (C) UMAP representation of the CD8 + T cells atlas colored by each experimental condition. (D) A dot plot showing the expression levels of marker genes in each CD8 + T cell subtype. The color of the dots indicates the average scaled expression level, and the size of the dots indicates the percentage of cells expressing the gene in each subtype. (E) Indicated expression of gene marker associated with memory ( Tcf7 , Il7r , Sell , and Ccl5 ), exhaustion ( Pdcd1 , Tigit , and Lag3 ), and cytotoxicity ( Gzmb , Ifng , and Prf1 ) in the UMAP plot. (F) A dot plot showing the expression levels of marker genes in E. The color of the dots indicates the average scaled expression level, and the size of the dots indicates the percentage of cells expressing the gene in each subtype. (G) Volcano plots of log 2 (fold change) and log 10 (adjusted FDR value) of differentially expressed genes in proliferating T and Gzmk + T rm from mice treated with ACT monotherapy and combination therapy with mBim@NPs. (H) Shannon entropy of the T ex , proliferating T, T naive , and Gzmk + T rm subsets across different treatment groups (T: ACT monotherapy; TP: ACT + mPuma@NPs; TB: ACT + mBim@NPs). (I) Shannon entropy of total CD8 + T cells and CD4 + T cells across different treatment groups. (J) A heatmap of normalized Shannon entropy for CD8 + T cell subsets across different treatment groups. See also .

Article Snippet: Human CD8 + T cell Negative Selection , MedChemExpress , Cat#HY-KO351.

Techniques: Isolation, Expressing, Marker

Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: mBH3@NPs induced effective antitumor immunity and remodeled the tumor microenvironment (A) Experimental timeline for mBH3@NPs administration in B16-F10-tumor-bearing mice. Mice were administered with mBH3@NPs three times, with a dosing interval of every 2 days (B) Average tumor volume curves for mice treated in the melanoma model ( n = 8). (C) Individual tumor volume curves for mice treated in the melanoma model ( n = 8). (D) Western blot analysis of Bax, Bak, Bcl-2, Bcl-x L , Mcl-1, Puma, Bim, caspase-3, and caspase-9 expression following different treatments. β-Actin was used as a loading control ( n = 3). (E) Representative images of tumor immunostaining and quantification for TUNEL (green)/DAPI (blue), CD8 (red)/DAPI (blue), and CRT (green)/DAPI (blue) at day 19 following the indicated treatments in the melanoma model. Scale bars, 200 μm. (F) HMGB1 expression in tumor tissues analyzed by ELISA following different treatments ( n = 5). (G) Heatmap of cytokine expression levels (GrB, IFN-γ, TNF-α, IL-12, and IL-6) in tumor tissues following different treatments ( n = 5). (H) Flow cytometric analysis of immune cell populations (T cells, CD8 + T cells, CD4 + T cells, Treg cells, NK cells, macrophages, M1-like TAMs, M2-like TAMs, and MDSCs) within tumors and CD11c + CD80 + CD86 + mature dendritic cells in lymph nodes ( n = 5). One-way ANOVA with Tukey’s multiple comparisons test was used for all statistical analyses. Data are presented as the mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant. See also and .

Article Snippet: Human CD8 + T cells were isolated from PBMCs using the Human CD8 + T cell Negative Selection Kit (MedChemExpress, #HY-KO351).

Techniques: Western Blot, Expressing, Control, Immunostaining, TUNEL Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: mBH3@NPs promoted the recruitment of adoptively transferred T cells (A) Schematic of transwell migration assay using B16-OVA cells and preactivated OT-1 T cells. (B) Transwell migration assay of OT-I T cells (CFDA-labeled). Representative flow cytometry graphs and quantification of T cell infiltration relative fold changes (calculated as the ratio of bottom well-located OT-I T cells to counting beads) ( n = 3). (C) Experimental timeline for mBH3@NPs administration combined with adoptively transferred T cells in B16-OVA tumor-bearing mice. (D) Serial IVIS images and quantification of infiltrating Cy5-T cell signals in B16-OVA tumor-bearing mice ( n = 5). (E) Quantification of tumor-infiltrating endogenous T cells and transferred CD8 + T cells per million cells ( n = 5). (F) Heatmap of RNA-seq data for key interleukin family member, chemokine family member, and granzyme family member from harvested tumor tissues, plotted as Z score of normalized gene expression for each gene ( n = 3). (G) Heatmap of the differences in pathway activities scored by GSVA from harvested tumor tissues, plotted as Z score of normalized gene expression for each gene ( n = 3). (H) Validation of key cytokines and chemokines expression by multiplex cytokine assay. Tumor tissue was homogenized, and the cell supernatant of each treatment group was collected ( n = 4). One-way ANOVA with Tukey’s multiple comparisons test was used for all statistical analyses. Data are presented as the mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant. See also .

Article Snippet: Human CD8 + T cells were isolated from PBMCs using the Human CD8 + T cell Negative Selection Kit (MedChemExpress, #HY-KO351).

