Journal: Nature Communications

Article Title: Fasting mimicking diet in mice delays cancer growth and reduces immunotherapy-associated cardiovascular and systemic side effects

doi: 10.1038/s41467-023-41066-3

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD4, VioBright FITC (REA604) , Miltenyi Biotec , 130-118-692.

Techniques: In Vivo

Journal: EMBO Molecular Medicine

Article Title: PD-1/LAG-3 co-signaling profiling uncovers CBL ubiquitin ligases as key immunotherapy targets

doi: 10.1038/s44321-024-00098-y

Figure Lengend Snippet: ( A ) Single-cell sequencing analysis of biopsies from non-small cell lung cancer (NSCLC) patients. Panels indicate the expression of PDCD1 , LAG3 and CBLB and CBLC analyzed from the single-cell lung cancer extended atlas (LuCA) (Salcher et al, ) repository as indicated. ( B ) Dot plot with the percentage of CD4 and CD8 T-cells that co-express PD-1 and LAG-3 after ex vivo activation, from healthy donors ( n = 8) and NSCLC patients ( n = 10). Statistical comparisons were performed by the Mann–Whitney test. Error bars correspond to ±SD ( C ) CBL-B expression by mean fluorescent intensities in CD4 and CD8 T-cells from a sample of non-responder NSCLC patients ( n = 4), activated ex vivo in the presence of the indicated treatments. Shown data from total CD4 and CD8 gated populations. Statistical comparisons were carried out by a two-way ANOVA to eliminate inter-patient variability followed by pair-wise Tukey tests. Box and whiskers with min to max values are plotted, computing the minimum, maximum, median and quartiles. The box extends from the 25th to 75th percentiles. The whiskers go down to the smallest value and up to the largest. ( D ) Same as ( C ) but for C-CBL expression. Box and whiskers with min to max values are plotted, computing the minimum, maximum, median and quartiles. The box extends from the 25th to 75th percentiles. The whiskers go down to the smallest value and up to the largest. ( E ) Percentage of proliferating CD4 T cells (left) and CD8 T cells (right) from a sample of high PD-1/LAG-3 co-expression patients before starting immunotherapy, activated ex vivo by A549-SC3 cells in the presence of the indicated antibodies. Statistical comparisons were carried out by a two-way ANOVA to eliminate inter-patient variability followed by pair-wise Tukey tests ( n = 5). Box and whiskers with min to max values are plotted, computing the minimum, maximum, median and quartiles. The box extends from the 25th to 75th percentiles. The whiskers go down to the smallest value and up to the largest. ( F ) Flow cytometry histograms of SATB1, Phospho SMAD 2/3, LCK and ZAP70 expression. Gates were established according to unstained controls in T-cells from a sample of non-responder NSCLC patients. Percentage of expression and Mean Fluorescence Intensity values are indicated. Data information: Statistical comparisons are shown in the graph as indicated in Methods. Briefly, for ( B ) statistical comparisons were performed by the Mann–Whitney test. For ( C – E ), statistical comparisons were carried out by a two-way ANOVA to eliminate inter-patient variability followed by pair-wise Tukey tests. Error bars correspond to ±SD. **, ***, ****, indicate P < 0.01, P < 0.001 and P < 0.0001 differences. .

Article Snippet: The following antibodies were used at 1:50 dilution unless otherwise stated: CD4-APC-Vio770 (clone M-T466, Miltenyi), CD3-APC (clone REA613, Milenyi Biotec), CD28-PECy7 (clone CD28.2, Biolegend), PD-1-PE (clone EH12.2H7, Biolegend), CD8-FITC (clone SDK1, Biolegend), LAG3-PE (clone 11C3C65, Biolegend), LAG3-PerCP-Cy5.5 (clone 11C3C65, Biolegend), CD4-FITC (clone REA623, Milteny), CD3-PerCP-Cy5.5 (clone T100, TONBO), CD4-PE-Vio770 (clone REA261, Milteny), CD223-PerCP-Cy5 (clone 11C3C65, Biolegend), PD-1-Pacific Blue (clone EH12.2H7, Biolegend).

Techniques: Sequencing, Expressing, Ex Vivo, Activation Assay, MANN-WHITNEY, Flow Cytometry, Fluorescence

Journal: EMBO Molecular Medicine

Article Title: PD-1/LAG-3 co-signaling profiling uncovers CBL ubiquitin ligases as key immunotherapy targets

doi: 10.1038/s44321-024-00098-y

Figure Lengend Snippet: ( A ) Schematic design of the experiment. BALB/c female mice were randomly allocated and subcutaneously injected with 2 × 10 6 Lung adenocarcinoma (Lacun3) cells per animal. 100 µg of anti-PD-1, 100 µg of anti-LAG3, 30 mg/kg of CBL-Bi and the corresponding depletion antibodies were administered intraperitoneally at days 0, 2, 6, 9, 13 and 15 as indicated in the figure. NK, CD4, and CD8 T‐cell depletions were carried out by intraperitoneal administration of 100 μg of anti‐mouse CD8a, CD4 or NK1.1 antibody. Mice were humanely sacrificed when tumor size reached ~150–200 mm 2 , or when tumor ulceration or discomfort were observed. ( B ) Kaplan–Meier survival plot of mice under the indicated treatments or depletion (percent). Statistical significance was tested with the Log-rank test. ( C ) Evolution of mean tumor size following the indicated treatments (left). Tumor volumes 9 days after treatment initiation (right). Error bars correspond to ±SEM (left) and box and whiskers with min to max values (right), computing the minimum, maximum, median and quartiles for 25th and 75th percentiles. The whiskers go down to the smallest value and up to the largest ( n = 6 mice per group). Data information: Statistical comparisons were carried out by a two-way ANOVA followed by pair-wise Tukey tests. ( D ) Tumor growth of individual mice in the indicated treatment groups ( n = 6 mice per group). Statistical comparisons are shown in the graph as indicated in Methods. Data information: Briefly, for ( B ), survival was represented by Kaplan–Meier plots and analyzed by log-rank test. For ( C ), statistical comparisons were carried out by a two-way ANOVA followed by pair-wise Tukey tests. *, **, ****, indicate P < 0.05, P < 0.01, and P < 0.0001 differences. .

