Journal: Cell Reports Medicine

Article Title: TIMEPOINT, a phase 1 study combining MTL-CEBPA with pembrolizumab, supports the immunomodulatory effect of MTL-CEBPA in solid tumors

doi: 10.1016/j.xcrm.2025.102041

Figure Lengend Snippet: MTL-CEBPA and pembrolizumab combination treatment is associated with changes across tumor types, consistent with immune inflammation and activation (A) Fold change of CEBPA RNA at C1 D2 normalized to pre-treatment values (C1 D1). Bar is at median with 95% confidence error bars, each point is one patient. Significance was determined by one sample Wilcoxon test with hypothetical median value of 1. (B) Fast gene set enrichment analysis (FGSEA) analysis of differentially expressed genes between pre- and post-treatment time points across all patients, testing Nanostring pathways of the IO Pan-cancer panel. Pathways in red reached significance ( p adjust <0.05), with significance determined and adjusted for multiple testing by the fgsea algorithm. (C) Quantification of CD3 + CD8 + T cell numbers (left) and CD3 + CD8+Ki67+GZMB+ T cell numbers (right) between pre- (screening) and post treatment (C2D16) of 23 paired biopsies as determined by IHC. p values are non-adjusted and are determined by paired two-sided Wilcoxon test. (D) Quantification of CD11b+CD68 + CD64 − CD206+CD163 − cells as a proportion of total macrophages (CD11b+CD68 + cells) (left) and stromal CD11b+CD68 + CD64 − CD206+CD163 − cell numbers (right) between pre- (screening) and post-treatment (C2D16) of 23 paired biopsies as determined by IHC. p values are non-adjusted and are determined by paired two-sided Wilcoxon test. (E) Quantification of parenchymal CD11b+CD15 + CD14 − HLA-DR − LOX1+ (PMN-MDSC) cells as a proportion of total myeloid cells (CD11b+ cells) (left), between pre- (screening) and post treatment (C2D16) of 23 paired biopsies as determined by IHC. Right, parenchymal PMN-MDSC cell numbers in patients with at least 1 PMN-MDSC per cell/mm 2 ( N = 18). p values are non-adjusted and are determined by paired two-sided Wilcoxon test. The boxplots in (C), (D), and (E) show the data distribution, where the line denotes the median, the box edges show the interquartile range, and each dot is one patient. (F) Flow cytometric detection of PMN-MDSCs in the blood of TIMEPOINT patients. % change refers to change of PMN-MDSC as a proportion of total live cells (see gating strategy in Figure S2 D) compared to C1 D1. Each point is one patient, line is at mean with SD error bars. Significance was determined by Wilcoxon test compared to expected value of 100.

Article Snippet: PerCP-Vio700-conjugated CD15 Antibody, anti-human , Miltenyi Biotec , Catalog #130-113-487, clone VIMC6 , .

Techniques: Activation Assay, Paraffin-embedded Immunohistochemistry

Journal: Cell Reports Medicine

Article Title: TIMEPOINT, a phase 1 study combining MTL-CEBPA with pembrolizumab, supports the immunomodulatory effect of MTL-CEBPA in solid tumors

doi: 10.1016/j.xcrm.2025.102041

Figure Lengend Snippet: MTL-CEBPA/pembrolizumab combination treatment is associated with immunomodulatory changes in the immune-desert TME, converting to immune-inflamed (A) Fold change in CD3 + CD8 + T cells (left) and CD3 + CD8 + GZMB+ T cells (right) at C2D16 compared to screening, between patients with low or high levels of TIL at baseline using the median as threshold at screening. p value was determined by an unpaired two-sided Wilcoxon test. (B) Quantification of Immunosign, IS21 between pre- (screening) and post treatment (C2D16) between cold and hot tumors, left and right, respectively. p values are non-adjusted and were determined by paired two-sided Wilcoxon test. (C) IHC quantification of parenchymal CD3 + CD8 + Ki67+ T cells (left) and CD3 + CD8 + Ki67+GZMB+ T cells (right) between pre- (screening) and post treatment (C2D16) of 9 paired biopsies with an immune-desert TME (IS21 < 10). p value is determined by paired two-sided Wilcoxon test. (D) IHC quantification of CD11b+CD14 + CD15 − HLA-DR+ cells in the whole tumor (left) or stroma (right) between pre- (screening) and post treatment (C2D16) of 9 paired biopsies with an immune-desert TME (IS21 < 10). p value is determined by paired two-sided Wilcoxon test. The boxplots in (A)–(D) show the data distribution, where the line denotes the median, the box edges show the interquartile range, and each dot is one patient. (E) Differential gene expression calculated between pre- and post-treatment time points of 9 paired immune-desert patient tumor biopsies. p values on y axis refer to adjusted p values by DESeq2 algorithm. (F) FGSEA analysis of differentially expressed gene sets between pre- (screening) and post treatment (C2D16) of 9 paired biopsies with an immune-desert TME (IS21 < 10), using supplied Nanostring pathways of the IO Pan-cancer panel. Pathways in red reached significance ( p adjust <0.05), adjusted by fgsea algorithm.

