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mouse spleen lymphocyte isolation solution kit (Beijing Solarbio Science)

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Beijing Solarbio Science mouse spleen lymphocyte isolation solution kit
Mouse Spleen Lymphocyte Isolation Solution Kit, supplied by Beijing Solarbio Science, used in various techniques. Bioz Stars score: 96/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/lymphocyte+isolation+kit/Mouse+Spleen+Lymphocyte+Isolation+Solution+Kit/pmc13045921-159-12-18
Average 96 stars, based on 136 article reviews

mouse spleen lymphocyte isolation solution kit - by Bioz Stars, 2026-10

96/100 stars

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Isolation:

Article Title: Nanospheres as the delivery vehicle: novel application of Toxoplasma gondii ribosomal protein S2 in PLGA and chitosan nanospheres against acute toxoplasmosis
Article Snippet: The secretion of cytokines interferon-γ (IFN-γ), transforming growth factor β (TGF-β), IL-4, IL-6, IL-10, and IL-17 in mice serum was determined using a commercial ELISA kit (Enzyme Linked Biotechnology Co., Ltd, Shanghai, China) according to the instructions. .. After 10 days of the first and the booster immunization, respectively, five mice from each group were sacrificed before immunization or challenge, and splenic lymphocytes were collected using a lymphocyte isolation kit (Solarbio, Beijing, China). ..

Article Title: Supporting Information
Article Snippet: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[thiol (polyethylene glycol)](DSPEPEG5000-SH) was purchased from Ponsure Biological (Shanghai, China). .. Mouse IgG2b Protein (catalog number, 51095-MNAH) and tumor-infiltrating lymphocyte Isolation Kit was obtained from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). .. 1,2- Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from CordenPharma company (Liestal, Switzerland).

Article Title: Restoring Antitumor Immunity by Reprogramming Abnormal Lipid Metabolism in the Tumor Microenvironment Using Irisin-Manganese Co-loaded Nanoparticles.
Article Snippet: Targeting dysregulated lipid metabolism within the tumor microenvironment (TME) has emerged as a promising strategy for restoring anti-tumor immunity and reversing immune suppression to improve therapeutic efficiency.. This study reports a nanomaterial platform for co-delivery of irisin and manganese ions (Mn2+) to exert cumulative effects of modulating lipid metabolism dysregulation and ameliorating the immunosuppressive TME in triple-negative breast cancer (TNBC).. Irisin stimulates lipolysis and inhibits lipogenesis, whereas Mn2+ strengthen irisin binding to integrin αVβ5 over-expressed in various cancers.

Article Title: Improved protection against H9N2 avian influenza virus challenge in chickens by the presence of LTB adjuvant on the 3M2e-NP nanoparticle vaccine delivered by sifA deficient Salmonella.
Article Snippet: The H9N2 avian influenza virus is a still a great threat to poultry production and public health due to its variation.. The highly conserved extracellular domain, M2 ion channel (M2e), and the nuclear protein (NP) are usually considered to be potential targets for a broad-spectrum influenza vaccine.. In this study, we took use of a regulated delayed lysis Salmonella χYL56 lacking the sifA gene as a delivery vector to deliver a lumazine synthase (LS) based nanoparticle vaccine decorating with three copies of M2e (H9N2, H5N1 and H1N1 subtype, 3M2e) and NP protein, without or with LTB adjuvant, yielding S311 and S325, respectively.

Article Title: PD-1-engineered manganese-based nanoplatform loaded with CXCL9 induces ICD and intensifies T-cell infiltration for melanoma treatment
Article Snippet: .. Created in https:// BioRe nder. com lymphocyte isolation kit, JC-1 probe, collagenase IV, and DNase I (Solarbio); Calcein AM/PI Kit and DCFH-DA (Beyotime); CRT and HMGB1 (Immunoway); PE antimouse CD274/PD-L1 (Elabscience); and flow antibodies (CD8a-FITC, CD3-PerCP/Cy5.5, CD4-PE, CD86-FITC, CD80-APC, CD11c-PE) from BioLegend. .. Hydrodynamic size distribution and zeta potential were performed on Malvern particle size analyzer (ZS90, Malvern).

Article Title: Napabucasin transforms liver microenvironment and boosts immunotherapy efficacy by converting potential metastases into “hot” tumors
Article Snippet: .. Mouse lymphocytes were isolated by excising the spleen, filtering the cells through a 70 μm cell strainer, and further purifying them using a lymphocyte isolation kit for mouse spleen (Solarbio, Beijing, China).14 For in vitro cell treatment, Napabucasin was dissolved in DMSO (Solarbio, Beijing, China). ..