Article Snippet: The following antibodies were used at 1:50 dilution unless otherwise stated: CD4-APC-Vio770 (clone M-T466, Miltenyi), CD3-APC (clone REA613, Milenyi Biotec), CD28-PECy7 (clone CD28.2, Biolegend), PD-1-PE (clone EH12.2H7, Biolegend), CD8-FITC (clone SDK1, Biolegend), LAG3-PE (clone 11C3C65, Biolegend), LAG3-PerCP-Cy5.5 (clone 11C3C65, Biolegend), CD4-FITC (clone REA623, Milteny), CD3-PerCP-Cy5.5 (clone T100, TONBO), CD4-PE-Vio770 (clone REA261, Milteny), CD223-PerCP-Cy5 (clone 11C3C65, Biolegend), PD-1-Pacific Blue (clone EH12.2H7, Biolegend).

Techniques: 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, 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: Molecular Neurodegeneration

Article Title: Gut-first Parkinson’s disease is encoded by gut dysbiome

doi: 10.1186/s13024-024-00766-0

Figure Lengend Snippet: Gut immunity remodeling in PD. A Representative immunofluorescence images of transverse mouse ileum sections stained with anti-CD11b. B Quantification of CD11b + cells per mm 2 in the ileum ( n = 5–9 mice per group). C , D Measurement of specific inflammatory cytokines by ELISA. C TNF ( n = 4–13 mice per group), ( D ) IL-6 ( n = 4–5 mice per group). E Representative immunofluorescence images of human terminal ileum sections stained with anti-CD11b. F Quantification of CD11b + cells per mm 2 in the ileum ( n = 4–5). G Representative photomicrographs images of transverse ileum sections stained with anti-CD4. H Quantification of CD4 + cells per mm 2 in the ileum ( n = 5–8 mice per group). I Representative immunofluorescence images of Th17 cells (CD4 + /IL17 + ) in transverse sections of the ileum. J Quantification of Th17 cells (CD4 + /IL17 + ) cells per mm 2 in the ileum ( n = 4–5 mice per group). K IL-17 levels (pg/mL) in the ileum ( n = 6–15 mice per group) measured by ELISA. L Representative images of human terminal ileum sections stained with anti-IL-17 and CD4. M Quantification of CD4 + /IL - 17 + cells per mm. 2 in human ileum ( n = 4–5). Data for histological analysis and IL - 17 determination were obtained from different animal cohorts. * p < 0.05, ** p < 0.01, *** p < 0.001, using one-way ANOVA with Dunnet´s test ( B , D , H and J , K ) or Kruskal–Wallis with Dunn´s test ( C ) and unpaired Student´s t - test ( F and M ). Data are expressed as mean ± SEM.. Scale bars are 50 µm. See also Tables S3-S4 and Figures S3

Article Snippet: PBMC pellet was incubated with anti-mouse CD45 PerCP (clone 30F11), anti-mouse CD3 FITC (clone REA641), anti-mouse CD4 APC (clone REA604) and anti-mouse CD8 PE (clone REA601) (1/50) (Miltenyi biotec) for 10 min at 4 oC.

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Molecular Neurodegeneration

Article Title: Gut-first Parkinson’s disease is encoded by gut dysbiome

doi: 10.1186/s13024-024-00766-0

Figure Lengend Snippet: Systemic inflammation and permeabilization of the blood–brain barrier. A Representative dot plots of CD45 + CD3 + CD4 + and CD45 + CD3 + CD8 + populations in serum samples by flow cytometry. B Quantification of the CD4/CD8 ratio ( n = 7–9 mice per group). C - E Measurement of specific inflammatory cytokines in mouse plasma by ELISA. (C) IFNγ ( n = 4–6 mice per group), ( D ) IL-6 levels ( n = 4–6 mice per group) and ( E ) IL - 17 levels ( n = 3–8 mice per group). F Representative immunohistological images of SN coronal sections stained with IgG. (G) Quantification of IgG-positive microvascular leakage per mm 2 in the SN ( n = 5 mice per group). * p < 0.05, ** p < 0.01, using one-way ANOVA with Dunnet´s test ( C - E and G ) or Kruskal–Wallis with Dunn´s test ( B ). Data are mean ± SEM. Scale bars are 50 µm and 500 µm (upper panel). See also Figure S6

Article Snippet: PBMC pellet was incubated with anti-mouse CD45 PerCP (clone 30F11), anti-mouse CD3 FITC (clone REA641), anti-mouse CD4 APC (clone REA604) and anti-mouse CD8 PE (clone REA601) (1/50) (Miltenyi biotec) for 10 min at 4 oC.

Techniques: Flow Cytometry, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Staining