Article Snippet: PerCP-Vio700-conjugated CD15 Antibody, anti-human , Miltenyi Biotec , Catalog #130-113-487, clone VIMC6 , .

Techniques: Gene Expression

Journal: Cell Reports Medicine

Article Title: TIMEPOINT, a phase 1 study combining MTL-CEBPA with pembrolizumab, supports the immunomodulatory effect of MTL-CEBPA in solid tumors

doi: 10.1016/j.xcrm.2025.102041

Figure Lengend Snippet: Characteristics linking to MTL-CEBPA mode of action are enriched in patients with stable disease after combination treatment (A) Fold change of CEBPA RNA at C1 D2 compared to pre-treatment, C1 D1 in patients with HCC from the OUTREACH clinical trial. Bar is at median with 95% confidence interval error bars, each point is one patient. Significance was tested with Kruskal-Wallis test with p value determined 0.1375. (B) Fold change of CEBPA RNA at C1 D2 normalized to pre-treatment, C1 D1 in iCCA patients from TIMEPOINT. Bar is at median with 95% confidence interval error bars, each point is one patient. Significance was not tested due to low patient numbers. For (A) and (B), clinical outcome refers to clinical response by RECIST or if unavailable, at C2 D22 determined by site. (C) Change in stromal CD11b+CD14 + CD15 − HLA-DR+ APC-like cells between pre- (screening) and post treatment (C2D16) calculated independently for patients with disease stabilization and progressive disease. Significance was determined by paired two-sided Wilcoxon test and p values are unadjusted. (D) Change in numbers of parenchymal CD8 T cells (CD3 + CD8 + ) between pre- (screening) and post treatment (C2D16) in patients with disease stabilization and progressive disease independently calculated. Significance was determined by paired two-sided Wilcoxon test and p values are unadjusted. (E) Change in proportion of CD11b+CD68 + CD64 − CD206+CD163 − cells as a proportion of total macrophages (CD11b+CD68 + cells) between pre- (screening) and post treatment (C2D16) in patients with disease stabilization and progressive disease independently calculated. Significance was determined by paired two-sided Wilcoxon test and p values are unadjusted. (F) Change in proportion of parenchymal CD11b+CD14 − CD15+HLA-DR-LOX1+ cells (PMN-MDSCs) as a percentage of total myeloid cells (CD11b+) between pre- (screening) and post treatment (C2D16) in patients with disease stabilization and progressive disease calculated independently. Significance was determined by paired two-sided Wilcoxon test and p values are unadjusted. The boxplots in (C)–(F) show the data distribution, where the line denotes the median, the box edges show the interquartile range, and each dot is one patient ( N = 23). Clinical outcome of PD or non-PD refers to the clinical response determined by site at C2 D22.

Article Snippet: PerCP-Vio700-conjugated CD15 Antibody, anti-human , Miltenyi Biotec , Catalog #130-113-487, clone VIMC6 , .

Techniques:

Journal: Cell Reports Medicine

Article Title: TIMEPOINT, a phase 1 study combining MTL-CEBPA with pembrolizumab, supports the immunomodulatory effect of MTL-CEBPA in solid tumors

doi: 10.1016/j.xcrm.2025.102041

Figure Lengend Snippet:

Article Snippet: PerCP-Vio700-conjugated CD15 Antibody, anti-human , Miltenyi Biotec , Catalog #130-113-487, clone VIMC6 , .