Article Title: Radiotherapy‐resistant prostate cancer cells escape immune checkpoint blockade through the senescence‐related ataxia telangiectasia and Rad3‐related protein
Article Snippet: .. Briefly, fresh lymphocytes were flushed from the spleens of 6‐week‐old C57BL/6 male WT mice with PBS and purified using a lymphocyte isolation kit (P8860, Solarbio). .. T cells were expanded in a 15 mL centrifuge tube with RPMI 1640 medium containing 10% FBS and 150 U/mL Il‐2 (HY‐P7077, MedChemExpress) for 24 h. Then, the T cells were subjected to isotype antigen stimulation twice every 7 days before being used.

Article Title: Antibody-Dependent Cellular Cytotoxicity Elicited by the Antibodies Against the E120R Protein of African Swine Fever Virus
Article Snippet: .. PBMCs were obtained from EDTA-anticoagulated whole blood via density gradient centrifugation using a porcine-specific lymphocyte isolation kit (Solarbio, Beijing, China, P6230) and used directly without cryopreservation or thawing to preserve the cellular viability and function. ..

In Vitro:

Purification:

Gradient Centrifugation:

Image Search Results

a-b , Following the schedule as in Extended Data Fig. , BALB/c mice ( a ) or OT-II mice ( b ) were fasted or fed. OCR traces of a mitochondrial stress test of magnetically purified pooled CD8 + T cells from fasted or fed group of BALB/c mice ( a ; n = 3 wells) or purified pooled CD4 + T cells from fasted or fed group of OT-II mice ( b ; n = 5 wells). ECAR was also measured (lower panel). OCR and ECAR associated parameters were tabulated. c-d , Following the scheme as in Extended Data Fig. , C57BL/6 wild type mice were fasted or fed. The magnetically purified CD8 + T cells were stimulated for 24 h followed by expansion in either effector skewing condition containing mIL2 ( c ) or in memory skewing condition containing mIL15 + mIL7 cytokines ( d ) for 7 days (n = 5 mice per group). Cell numbers were counted and plotted. e-f , pooled memory skewed CD8 + T cells from d , were subjected to OCR ( e ) and ECAR measurement ( f ) (n = 8 wells). OCR and ECAR associated parameters were tabulated (right). g-l , OT-I mice on Rag2 −/− background fasted or fed as per scheme in Extended Data Fig. (n = 3 mice per group). g , Lymphocyte absolute number was compared ex vivo among fasted and fed mice. h-i , OCR ( h ) and ECAR ( i ) were measured for ex vivo fasted and fed purified OT-I CD8 + T cells (n = 3 (for fasted) or 5 (for fed) wells). OCR and ECAR parameters were calculated and shown in bar graph (right). j , Representative cytogram of ex vivo CD8 + T cells subpopulations based on the intensity of CD62L and CD44 staining and stacked bar graph of different subpopulations are shown. k-l , ex vivo CD8 + T cells were acutely stimulated with PMA+Ionomycin for overnight and transport inhibitor was added for 4 h before cells collection for staining. Further, cytokines were assessed intracellularly. Representative cytogram of TNF ( k ) and IL2 ( l ) staining and the bar graph of their gMFI are shown on right. For figure a, b, e , f, h, i , T cells were pooled from one group of mice, Means ± SEM, n is number of technical replicate wells of pooled T cells from respective group, one-way ANOVA analysis. For figure c , d , Means ± SEM, n = 3 mice/group, two-tailed unpaired Student’s t -test analysis. For figure g, j-l , each dot represents a mice, Means ± SEM, two-tailed unpaired Student’s t -test analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns=not significant. Data are representative of three independent experiments (with a total of n = 10 mice in each fasted and fed condition).

Journal: Nature

Article Title: Postprandial lipid metabolism durably enhances T cell immunity

doi: 10.1038/s41586-026-10432-8

Figure Lengend Snippet: a-b , Following the schedule as in Extended Data Fig. , BALB/c mice ( a ) or OT-II mice ( b ) were fasted or fed. OCR traces of a mitochondrial stress test of magnetically purified pooled CD8 + T cells from fasted or fed group of BALB/c mice ( a ; n = 3 wells) or purified pooled CD4 + T cells from fasted or fed group of OT-II mice ( b ; n = 5 wells). ECAR was also measured (lower panel). OCR and ECAR associated parameters were tabulated. c-d , Following the scheme as in Extended Data Fig. , C57BL/6 wild type mice were fasted or fed. The magnetically purified CD8 + T cells were stimulated for 24 h followed by expansion in either effector skewing condition containing mIL2 ( c ) or in memory skewing condition containing mIL15 + mIL7 cytokines ( d ) for 7 days (n = 5 mice per group). Cell numbers were counted and plotted. e-f , pooled memory skewed CD8 + T cells from d , were subjected to OCR ( e ) and ECAR measurement ( f ) (n = 8 wells). OCR and ECAR associated parameters were tabulated (right). g-l , OT-I mice on Rag2 −/− background fasted or fed as per scheme in Extended Data Fig. (n = 3 mice per group). g , Lymphocyte absolute number was compared ex vivo among fasted and fed mice. h-i , OCR ( h ) and ECAR ( i ) were measured for ex vivo fasted and fed purified OT-I CD8 + T cells (n = 3 (for fasted) or 5 (for fed) wells). OCR and ECAR parameters were calculated and shown in bar graph (right). j , Representative cytogram of ex vivo CD8 + T cells subpopulations based on the intensity of CD62L and CD44 staining and stacked bar graph of different subpopulations are shown. k-l , ex vivo CD8 + T cells were acutely stimulated with PMA+Ionomycin for overnight and transport inhibitor was added for 4 h before cells collection for staining. Further, cytokines were assessed intracellularly. Representative cytogram of TNF ( k ) and IL2 ( l ) staining and the bar graph of their gMFI are shown on right. For figure a, b, e , f, h, i , T cells were pooled from one group of mice, Means ± SEM, n is number of technical replicate wells of pooled T cells from respective group, one-way ANOVA analysis. For figure c , d , Means ± SEM, n = 3 mice/group, two-tailed unpaired Student’s t -test analysis. For figure g, j-l , each dot represents a mice, Means ± SEM, two-tailed unpaired Student’s t -test analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns=not significant. Data are representative of three independent experiments (with a total of n = 10 mice in each fasted and fed condition).