Techniques: Transwell Migration Assay, Labeling, Flow Cytometry, RNA Sequencing, Gene Expression, Biomarker Discovery, Expressing, Multiplex Assay, Cytokine Assay

Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: mBH3@NPs combined with adoptive T cell therapy improved the therapeutic effect (A) Experimental timeline for mBH3@NPs administration combined with adoptively transferred T cells from PMEL mice in B16-F10-tumor-bearing mice. (B) The average tumor volume curves for mice treated in the B16-F10-tumor-bearing mice model ( n = 7). (C) Survival analysis for mice treated in B16-F10-tumor-bearing mice model ( n = 7). Mice were humanely euthanized when tumor volume reached the predefined ethical endpoint (15 mm × 15 mm). The x axis indicates days post-mBH3@NPs administration. (D) Experimental timeline for mBH3@NPs administration combined with adoptive transfered T cells from OT-1 mice in B16-OVA-tumor-bearing mice. (E) The average tumor volume curves for mice treated in B16-OVA-tumor-bearing mice model ( n = 7). (F) Survival analysis for mice treated in B16-OVA-tumor-bearing mice model ( n = 7). Mice were humanely euthanized when tumor volume reached the predefined ethical endpoint (15 mm × 15 mm). (G) Representative flow cytometry plot and quantification of CD44 and CD62L expression levels gated on total CD8 + T cells (upper panel) and the percentage of endogenous (CD90.2) CD8 + T cells and transferred PMEL (CD90.1) CD8 + T cells gated on total CD8 + T cells (lower panel) ( n = 5). (H) Representative flow cytometry plot and quantification of the expression level of granzyme B (GrB) and perforin in total CD8 + T cells from tumors and spleens ( n = 5). (I) Representative flow cytometry plot and quantification of the expression level of PD-1 and LAG-3 in both endogenous and transferred PMEL CD8 + T cells from tumors ( n = 5). (J) Immunofluorescence evaluation of CD8 + T cell exhaustion, effector, and memory function. Upper panel: representative immunofluorescence images of tumors from different groups for analyzing the exhaustion (PD-1 + , cyan) and effector function (IFN-γ + , green) of CD8 + T cells (CD8 + , pink). Scale bars, 100 μm. Lower panel: representative immunofluorescence images of tumor-infiltrating CD8 + T cells (CD8 + , green) along with markers for activation (CD69 + , red) and memory differentiation (CD44 + , pink; CCR7 + , cyan). Scale bars, 200 μm ( n = 3). One-way ANOVA with Tukey’s multiple comparisons test was used for all statistical analyses. Data are presented as the mean ± SD. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant. See also .

Article Snippet: Human CD8 + T cells were isolated from PBMCs using the Human CD8 + T cell Negative Selection Kit (MedChemExpress, #HY-KO351).

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Activation Assay

Journal: Cell Reports Medicine

Article Title: mRNA lipid-nanoparticle-mediated mitochondrial apoptosis augments adoptive T cell immunotherapy

doi: 10.1016/j.xcrm.2026.102706

Figure Lengend Snippet: Combination therapy drove CD8 + T cells toward memory-like states with expanded TCR diversity (A) Experimental design for scRNA-seq. Mice were treated with ACT alone or combined with mBim@NPs and mPuma@NPs as shown ( n = 3); CD3 + T cell isolated from mice for scRNA-seq. (B) UMAP representation and distribution of 68,176 cells of the CD8 + T cell atlas in each experimental condition, colored by 12 CD8 + T cell subtypes annotated in this study. (C) UMAP representation of the CD8 + T cells atlas colored by each experimental condition. (D) A dot plot showing the expression levels of marker genes in each CD8 + T cell subtype. The color of the dots indicates the average scaled expression level, and the size of the dots indicates the percentage of cells expressing the gene in each subtype. (E) Indicated expression of gene marker associated with memory ( Tcf7 , Il7r , Sell , and Ccl5 ), exhaustion ( Pdcd1 , Tigit , and Lag3 ), and cytotoxicity ( Gzmb , Ifng , and Prf1 ) in the UMAP plot. (F) A dot plot showing the expression levels of marker genes in E. The color of the dots indicates the average scaled expression level, and the size of the dots indicates the percentage of cells expressing the gene in each subtype. (G) Volcano plots of log 2 (fold change) and log 10 (adjusted FDR value) of differentially expressed genes in proliferating T and Gzmk + T rm from mice treated with ACT monotherapy and combination therapy with mBim@NPs. (H) Shannon entropy of the T ex , proliferating T, T naive , and Gzmk + T rm subsets across different treatment groups (T: ACT monotherapy; TP: ACT + mPuma@NPs; TB: ACT + mBim@NPs). (I) Shannon entropy of total CD8 + T cells and CD4 + T cells across different treatment groups. (J) A heatmap of normalized Shannon entropy for CD8 + T cell subsets across different treatment groups. See also .

Article Snippet: Human CD8 + T cells were isolated from PBMCs using the Human CD8 + T cell Negative Selection Kit (MedChemExpress, #HY-KO351).

Techniques: Isolation, Expressing, Marker