Techniques: Purification, Polymer, Derivative Assay, Clinical Proteomics, Extraction, Immunohistochemistry, Gene Expression, Inhibition, Software

Journal: Nature cancer

Article Title: Autophagy promotes growth of tumors with high mutational burden by inhibiting a T-cell immune response

doi: 10.1038/s43018-020-00110-7

Figure Lengend Snippet: a, Serum cytokine and chemokine profiling of female and male Atg7+/+ (n = 4 mice) and Atg7Δ/Δ (n = 8 mice) hosts without (w/o) tumors, showing those with significant differences among 26 cytokines and chemokines. b, Serum cytokine and chemokine profiling of female and male Atg7+/+ and Atg7Δ/Δ hosts with (w/) MB49 tumors (n = 12 tumors each) showing those with significant differences. c, CD3+, CD4+ and CD8+ cells (percentage) in high-TMB tumors (MB49) grown on male (n = 5 mice each) and female (n = 7 mice each) Atg7+/+ and Atg7Δ/Δ hosts analyzed by flow cytometry. These data are representative of three independent experiments. d,e, Representative IHC images and quantification of CD3+, CD4+ and CD8+ T cells in MB49 tumors from male (d) and female (e) Atg7+/+ and Atg7Δ/Δ hosts (n = 5 mice each). All data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.01, ****P < 0.0001 using a two-sided Student’s t-test. Source data are available for a–e.

Article Snippet: For T-cell exhaustion, the following antibodies were used: CD45 (REA737, 1:50 dilution, 130–110-802), CD4 (REA604, 1:50 dilution, 130–118-852), CD8 (REA601, 1:10 dilution, 130–109-247; Miltenyi Biotec); CD3 (500A2, 1:500 dilution 152315), PD1 (29F.1A12, 1:300 dilution, 135213), TIM3 (RMT3–23, 1:100 dilution, 119721) and LAG3 (C9B7W, 1:50 dilution, 125219; BioLegend).

Techniques: Flow Cytometry

Journal: Nature cancer

Article Title: Autophagy promotes growth of tumors with high mutational burden by inhibiting a T-cell immune response

doi: 10.1038/s43018-020-00110-7

Figure Lengend Snippet: a, Serum and b, liver cytokine and chemokine profiling of female and male hosts bearing MB49 tumors with or without liver-specific deletion of Atg7 (n = 6 mice each), showing those with significant differences among 26. c, experimental design to induce liver-specific Fip200 deletion (liver Fip200Δ/Δ) and wild-type controls (liver Fip200+/+). Fip200flox/flox or C57BL/6J mice were injected with AAV-TBG-iCre and MB49 cells were then injected subcutaneously and tumor growth was monitored over three weeks. d, Cropped Western blotting showing expression of Fip200 in livers, kidneys and brains (n = 3 each) from one experiment. e, Comparison of MB49 tumor growth on male liver Fip200+/+(n = 8 tumors) and liver Fip200Δ/Δ (n = 6 tumors) hosts. f, experimental design to induce conditional Fip200 deletion (Fip200Δ/Δ) and wild-type (Fip200+/+) controls. C57BL/6J mice were injected with lentiCRISPR scramble or Fip200 to delete Fip200 and were then injected subcutaneously with tumor cells. Tumor growth was monitored over three weeks. g, Representative surveyor assay for Fip200 gRNA screening using liver gDNA from Fip200+/+ and Fip200Δ/Δ hosts (n = 10 mice each) from one experiment. h, Representative IHC images of FIP200+ and p62+ cells in liver from Fip200+/+ and Fip200Δ/Δ hosts (n = 10 mice each). i, j, Comparison of MB49 tumor growth on female Fip200+/+ and Fip200Δ/Δ hosts with or without CD4/8 depletion (n = 10 tumors each). Tumor pictures (i), and tumor weight and ratio (antibody treated/control) (j) from the experimental endpoint. Data are mean +/− s.e.m. *P < 0.05, **P < 0.01, ***P<0.001, ****P < 0.0001 using two-sided Student’s t-test. Source data available for a, b, e, j.

Article Snippet: For T-cell exhaustion, the following antibodies were used: CD45 (REA737, 1:50 dilution, 130–110-802), CD4 (REA604, 1:50 dilution, 130–118-852), CD8 (REA601, 1:10 dilution, 130–109-247; Miltenyi Biotec); CD3 (500A2, 1:500 dilution 152315), PD1 (29F.1A12, 1:300 dilution, 135213), TIM3 (RMT3–23, 1:100 dilution, 119721) and LAG3 (C9B7W, 1:50 dilution, 125219; BioLegend).