Article Snippet: Naive (CD62L high CD44 low ) CD8 + T cells were then purified from total pooled lymphocytes according to the manufacturer’s instructions (Miltenyi Biotec, 130-096-543).

Techniques: Purification, Ex Vivo, Staining, Two Tailed Test

Journal: Nature

Article Title: Postprandial lipid metabolism durably enhances T cell immunity

doi: 10.1038/s41586-026-10432-8

Figure Lengend Snippet: a , Schema; in short, bone marrow cells from B6 WT (Thy1.1) and LDLR –/– (Thy1.2) were co-transferred in equal number in an irradiated RAG2 –/– mouse. b , Reconstitution was confirmed in bone marrow chimera mice after 4-6 weeks of adoptive transfer of mixed bone marrow. Thy1.1 (WT) and Thy1.2 (LDLR –/– ) lymphocytes (left) and different immune T cells subsets were quantified (right) after gating on WT or LDLR –/– lymphocytes. c , Metabolic parameters of CD8 + T cells were compared ex vivo from fasted or fed bone marrow chimera mice. Each dot represents an individual mouse. Histogram of Glucose-Cy3 (left), BODIPY-NL (493/503) (middle), and mito mass (right), are shown and their gMFI are plotted in bar graphs (n = 5 mice for each fasted and fed group) (lower panel). d , OCR was measured for ex vivo purified pooled Thy1.1 B6 WT ctrl CD8 + T cells (left) or Thy1.2 LDLR –/– CD8 + T cells (middle) from fasted or fed bone marrow chimera mice and the OCR associated parameters are tabulated (lower). e , Cytokine profile from in vitro expanded Thy1.1 (WT) and Thy1.2 (LDLR –/– ) lymphocytes. Bar graph of gMFI of IFN-γ and IL2 are shown. For figure d , Means ± SEM, n = 3-10 wells. For figure b, c , and e , Means ± SEM, n = 5 mice/group, two-way ANOVA analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant. Data are representative of total 14 fasted and 14 fed bone marrow chimera mice from three independent experiments.

Article Snippet: Naive (CD62L high CD44 low ) CD8 + T cells were then purified from total pooled lymphocytes according to the manufacturer’s instructions (Miltenyi Biotec, 130-096-543).

Techniques: Irradiation, Adoptive Transfer Assay, Ex Vivo, Purification, In Vitro

DADA inhibits tumor growth by potentiating CD8 + T cell anti‐tumor immune responses. (A) Schematic experimental procedure in (B–E): WT mice were fed with DADA‐containing or normal water from day ‐14 until the experimental endpoints or until day 0, and were injected (subcutaneously [s.c.]) with 16‐F10 cells on day 0. (B,C) Tumor growth curves (B) and tumor weights (C) at 17 days after B16‐F10 inoculation; n = 6. The experiment was repeated three times. (D,E) Representative flow cytometry plots and quantification of CD8 + T (D), IFN‐γ + CD8 + T and TNF‐α + CD8 + T (E) cells from tumor; n = 6. The experiment was repeated twice. (F) Schematic experimental procedure for (G–J): WT mice were fed with DADA‐containing or normal water from day ‐14 until the experimental endpoints or until day 0, and were injected (s.c.) with MC38 cells on day 0. (G and H) Tumor growth curves (G) and tumor weights (H) at 17 days after MC38 inoculation; n = 6. The experiment was repeated three times. (I,J) Representative flow cytometry plots and quantification of CD8 + T (I), IFN‐γ + CD8 + T, TNF‐α + CD8 + T and GZMB + CD8 + T (J) cells from tumor; n = 6. The experiment was repeated twice. (K) Schematic experimental procedure for (K–O): WT mice were fed with DADA‐containing or normal water from day ‐14 until the experimental endpoints or until day 0, and were injected (intravenously [i.v.]) with B16‐F10 cells on day 0. (L,M) Appearance of lungs (L) and the number of tumor nodules (M) at 17 days after B16‐F10 inoculation; n = 7. The experiment was repeated twice. (N and O) Representative flow cytometry plots and quantification of CD8 + T (N), IFN‐γ + CD8 + T, TNF‐α + CD8 + T and GZMB + CD8 + T (O) cells from the lung; n = 7. The experiment was repeated twice. (P) Schematic experimental procedure for (Q–S): WT mice were fed with DADA‐containing or normal water for 14 days and were injected (intraperitoneally [i.p.]) with 100 µg of anti‐CD8α antibody weekly. The mice were injected (s.c.) with MC38 cells on day 0. (Q–S) Tumor growth curves (Q), tumor weights (R), and representative flow cytometry plots of CD8 + T cells (S) at 17 days after MC38 inoculation; n = 7. The experiment was repeated three times. Data are presented as mean ± SD and are analyzed by two‐way ANOVA (B, G, and Q) and one‐way ANOVA (C–E, H–J, M–O, and R); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Journal: Advanced Science

