easysep mouse naive cd8+ t cell negative selection kit  (STEMCELL Technologies Inc)

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

Journal: Journal for immunotherapy of cancer

Article Title: Oxaliplatin, ATR inhibitor and anti-PD-1 antibody combination therapy controls colon carcinoma growth, induces local and systemic changes in the immune compartment, and protects against tumor rechallenge in mice.

doi: 10.1136/jitc-2024-010791

Figure Lengend Snippet: Figure 3 The Vox+anti-PD-1 antibody combination reduces neutrophil, CD206+ macrophages and regulatory T cell infiltration in PM nodules. (A) Number per mg of tumor of the indicated immune cell populations measured by quantitative flow cytometry in GFP-Luc-CT26 PM samples harvested from untreated controls (Ctr n=7) and mice treated with the anti-PD-1 antibody (n=8) alone and with Vox (n=9) for 1 week. (B) Histograms showing the percentage among all tumor- infiltrated CD45+ immune cells of the indicated immune cell populations from the same experiment described in A. (C) Representative images of CD8 (purple) and FOXP3 (brown) double staining in GFP-Luc-CT26 PM samples from mice treated with the anti-PD-1 alone (n=15) and with Vox (n=13). (D) Histograms showing the ratio of CD8+ T cells and FOXP3+ cells over the total cell number per tumor from the IHC staining images. *p=0.05; **p=0.01; ***p=0.001; ****p=0.0001; ns, not significant (one-way analysis of variance for A and B and Student’s t-test for D). aPD-1, anti-programmed-cell death receptor-1; Ctr, control; IHC, immunohistochemistry; PM, peritoneal metastases; Vox, VE-822+oxaliplatin.

Article Snippet: Briefly, mouse spleen cell suspensions were enriched in CD8+ T cells using CD8+ T- cell negative selection from Miltenyi (130- 104- 075) according to provider indications with the MultiMacs 24 cell separator.

Techniques: Flow Cytometry, Double Staining, Immunohistochemistry, Control

Journal: Journal for immunotherapy of cancer

Article Title: Oxaliplatin, ATR inhibitor and anti-PD-1 antibody combination therapy controls colon carcinoma growth, induces local and systemic changes in the immune compartment, and protects against tumor rechallenge in mice.

doi: 10.1136/jitc-2024-010791

Figure Lengend Snippet: Figure 6 Vox treatment leads to the emergence of Ly- 6C+PD-1+ CD8+ T cells in blood and spleen. (A and B) Number per mL of blood (A) and percentage relative to immune cells (B) of the indicated CD8+ T-cell populations from GFP-Luc-CT26 PM mice untreated (Ctr n=5), or treated with Vox (n=6), anti-PD-1 antibody (n=6) and Vox+anti- PD-1 antibody (n=5). (C) Number per spleen of CD8+ T cells with the indicated phenotypes from GFP-Luc-CT26 PM mice untreated (Ctr n=7), or treated with the anti-PD-1 antibody (n=16) or with Vox+anti-PD-1 antibody (n=10). (D) Histogram showing the percentage of Ki67+ cells among the indicated CD8+ T-cell subpopulations from GFP-Luc-CT26 PM mice untreated (Ctr n=6) or treated with the anti-PD-1 antibody (n=6) or Vox+anti-PD-1 antibody (n=5). (E) Relative messenger RNA expression of the indicated genes in the indicated CD8+ T-cell subpopulations purified from the spleen of Vox-treated GFP-Luc-CT26 PM mice (n=5). (F) CXCR3, Eomes and CD62L expression in CD8+ T cells (flow cytometry analysis) from the spleen of Vox-treated GFP-Luc-CT26 PM mice. *p=0.05; **p=0.01; ***p=0.001; ****p=0.0001; ns, not significant (one-way analysis of variance). aPD-1, anti- programmed-cell death receptor-1; Ctr, control; GFP, green fluorescent protein; PM, peritoneal metastases; Vox, VE- 822+oxaliplatin.

Article Snippet: Briefly, mouse spleen cell suspensions were enriched in CD8+ T cells using CD8+ T- cell negative selection from Miltenyi (130- 104- 075) according to provider indications with the MultiMacs 24 cell separator.

Techniques: RNA Expression, Purification, Expressing, Flow Cytometry, Control

Journal: Journal for immunotherapy of cancer

Article Title: Oxaliplatin, ATR inhibitor and anti-PD-1 antibody combination therapy controls colon carcinoma growth, induces local and systemic changes in the immune compartment, and protects against tumor rechallenge in mice.

doi: 10.1136/jitc-2024-010791

Figure Lengend Snippet: Figure 7 The Ly-6C+PD-1+ CD8+ T-cell population contains a strong proportion of tumor antigen-specific T cells. (A) Histogram showing the percentage of BCL6+CD122+ cells among PD-1+Ly-6C+ T cells (black) and PD-1+Ly-6C− CD8+ T cells (white) from the spleen of GFP-Luc-CT26 PM mice treated with the anti-PD-1 antibody (n=8) or Vox+anti-PD-1 antibody (n=11). (B) Plots showing the gating strategy to identify GFP-specific T-cell receptor-positive cells after Dextramer staining and Ly-6C+ and PD-1+ cells among them in the spleen of Vox+anti-PD-1 antibody-treated GFP-Luc-CT26 PM mice. (C) Pie chart (top) and histogram (bottom) showing the distribution of dextran-positive (right) and dextran-negative (left) cells in the different CD8+ T-cell subpopulations from the spleen of Vox+anti-PD-1 antibody-treated GFP-Luc-CT26 PM mice (n=5). *p=0.05; **p=0.01 (one-way analysis of variance). PD-1, anti-programmed-cell death receptor-1; GFP, green fluorescent protein; PM, peritoneal metastases; Vox, VE-822+oxaliplatin.

Article Snippet: Briefly, mouse spleen cell suspensions were enriched in CD8+ T cells using CD8+ T- cell negative selection from Miltenyi (130- 104- 075) according to provider indications with the MultiMacs 24 cell separator.

Techniques: Staining