Techniques: Injection, Western Blot, Expressing, Comparison, Control

Journal: Nature cancer

Article Title: Autophagy promotes growth of tumors with high mutational burden by inhibiting a T-cell immune response

doi: 10.1038/s43018-020-00110-7

Figure Lengend Snippet: a, experimental design to induce liver hepatocyte-specific Atg7 deletion (liver Atg7Δ/Δ) and wild-type controls (liver Atg7+/+). Atg7flox/flox mice were either injected with adeno-associated virus (AAV)–thyroxine binding globulin (TBG) promoter–GFP or AAV–TBG–iCre and MB49 cells were then injected subcutaneously and tumor growth was monitored over 3 weeks. b,c, Comparison of MB49 tumor growth on male (b) liver Atg7Δ/Δ and liver Atg7+/+ (n = 14 tumors each) and female (c) liver Atg7Δ/Δ and liver Atg7+/+ (n = 10 and 14 tumors, respectively). Tumor pictures and weight from the experimental end point. d, Representative IHC images and quantification of CD3+, CD4+ and CD8+ T cells in MB49 tumors from male liver Atg7+/+ and liver Atg7Δ/Δ hosts (n = 5 mice each). e, experimental design to induce conditional liver-specific liver Atg7Δ/Δ and liver Atg7+/+ controls with loss of Ifnγ. The Ifnγ−/−;Atg7flox/flox were either injected with AAV–TBG–GFP or AAV–TBG–iCre and MB49 cells were then injected subcutaneously and tumor growth was monitored over 3 weeks. f–i, Comparison of MB49 tumor growth on male (f,g) liver Atg7+/+ (n = 8 tumors), Ifnγ−/−;liver Atg7+/+ (n = 10 tumors), liver Atg7Δ/Δ (n = 8 tumors) and liver Ifnγ−/−;Atg7Δ/Δ (n = 10 tumors) and female (h,i) liver Atg7+/+ (n = 12 tumors), Ifnγ−/−;liver Atg7+/+ (n = 12 tumors), liver Atg7Δ/Δ (n = 12 tumors) and liver Ifnγ−/−;Atg7Δ/Δ (n = 10 tumors). Tumor pictures (f,h) and tumor weight and ratio (antibody treated/control) (g,i) from the experimental end point. All data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using a two-sided Student’s t-test. Source data are available for b–d,g,i.

Article Snippet: For T-cell exhaustion, the following antibodies were used: CD45 (REA737, 1:50 dilution, 130–110-802), CD4 (REA604, 1:50 dilution, 130–118-852), CD8 (REA601, 1:10 dilution, 130–109-247; Miltenyi Biotec); CD3 (500A2, 1:500 dilution 152315), PD1 (29F.1A12, 1:300 dilution, 135213), TIM3 (RMT3–23, 1:100 dilution, 119721) and LAG3 (C9B7W, 1:50 dilution, 125219; BioLegend).

Techniques: Injection, Virus, Binding Assay, Comparison, Control

Journal: Nature cancer

Article Title: Autophagy promotes growth of tumors with high mutational burden by inhibiting a T-cell immune response

doi: 10.1038/s43018-020-00110-7

Figure Lengend Snippet: a, experimental design to induce conditional whole-body Atg7 deletion (Atg7Δ/Δ) and wild-type (Atg7+/+) controls with loss of Ifnγ. Ifnγ−/−;Ubc-CreERT2/+;Atg7+/+ and Ifnγ−/−;Ubc-CreERT2/+;Atg7flox/flox mice were injected with TAM to delete Atg7 and were then injected subcutaneously with tumor cells. Tumor growth was monitored over 3 weeks. b–e, Comparison of MB49 tumor growth on male (b,c) Atg7+/+, Ifnγ−/−;Atg7+/+, Atg7Δ/Δ and Ifnγ−/−;Atg7Δ/Δ (n = 12 tumors each) and female (d,e) Atg7+/+ (n = 10 tumors), Ifnγ−/−;Atg7+/+ (n = 8 tumors), Atg7Δ/Δ (n = 6 tumors) and Ifnγ−/−;Atg7Δ/Δ (n = 10 tumors). Tumor pictures (b,d) and tumor weight and ratio (antibody treated/control) (c,e) from the experimental end point. f, Percentage of CD4+PD1+, CD4+TIM3+ and CD4+LAG3+ cells in tumors in male Atg7+/+, Ifnγ−/−;Atg7+/+, Atg7Δ/Δ and Ifnγ−/−;Atg7Δ/Δ hosts (n = 3 tumors each) analyzed by flow cytometry. g, Percentage of CD8+PD1+, CD8+TIM3+ and CD8+LAG3+ cells in tumors in male Atg7+/+, Ifnγ−/−;Atg7+/+, Atg7Δ/Δ and Ifnγ−/−;Atg7Δ/Δ hosts (n = 3 tumors each) analyzed by flow cytometry. h, NanoString expression analysis of genes involved in the MHC and antigen presentation pathway in MB49 tumors from female Atg7+/+ and Atg7Δ/Δ hosts with or without anti-CD4/8 (n = 2 tumors each). Red genes indicate MHC pathway. Red: high expression level; blue: low expression level. i, Comparison of MB49 shC and shB2m tumor growth on female Atg7+/+ (n = 10–12 tumors) and Atg7Δ/Δ (n = 10 tumors) hosts. Tumor pictures and tumor weight from the experimental end point. All data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.01, ****P < 0.0001 using a two-sided Student’s t-test. Source data are available for c,e–g,i.