Article Title: DADA Enhances CD8 + T Cell Stemness to Improve Anti‐Tumor Immunity and Immunotherapy Efficacy

doi: 10.1002/advs.202519765

Figure Lengend Snippet: DADA inhibits tumor growth by potentiating CD8 + T cell anti‐tumor immune responses. (A) Schematic experimental procedure in (B–E): WT mice were fed with DADA‐containing or normal water from day ‐14 until the experimental endpoints or until day 0, and were injected (subcutaneously [s.c.]) with 16‐F10 cells on day 0. (B,C) Tumor growth curves (B) and tumor weights (C) at 17 days after B16‐F10 inoculation; n = 6. The experiment was repeated three times. (D,E) Representative flow cytometry plots and quantification of CD8 + T (D), IFN‐γ + CD8 + T and TNF‐α + CD8 + T (E) cells from tumor; n = 6. The experiment was repeated twice. (F) Schematic experimental procedure for (G–J): WT mice were fed with DADA‐containing or normal water from day ‐14 until the experimental endpoints or until day 0, and were injected (s.c.) with MC38 cells on day 0. (G and H) Tumor growth curves (G) and tumor weights (H) at 17 days after MC38 inoculation; n = 6. The experiment was repeated three times. (I,J) Representative flow cytometry plots and quantification of CD8 + T (I), IFN‐γ + CD8 + T, TNF‐α + CD8 + T and GZMB + CD8 + T (J) cells from tumor; n = 6. The experiment was repeated twice. (K) Schematic experimental procedure for (K–O): WT mice were fed with DADA‐containing or normal water from day ‐14 until the experimental endpoints or until day 0, and were injected (intravenously [i.v.]) with B16‐F10 cells on day 0. (L,M) Appearance of lungs (L) and the number of tumor nodules (M) at 17 days after B16‐F10 inoculation; n = 7. The experiment was repeated twice. (N and O) Representative flow cytometry plots and quantification of CD8 + T (N), IFN‐γ + CD8 + T, TNF‐α + CD8 + T and GZMB + CD8 + T (O) cells from the lung; n = 7. The experiment was repeated twice. (P) Schematic experimental procedure for (Q–S): WT mice were fed with DADA‐containing or normal water for 14 days and were injected (intraperitoneally [i.p.]) with 100 µg of anti‐CD8α antibody weekly. The mice were injected (s.c.) with MC38 cells on day 0. (Q–S) Tumor growth curves (Q), tumor weights (R), and representative flow cytometry plots of CD8 + T cells (S) at 17 days after MC38 inoculation; n = 7. The experiment was repeated three times. Data are presented as mean ± SD and are analyzed by two‐way ANOVA (B, G, and Q) and one‐way ANOVA (C–E, H–J, M–O, and R); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Article Snippet: For isolation of mouse CD8 + T cells, cells were purified from splenic lymphocytes using through magnetic activated cell sorting (MACS) using the Mouse CD8 + T Lymphocyte Negative Selection Kit (Cat#130‐104‐075, Miltenyi Biotec, USA).

Techniques: Injection, Flow Cytometry

DADA prevents CD8 + T cells terminal exhaustion while promoting Tpex cell accumulation in the tumor microenvironment. (A) Schematic experimental procedure for (B–I): WT mice were fed with DADA‐containing or normal water for 31 days and were injected (s.c.) with MC38 cells on day 0. Tumor‐infiltrating CD45 + cells were harvested for 10× genomic scRNA‐seq. (B) Biaxial tSNE clustering plots showing tumor‐infiltrating CD45 + cells. (C) Dot plot showing the expression of representative genes for each cell type in (B). (D) Relative percentages of each cell type in (B). (E) Top 20 enriched GO terms in CD8 + T cells from DADA‐treated mice versus CD8 + T cells from control mice. (F) Biaxial tSNE plots showing secondary clusters of CD8 + T cells. (G) Dot plot showing the expression of representative genes for each cell subset in (F). (H) Relative percentages of each subset in (F). (I) Schematic experimental procedure for (J–M): WT mice were fed with DADA‐containing or normal water from day ‐14 until the experimental endpoints or until day 0, and were injected (s.c.) with MC38 cells on day 0. (J–M) Representative flow cytometry plots and quantification of PD‐1 + TIM‐3 + (J), TCF1 − TIM‐3 + , TCF1 + TIM‐3 − (K), TOX + (L), and Ki67 + (M) cells among CD8 + T cells from tumor; n = 6. The experiment was repeated three times. Data are presented as mean ± SD and are analyzed by one‐way ANOVA (J–M); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Journal: Advanced Science

