mouse spleen lymphocyte isolation solution kit  (Beijing Solarbio Science)

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

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

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.

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, Expressing, Control, Flow Cytometry

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.

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

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.

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: Membrane, Staining, Expressing

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.

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: Labeling, Activation Assay, Injection