Article Snippet: For T-cell exhaustion, the following antibodies were used: CD45 (REA737, 1:50 dilution, 130–110-802), CD4 (REA604, 1:50 dilution, 130–118-852), CD8 (REA601, 1:10 dilution, 130–109-247; Miltenyi Biotec); CD3 (500A2, 1:500 dilution 152315), PD1 (29F.1A12, 1:300 dilution, 135213), TIM3 (RMT3–23, 1:100 dilution, 119721) and LAG3 (C9B7W, 1:50 dilution, 125219; BioLegend).

Techniques: Injection, Comparison, Control, Flow Cytometry, Expressing, Immunopeptidomics

Journal: Nature cancer

Article Title: Autophagy promotes growth of tumors with high mutational burden by inhibiting a T-cell immune response

doi: 10.1038/s43018-020-00110-7

Figure Lengend Snippet: a, experimental design to induce host mice with conditional whole-body Atg7 deletion (Atg7Δ/Δ) and the wild-type control (Atg7+/+), without and with T-cell depletion. Ubc-CreERT2/+;Atg7+/+ and Ubc-CreERT2/+;Atg7flox/flox mice were injected with tamoxifen (TAM) to delete Atg7 and were then injected subcutaneously with tumor cells and intraperitoneally with anti-CD4/8. Tumor growth was monitored over 3 weeks, where all tumors establish similarly with antitumor effects becoming apparent beyond 2 weeks. b,c, Comparison of YUMM1.1 tumor growth on female Atg7+/+ (n = 12 tumors), Atg7+/+ + anti-CD4/8 (n = 12 tumors), Atg7Δ/Δ (n = 14 tumors) and Atg7Δ/Δ + anti-CD4/8 (n = 12 tumors). Tumor pictures (b), tumor weight and ratio (antibody treated/control) (c) from the experimental end point. d–g, Comparison of MB49 tumor growth on female (d,e) Atg7+/+ (n = 7 tumors), Atg7+/+ + anti-CD4/8 (n = 7 tumors), Atg7Δ/Δ (n = 7 tumors) and Atg7Δ/Δ + anti-CD4/8 (n = 10 tumors) and male (f,g) Atg7+/+ (n = 6 tumors), Atg7+/+ + anti-CD4/8 (n = 8 tumors), Atg7Δ/Δ (n = 8 tumors) and Atg7Δ/Δ + anti-CD4/8 (n = 6 tumors) hosts. Tumor pictures (d,f), tumor weight and ratio (antibody treated/control) (e,g) from the experimental end point. All data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.01, ****P < 0.0001 using a two-sided Student’s t-test. Source data are available for c,e,g.

Article Snippet: For T-cell exhaustion, the following antibodies were used: CD45 (REA737, 1:50 dilution, 130–110-802), CD4 (REA604, 1:50 dilution, 130–118-852), CD8 (REA601, 1:10 dilution, 130–109-247; Miltenyi Biotec); CD3 (500A2, 1:500 dilution 152315), PD1 (29F.1A12, 1:300 dilution, 135213), TIM3 (RMT3–23, 1:100 dilution, 119721) and LAG3 (C9B7W, 1:50 dilution, 125219; BioLegend).