Article Title: DADA Enhances CD8 + T Cell Stemness to Improve Anti‐Tumor Immunity and Immunotherapy Efficacy

doi: 10.1002/advs.202519765

Figure Lengend Snippet: DADA prevents CD8 + T cells terminal exhaustion while promoting Tpex cell accumulation in the tumor microenvironment. (A) Schematic experimental procedure for (B–I): WT mice were fed with DADA‐containing or normal water for 31 days and were injected (s.c.) with MC38 cells on day 0. Tumor‐infiltrating CD45 + cells were harvested for 10× genomic scRNA‐seq. (B) Biaxial tSNE clustering plots showing tumor‐infiltrating CD45 + cells. (C) Dot plot showing the expression of representative genes for each cell type in (B). (D) Relative percentages of each cell type in (B). (E) Top 20 enriched GO terms in CD8 + T cells from DADA‐treated mice versus CD8 + T cells from control mice. (F) Biaxial tSNE plots showing secondary clusters of CD8 + T cells. (G) Dot plot showing the expression of representative genes for each cell subset in (F). (H) Relative percentages of each subset in (F). (I) Schematic experimental procedure for (J–M): WT mice were fed with DADA‐containing or normal water from day ‐14 until the experimental endpoints or until day 0, and were injected (s.c.) with MC38 cells on day 0. (J–M) Representative flow cytometry plots and quantification of PD‐1 + TIM‐3 + (J), TCF1 − TIM‐3 + , TCF1 + TIM‐3 − (K), TOX + (L), and Ki67 + (M) cells among CD8 + T cells from tumor; n = 6. The experiment was repeated three times. Data are presented as mean ± SD and are analyzed by one‐way ANOVA (J–M); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Techniques: Injection, Expressing, Control, Flow Cytometry

DADA enhances CD8 + T cell stemness in a PDK1‐dependent manner. (A) Schematic experimental procedure for the generation of mouse exhausted CD8 + T cells for (B–E): purified splenic CD8 + T cells were stimulated with anti‐CD3/CD28 mAbs and IL‐2 from for 6 days, with the addition of 40 µM DADA staring from day 3. (B–E) Representative flow cytometry plots and histogram, and quantification of PD‐1 + TIM‐3 + (B), TCF1 − TIM‐3 + , TCF1 + TIM‐3 − (C), Ly108 expression (D), TNF‐α + , IFN‐γ + , and GZMB + (E) cells among CD8 + T cells; n = 3. The experiment was repeated three times. (F) Schematic experimental procedure for the generation of mouse exhausted OT‐1 CD8 + T cells for (G–J): splenic cells from OT‐1 mice were activated with ovalbumin (OVA) peptide and IL‐2 for 2 days, followed by stimulation with anti‐CD3/CD28 mAbs and IL‐2 in the presence of 40 µM DADA from day 3 to day 6. (G–J) Representative flow cytometry plots and histogram, and quantification of PD‐1 + TIM‐3 + (G), TCF1 − TIM‐3 + , TCF1 + TIM‐3 − (H), Ly108 expression (I), TNF‐α + and IFN‐γ + (J) cells among CD8 + T cells; n = 3. The experiment was repeated twice. (K) DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells were collected for RNA‐seq. Heatmap showing the expression of selected genes. (L) mRNA levels of the indicated molecules in DADA‐ or DMSO‐treated mouse exhausted CD8 + T cells; n = 3. The experiment was repeated twice. (M) GSEA of DADA‐ versus DMSO‐treated mouse exhausted CD8 + T cells in indicated gene sets. NES, normalized enrichment score. (N) Heatmap showing the expression of Pdk1 , Pdk2 , Pdk3 , and Pdk4 in DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells. (O) Mouse Pdk1 knockout CD8 + T cells induced to exhaustion subjected to DADA treatment were collected. Quantification of TCF1 − TIM‐3 + , TCF1 + TIM‐3 − , Ly108 expression, TNF‐α + , IFN‐γ + and GZMB + cells among CD8 + T cells; n = 3. Data are presented as mean ± SD and are analyzed by unpaired t test (B‐E, G‐J, and L) and two‐way ANOVA (O); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Journal: Advanced Science