Techniques: Control, Injection, Comparison

Journal: Nature cancer

Article Title: Autophagy promotes growth of tumors with high mutational burden by inhibiting a T-cell immune response

doi: 10.1038/s43018-020-00110-7

Figure Lengend Snippet: a, CD3+, CD4+, CD8+ T cells (percentage) in high TMB tumors (MB49) grown on female Atg7+/+ and Atg7Δ/Δ (n = 5 mice each) hosts following αCD4/8, analyzed by flow cytometry. b, Representative IHC images and quantification of CD3+, CD4+ and CD8+ T cells in YUMM1.3 tumors from male Atg7+/+ and Atg7Δ/Δ hosts (n = 5 mice each). c, d, e, f, Comparison of YUMM1.3 tumor growth on male (c, d) Atg7+/+, Atg7+/+ + αCD4/8, Atg7Δ/Δ and Atg7Δ/Δ + αCD4/8 (n = 10 tumors each) and female (e, f) Atg7+/+(n = 6 tumors), Atg7+/+ + αCD4/8 (n = 12 tumors), Atg7Δ/Δ (n = 12 tumors) and Atg7Δ/Δ + αCD4/8 (n = 12 tumors). Tumor pictures (c, e), and tumor weight and ratio (antibody treated/control) (d, f) from the experimental endpoint. g, h, Comparison of MB49 tumor growth on female Atg7+/+(n = 14 tumors), Atg7+/+ + αCD4 (n = 12 tumors), Atg7+/+ + αCD8 (n = 12 tumors), Atg7+/+ + αCD4/8 (n = 8 tumors), Atg7Δ/Δ (n = 12 tumors), Atg7Δ/Δ + αCD4 (n = 14 tumors), Atg7Δ/Δ + αCD8 (n = 14 tumors) and Atg7Δ/Δ + αCD4/8 (n = 12 tumors). Tumor pictures (g), and tumor weight and ratio (antibody treated/control) (h) from the experimental endpoint. All data are mean +/− s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001 using two-sided Student’s t-test. Source data available for a, b, d, f, h.

Article Snippet: For T-cell exhaustion, the following antibodies were used: CD45 (REA737, 1:50 dilution, 130–110-802), CD4 (REA604, 1:50 dilution, 130–118-852), CD8 (REA601, 1:10 dilution, 130–109-247; Miltenyi Biotec); CD3 (500A2, 1:500 dilution 152315), PD1 (29F.1A12, 1:300 dilution, 135213), TIM3 (RMT3–23, 1:100 dilution, 119721) and LAG3 (C9B7W, 1:50 dilution, 125219; BioLegend).

Techniques: Flow Cytometry, Comparison, Control

Journal: Nature cancer

Article Title: Autophagy promotes growth of tumors with high mutational burden by inhibiting a T-cell immune response

doi: 10.1038/s43018-020-00110-7

Figure Lengend Snippet: a, experimental design to induce conditional whole-body Atg5 deletion (Atg5Δ/Δ), and wild-type (Atg5+/+) controls without and with T cell depletion. Ubc-CreERT2/+;Atg5+/+ and Ubc-CreERT2/+;Atg5flox/flox mice were injected with TAM to delete Atg5 and were then injected subcutaneously with tumor cells and intraperitoneally with αCD4/8. Tumor growth was monitored over three weeks. b, c, d, e, Comparison of MB49 tumor growth on male (b, c) Atg5+/+(n = 8 tumors), Atg5+/+ + αCD4/8 (n = 6 tumors), Atg5Δ/Δ (n = 8 tumors) and Atg5Δ/Δ + αCD4/8 (n = 8 tumors) and female (d, e) Atg5+/+(n = 12 tumors), Atg5+/+ + αCD4/8 (n = 10 tumors), Atg5Δ/Δ (n = 12 tumors) and Atg5Δ/Δ + αCD4/8 (n = 10 tumors). Tumor pictures (b, d), and tumor weight and ratio (antibody treated/control) (c, e) from the experimental endpoint. f, Representative IHC images and quantification of CD3+, CD4+ and CD8+ T cells in MB49 tumors from male Atg5+/+ and Atg5Δ/Δ hosts (n = 5 mice each). g, h, Comparison of UV YUMM1.1–9 tumor growth on male (g) Atg7+/+ (n = 10 tumors) and Atg7Δ/Δ (n = 8 tumors) and female (h) Atg7+/+ (n = 16 tumors) and Atg7Δ/Δ (n = 12 tumors) hosts. Tumor pictures and tumor weight from the experimental endpoint. All data are mean +/− s.e.m. *P < 0.05, ***P < 0.001, ****P < 0.0001 using two-sided Student’s t-test. Source data available for c, e, f, g, h.

Article Snippet: For T-cell exhaustion, the following antibodies were used: CD45 (REA737, 1:50 dilution, 130–110-802), CD4 (REA604, 1:50 dilution, 130–118-852), CD8 (REA601, 1:10 dilution, 130–109-247; Miltenyi Biotec); CD3 (500A2, 1:500 dilution 152315), PD1 (29F.1A12, 1:300 dilution, 135213), TIM3 (RMT3–23, 1:100 dilution, 119721) and LAG3 (C9B7W, 1:50 dilution, 125219; BioLegend).

Techniques: Injection, Comparison, Control

Journal: Nature cancer

Article Title: Autophagy promotes growth of tumors with high mutational burden by inhibiting a T-cell immune response

doi: 10.1038/s43018-020-00110-7

Figure Lengend Snippet: a–d, Comparison of UV YUMM1.1–9 tumor growth on male (a,b) Atg7+/+ (n = 14 tumors), Atg7+/+ + anti-CD4/8 (n = 14 tumors), Atg7Δ/Δ (n = 12 tumors) and Atg7Δ/Δ + anti-CD4/8 (n = 12 tumors) and female (c,d) Atg7+/+ (n = 12 tumors), Atg7+/+ + anti-CD4/8 (n = 12 tumors), Atg7Δ/Δ (n = 12 tumors) and Atg7Δ/Δ + anti-CD4/8 (n = 8 tumors). Tumor pictures (a,c), tumor weight and ratio (antibody treated/control) (b,d) from the experimental end point. e,f, NanoString expression analysis of genes involved in signature immune pathways (e) or immune cell type (f) on female Atg7+/+ and Atg7Δ/Δ hosts with or without anti-CD4/8 (n = 2 mice each). Red, high expression level; blue, low expression level. TNF, tumor necrosis factor; TLR, Toll-like receptor; NK, natural killer; MHC, major histocompatibility complex; TH1, type 1 helper T cells. g, experimental design to induce conditional whole-body Atg7 deletion (Atg7Δ/Δ) and wild-type (Atg7+/+) controls without and with Treg cell depletion. Ubc-CreERT2/+;Atg7+/+ and Ubc-CreERT2/+;Atg7flox/flox mice were injected with TAM to delete Atg7 and were then injected subcutaneously with tumor cells and intraperitoneally with anti-CD25. Tumor growth was monitored over 3 weeks. h–k, Comparison of MB49 tumor growth on male (h,i) Atg7+/+ (n = 12 tumors), Atg7+/+ + anti-CD25 (n = 10 tumors), Atg7Δ/Δ (n = 14 tumors) and Atg7Δ/Δ + anti-CD25 (n = 12 tumors) and female (j,k) Atg7+/+, Atg7+/+ + anti-CD25, Atg7Δ/Δ and Atg7Δ/Δ + anti-CD25 (n = 10 tumors each). Tumor pictures (h,j) and tumor weight (i,k) from the experimental end point. All data are mean ± s.e.m. *P < 0.05, ***P < 0.001, ****P < 0.0001 using a two-sided Student’s t-test. Source data are available for b,d,i,k.

Article Snippet: For T-cell exhaustion, the following antibodies were used: CD45 (REA737, 1:50 dilution, 130–110-802), CD4 (REA604, 1:50 dilution, 130–118-852), CD8 (REA601, 1:10 dilution, 130–109-247; Miltenyi Biotec); CD3 (500A2, 1:500 dilution 152315), PD1 (29F.1A12, 1:300 dilution, 135213), TIM3 (RMT3–23, 1:100 dilution, 119721) and LAG3 (C9B7W, 1:50 dilution, 125219; BioLegend).

Techniques: Comparison, Control, Expressing, Immunopeptidomics, Injection

Journal: Nature cancer

Article Title: Autophagy promotes growth of tumors with high mutational burden by inhibiting a T-cell immune response