Article Title: DADA Enhances CD8 + T Cell Stemness to Improve Anti‐Tumor Immunity and Immunotherapy Efficacy

doi: 10.1002/advs.202519765

Figure Lengend Snippet: DADA enhances CD8 + T cell stemness in a PDK1‐dependent manner. (A) Schematic experimental procedure for the generation of mouse exhausted CD8 + T cells for (B–E): purified splenic CD8 + T cells were stimulated with anti‐CD3/CD28 mAbs and IL‐2 from for 6 days, with the addition of 40 µM DADA staring from day 3. (B–E) Representative flow cytometry plots and histogram, and quantification of PD‐1 + TIM‐3 + (B), TCF1 − TIM‐3 + , TCF1 + TIM‐3 − (C), Ly108 expression (D), TNF‐α + , IFN‐γ + , and GZMB + (E) cells among CD8 + T cells; n = 3. The experiment was repeated three times. (F) Schematic experimental procedure for the generation of mouse exhausted OT‐1 CD8 + T cells for (G–J): splenic cells from OT‐1 mice were activated with ovalbumin (OVA) peptide and IL‐2 for 2 days, followed by stimulation with anti‐CD3/CD28 mAbs and IL‐2 in the presence of 40 µM DADA from day 3 to day 6. (G–J) Representative flow cytometry plots and histogram, and quantification of PD‐1 + TIM‐3 + (G), TCF1 − TIM‐3 + , TCF1 + TIM‐3 − (H), Ly108 expression (I), TNF‐α + and IFN‐γ + (J) cells among CD8 + T cells; n = 3. The experiment was repeated twice. (K) DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells were collected for RNA‐seq. Heatmap showing the expression of selected genes. (L) mRNA levels of the indicated molecules in DADA‐ or DMSO‐treated mouse exhausted CD8 + T cells; n = 3. The experiment was repeated twice. (M) GSEA of DADA‐ versus DMSO‐treated mouse exhausted CD8 + T cells in indicated gene sets. NES, normalized enrichment score. (N) Heatmap showing the expression of Pdk1 , Pdk2 , Pdk3 , and Pdk4 in DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells. (O) Mouse Pdk1 knockout CD8 + T cells induced to exhaustion subjected to DADA treatment were collected. Quantification of TCF1 − TIM‐3 + , TCF1 + TIM‐3 − , Ly108 expression, TNF‐α + , IFN‐γ + and GZMB + cells among CD8 + T cells; n = 3. Data are presented as mean ± SD and are analyzed by unpaired t test (B‐E, G‐J, and L) and two‐way ANOVA (O); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Techniques: Purification, Flow Cytometry, Expressing, RNA Sequencing, Knock-Out

DADA enhances CD8 + T cell stemness by triggering OXPHOS. (A) Acetyl‐CoA contents in DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells; n = 3. The experiment was repeated twice. (B) Top 10 pathways upregulated in DADA‐ versus DMSO‐treated exhausted CD8 + T cells. (C) GSEA of DADA‐ versus DMSO‐treated mouse exhausted CD8 + T cells in indicated gene sets. (D) mRNA levels of the indicated molecules in DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells; n = 3. The experiment was repeated twice. (E) OCR of DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells. Oligo, oligomycin; FCCP, carbonyl cyanide p‐trifluoromethoxyphenylhydrazone; R+A, rotenone and antimycin A; n = 3. The experiment was repeated twice. (F) Extracellular acidification rate (ECAR) of DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells. Glu, glucose; Oligo, oligomycin; 2‐DG, 2‐deoxyglucose; n = 3. The experiment was repeated twice. (G) The mitochondrial membrane potential (TMRM staining), mitochondrial mass (MitoTracker Deep Red) and mitochondrial superoxide (MitoSox Red) of DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells were measured. Representative histograms and quantification of TMRM, MitoTracker, and MitoSox in CD8 + T cells; n = 3. The experiment was repeated three times. (H) Representative histograms and quantification of TMRM, MitoTracker, and MitoSox in DADA‐ and DMSO‐treated mouse exhausted OT‐1 CD8 + T cells; n = 3. The experiment was repeated three times. (I) Representative histograms and quantification of TMRM, MitoTracker, and MitoSox in DADA‐ and DMSO‐treated human exhausted CD8 + T cells; n = 3. The experiment was repeated three times. (J) Mouse exhausted CD8 + T cells subjected to DADA and Oligomycin (1 µM) combined treatment or single treatment were collected. Quantification of PD‐1 + TIM‐3 + , TCF1 − TIM‐3 + , TCF1 + TIM‐3 − , Ly108 expression, TNF‐α + and IFN‐γ + cells among CD8 + T cells; n = 3. The experiment was repeated twice. (K) Mouse exhausted CD8 + T cells subjected to DADA and Acetyl‐CoA (2 mM) combined treatment or single treatment were collected. Quantification of PD‐1 + TIM‐3 + , TCF1 − TIM‐3 + , TCF1 + TIM‐3 − , Ly108 expression, TNF‐α + and IFN‐γ + cells among CD8 + T cells; n = 3. The experiment was repeated twice. (L) Mouse exhausted CD8 + T cells subjected to DADA and UK5099 (20 µM) combined treatment or single treatment were collected. Quantification of PD‐1 + TIM‐3 + , TCF1 − TIM‐3 + , TCF1 + TIM‐3 − , Ly108 expression, TNF‐α + and IFN‐γ + cells among CD8 + T cells; n = 3. The experiment was repeated twice. Data are presented as mean ± SD and are analyzed by unpaired t test (A, D, and E–H), paired t test (I) and two‐way ANOVA (J–L); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Journal: Advanced Science