doi: 10.1038/s43018-020-00110-7

Figure Lengend Snippet: a, Percentage of CD4+PD1+, CD4+TIM3+ and CD4+LAG3+ cells in tumors in male Atg7+/+ and Atg7Δ/Δ hosts (n = 3 tumors each) analyzed by flow cytometry. These data are representative of three independent experiments. MFI: mean fluorescent intensity. b, Percentage of CD8+PD1+, CD8+TIM3+ and CD8+LAG3+ cells in tumors in male Atg7+/+ and Atg7Δ/Δ hosts (n = 3 tumors each) analyzed by flow cytometry. c, NanoString expression analysis of genes involved in the IFN pathway in MB49 tumors from female Atg7+/+ and Atg7Δ/Δ hosts with or without anti-CD4/8 (n = 2 mice each). Blue genes indicate IFN-αβ, red genes indicate IFN-γ pathways. Red: high expression level; blue: low expression level. d, experimental design to induce conditional whole-body Atg7 deletion (Atg7Δ/Δ) and wild-type (Atg7+/+) controls with loss of Sting. Stinggt/gt;Ubc-CreERT2/+;Atg7+/+ and Stinggt/gt;Ubc-CreERT2/+;Atg7flox/flox mice were injected with TAM to delete Atg7 and were then injected subcutaneously with tumor cells. Tumor growth was monitored over 3 weeks. e–h, Comparison of MB49 tumor growth on male (e,f) Atg7+/+(n = 10 tumors), Stinggt/gt;Atg7+/+(n = 8 tumors), Atg7Δ/Δ (n = 8 tumors) and Stinggt/gt;Atg7Δ/Δ (n = 10 tumors) and female (g,h) Atg7+/+ (n = 10 tumors), Stinggt/gt;Atg7+/+ (n = 8 tumors), Atg7Δ/Δ (n = 6 tumors) and Stinggt/gt;Atg7Δ/Δ (n = 8 tumors). Tumor pictures (e,g) and tumor weight and ratio (antibody treated/control) (f,h) from the experimental end point. All data are mean ± s.e.m. *P < 0.05, ****P < 0.0001 using a two-sided Student’s t-test. Source data are available for a,b,f,h.

Article Snippet: For T-cell exhaustion, the following antibodies were used: CD45 (REA737, 1:50 dilution, 130–110-802), CD4 (REA604, 1:50 dilution, 130–118-852), CD8 (REA601, 1:10 dilution, 130–109-247; Miltenyi Biotec); CD3 (500A2, 1:500 dilution 152315), PD1 (29F.1A12, 1:300 dilution, 135213), TIM3 (RMT3–23, 1:100 dilution, 119721) and LAG3 (C9B7W, 1:50 dilution, 125219; BioLegend).

Techniques: Flow Cytometry, Expressing, Injection, Comparison, Control

Journal: Nature cancer

Article Title: Autophagy promotes growth of tumors with high mutational burden by inhibiting a T-cell immune response

doi: 10.1038/s43018-020-00110-7

Figure Lengend Snippet: a, Overall NanoString gene expression analysis (750 genes) from tumors on female Atg7+/+ and Atg7Δ/Δ hosts with or without αCD4/8 (n = 2 tumors each). Red: high, blue: low expression level. b, Representative IHC images and quantification of FOXP3+ cells in MB49 tumors from female Atg7+/+ and Atg7Δ/Δ hosts (n = 5 tumors each). c, FOXP3+ Treg cells, CD4+ and CD8+ T cells (percentage) in high TMB tumors (MB49) grown on female (n = 3 tumors) Atg7+/+ and Atg7Δ/Δ hosts following αCD25, analyzed by flow cytometry. d, Representative IHC images and quantification of FOXP3+ cells in MB49 tumors from female Atg7+/+ and Atg7Δ/Δ hosts following αCD25 (n = 3 tumors each). e, Survival analysis of Atg7+/+ (n = 19 mice), Atg7Δ/Δ (n = 19 mice), Stinggt/gt;Atg7+/+ (n = 15 mice) and Stinggt/gt;Atg7Δ/Δ (n = 13 mice) mice. f, scRNA-seq UMAP projection of cell cluster from tumors from female Atg7+/+ and Atg7Δ/Δ hosts (n = 3 tumors each). All data are mean +/− s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using two-sided Student’s t-test. Source data available for b, c, d.

Article Snippet: For T-cell exhaustion, the following antibodies were used: CD45 (REA737, 1:50 dilution, 130–110-802), CD4 (REA604, 1:50 dilution, 130–118-852), CD8 (REA601, 1:10 dilution, 130–109-247; Miltenyi Biotec); CD3 (500A2, 1:500 dilution 152315), PD1 (29F.1A12, 1:300 dilution, 135213), TIM3 (RMT3–23, 1:100 dilution, 119721) and LAG3 (C9B7W, 1:50 dilution, 125219; BioLegend).

Techniques: Gene Expression, Expressing, Flow Cytometry

Journal: Journal of Inflammation (London, England)

Article Title: Tissue-plasminogen activator effects on the phenotype of splenic myeloid cells in acute inflammation

doi: 10.1186/s12950-024-00375-0

Figure Lengend Snippet: References of flow cytometry antibodies used for immunophenotyping

Article Snippet: CD71 , REA627 , PerCP-Vio700 , 130–109-577 , human IgG1/REA293 , Miltenyi Biotec.

Techniques: Flow Cytometry, Clone Assay