Article Title: DADA Enhances CD8 + T Cell Stemness to Improve Anti‐Tumor Immunity and Immunotherapy Efficacy

doi: 10.1002/advs.202519765

Figure Lengend Snippet: DADA enhances CD8 + T cell stemness by triggering OXPHOS. (A) Acetyl‐CoA contents in DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells; n = 3. The experiment was repeated twice. (B) Top 10 pathways upregulated in DADA‐ versus DMSO‐treated exhausted CD8 + T cells. (C) GSEA of DADA‐ versus DMSO‐treated mouse exhausted CD8 + T cells in indicated gene sets. (D) mRNA levels of the indicated molecules in DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells; n = 3. The experiment was repeated twice. (E) OCR of DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells. Oligo, oligomycin; FCCP, carbonyl cyanide p‐trifluoromethoxyphenylhydrazone; R+A, rotenone and antimycin A; n = 3. The experiment was repeated twice. (F) Extracellular acidification rate (ECAR) of DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells. Glu, glucose; Oligo, oligomycin; 2‐DG, 2‐deoxyglucose; n = 3. The experiment was repeated twice. (G) The mitochondrial membrane potential (TMRM staining), mitochondrial mass (MitoTracker Deep Red) and mitochondrial superoxide (MitoSox Red) of DADA‐ and DMSO‐treated mouse exhausted CD8 + T cells were measured. Representative histograms and quantification of TMRM, MitoTracker, and MitoSox in CD8 + T cells; n = 3. The experiment was repeated three times. (H) Representative histograms and quantification of TMRM, MitoTracker, and MitoSox in DADA‐ and DMSO‐treated mouse exhausted OT‐1 CD8 + T cells; n = 3. The experiment was repeated three times. (I) Representative histograms and quantification of TMRM, MitoTracker, and MitoSox in DADA‐ and DMSO‐treated human exhausted CD8 + T cells; n = 3. The experiment was repeated three times. (J) Mouse exhausted CD8 + T cells subjected to DADA and Oligomycin (1 µM) combined treatment or single treatment were collected. Quantification of PD‐1 + TIM‐3 + , TCF1 − TIM‐3 + , TCF1 + TIM‐3 − , Ly108 expression, TNF‐α + and IFN‐γ + cells among CD8 + T cells; n = 3. The experiment was repeated twice. (K) Mouse exhausted CD8 + T cells subjected to DADA and Acetyl‐CoA (2 mM) combined treatment or single treatment were collected. Quantification of PD‐1 + TIM‐3 + , TCF1 − TIM‐3 + , TCF1 + TIM‐3 − , Ly108 expression, TNF‐α + and IFN‐γ + cells among CD8 + T cells; n = 3. The experiment was repeated twice. (L) Mouse exhausted CD8 + T cells subjected to DADA and UK5099 (20 µM) combined treatment or single treatment were collected. Quantification of PD‐1 + TIM‐3 + , TCF1 − TIM‐3 + , TCF1 + TIM‐3 − , Ly108 expression, TNF‐α + and IFN‐γ + cells among CD8 + T cells; n = 3. The experiment was repeated twice. Data are presented as mean ± SD and are analyzed by unpaired t test (A, D, and E–H), paired t test (I) and two‐way ANOVA (J–L); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Techniques: Membrane, Staining, Expressing

DADA improves the efficacy of ACT and ICB immunotherapies. (A) Schematic experimental procedure in (B–F): CTV‐labeled OT‐1 cells, which had undergone an initial activation with OVA peptide for 2 days and subsequent stimulation with anti‐CD3/CD28 mAbs and IL‐2 in the presence of DADA for 4 days, were transferred into MC38‐OVA tumor‐bearing mice. (B–D) Tumor growth curves (B), images of tumors (C), and tumor weights (D) at 15 days after MC38‐OVA inoculation; n = 6. (E,F) Quantification of transferred OT‐1 CD8 + T (E), Ki67 + CD8 + T, PD‐1 + TIM‐3 + CD8 + T, TCF1 − TIM‐3 + CD8 + T, TCF1 + TIM‐3 − CD8 + T, IFN‐γ + CD8 + T, TNF‐α + CD8 + T, and GZMB + CD8 + T (F) cells from tumor; n = 6. (G) Schematic experimental procedure in (H–L): WT mice were fed with DADA‐containing or normal water from day ‐14 until day 0, and were injected (s.c.) with MC38 cells on day 0. Anti‐PD‐1 mAb were injected (i.p.) on day 9, 12 and 15. (H–J) Tumor growth curves (H), images of tumors (I), and tumor weights (J) at 15 days after MC38 inoculation; n = 7. The experiment was repeated three times. (K,L) Quantification of CD8 + T (K), Ki67 + CD8 + T, PD‐1 + TIM‐3 + CD8 + T, TCF1 − TIM‐3 + CD8 + T, TCF1 + TIM‐3 − CD8 + T, IFN‐γ + CD8 + T, TNF‐α + CD8 + T, and GZMB + CD8 + T (L) cells from tumor; n = 7. The experiment was repeated three times. (M) Schematic experimental procedure in (N–R): WT mice were fed with DADA‐containing or normal water from day ‐14 until day 0, and were injected (s.c.) with B16‐F10 cells on day 0. Anti‐PD‐1 mAb were injected (i.p.) on day 9, 12 and 15. (N‐P) Tumor growth curves (N), images of tumors (O), and tumor weights (P) at 17 days after B16‐F10 inoculation; n = 7. The experiment was repeated three times. (Q,R) Quantification of CD8 + T (Q), Ki67 + CD8 + T, PD‐1 + TIM‐3 + CD8 + T, TCF1 − TIM‐3 + CD8 + T, TCF1 + TIM‐3 − CD8 + T, IFN‐γ + CD8 + T, and TNF‐α + CD8 + T (R)cells from tumor; n = 7. The experiment was repeated three times. Data are presented as mean ± SD and are analyzed by two‐way ANOVA (B, H, and N), unpaired t test (D–F), and one‐way ANOVA (J–L, and P‐R); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Journal: Advanced Science

Article Title: DADA Enhances CD8 + T Cell Stemness to Improve Anti‐Tumor Immunity and Immunotherapy Efficacy

doi: 10.1002/advs.202519765

Figure Lengend Snippet: DADA improves the efficacy of ACT and ICB immunotherapies. (A) Schematic experimental procedure in (B–F): CTV‐labeled OT‐1 cells, which had undergone an initial activation with OVA peptide for 2 days and subsequent stimulation with anti‐CD3/CD28 mAbs and IL‐2 in the presence of DADA for 4 days, were transferred into MC38‐OVA tumor‐bearing mice. (B–D) Tumor growth curves (B), images of tumors (C), and tumor weights (D) at 15 days after MC38‐OVA inoculation; n = 6. (E,F) Quantification of transferred OT‐1 CD8 + T (E), Ki67 + CD8 + T, PD‐1 + TIM‐3 + CD8 + T, TCF1 − TIM‐3 + CD8 + T, TCF1 + TIM‐3 − CD8 + T, IFN‐γ + CD8 + T, TNF‐α + CD8 + T, and GZMB + CD8 + T (F) cells from tumor; n = 6. (G) Schematic experimental procedure in (H–L): WT mice were fed with DADA‐containing or normal water from day ‐14 until day 0, and were injected (s.c.) with MC38 cells on day 0. Anti‐PD‐1 mAb were injected (i.p.) on day 9, 12 and 15. (H–J) Tumor growth curves (H), images of tumors (I), and tumor weights (J) at 15 days after MC38 inoculation; n = 7. The experiment was repeated three times. (K,L) Quantification of CD8 + T (K), Ki67 + CD8 + T, PD‐1 + TIM‐3 + CD8 + T, TCF1 − TIM‐3 + CD8 + T, TCF1 + TIM‐3 − CD8 + T, IFN‐γ + CD8 + T, TNF‐α + CD8 + T, and GZMB + CD8 + T (L) cells from tumor; n = 7. The experiment was repeated three times. (M) Schematic experimental procedure in (N–R): WT mice were fed with DADA‐containing or normal water from day ‐14 until day 0, and were injected (s.c.) with B16‐F10 cells on day 0. Anti‐PD‐1 mAb were injected (i.p.) on day 9, 12 and 15. (N‐P) Tumor growth curves (N), images of tumors (O), and tumor weights (P) at 17 days after B16‐F10 inoculation; n = 7. The experiment was repeated three times. (Q,R) Quantification of CD8 + T (Q), Ki67 + CD8 + T, PD‐1 + TIM‐3 + CD8 + T, TCF1 − TIM‐3 + CD8 + T, TCF1 + TIM‐3 − CD8 + T, IFN‐γ + CD8 + T, and TNF‐α + CD8 + T (R)cells from tumor; n = 7. The experiment was repeated three times. Data are presented as mean ± SD and are analyzed by two‐way ANOVA (B, H, and N), unpaired t test (D–F), and one‐way ANOVA (J–L, and P‐R); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Techniques: Labeling, Activation Assay, Injection

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