Journal: Cell Reports Medicine

Article Title: Cathepsin-D-mediated MHC class I degradation contributes to immune evasion in colorectal cancer

doi: 10.1016/j.xcrm.2025.102534

Figure Lengend Snippet: Elevated CTSD expression correlates with reduced immune cell infiltration in CRC (A) Venn diagram shows overlapping of upregulated genes from RNA-seq data comparing MSS versus MSI-H and focus on upregulated genes associated with immunotherapy response in MSS CRC. Based on log 2FC > 1 and p < 0.05. (B) Representative immunohistochemical staining images of CTSD in CRC tissues. Scale bars, 100 μm. (C) Histogram showing the overall response rate in patients with low and high CTSD expression levels in the refractory MSS CRC cohort. (D) Representative computed tomography (CT) images of patients with lung metastasis of CRC before and after anti-PD-1 mAb (PD-1) treatment. Red circles indicate the location of lung metastases. (E) Kaplan-Meier analysis of PFS comparing patients with low and high CTSD expression in the refractory MSS CRC cohort. (F and G) CTSD expression in CRC tissues stratified by invasion (F) and stage (G). (H) Overall survival curves were generated based on the protein levels of CTSD in CRC. (I–L) Multiplex immunofluorescence analysis of immune cell infiltration intensity between low and high CTSD expression groups. Tumor samples were stained with specific antibodies to CTSD in the red fluorescence channel, CD8 in celeste, CD163 in green, CD4 in purple, and cell nuclei in blue by DAPI staining (I). Scale bars, 50 μm. Statistics of CD8 (J), CD4 (K), and CD163 (L) cells based on CTSD expression. (M and N) Kaplan-Meier survival curves for OS and PFS analysis of patients based on relative expression of CTSD in the Kaplan-Meier plotter immunotherapy cohort showing pan-cancer samples with anti-PD-1 (pembrolizumab) or anti-CTLA4 (ipilimumab) treatment. Data are means ± SEM. p values were determined by log rank test (E, H, M, and N), unpaired Student’s t test (F, G, and J–L), and Fisher’s exact test (C).

Article Snippet: InVivoMAb anti-mouse CD4 , Bio X Cell , Cat# BE0003-1; RRID: AB_1107636.

Techniques: Expressing, RNA Sequencing, Immunohistochemical staining, Staining, Computed Tomography, Generated, Multiplex Assay, Immunofluorescence, Fluorescence

Journal: Cell Reports Medicine

Article Title: Cathepsin-D-mediated MHC class I degradation contributes to immune evasion in colorectal cancer

doi: 10.1016/j.xcrm.2025.102534

Figure Lengend Snippet: Suppression of CTSD boosts cytotoxic T cell activation and enhances anti-tumor immunotherapeutic responses (A) Primary tumors from mice were harvested for flow cytometry to determine the percentages of GZMB + and IFN-γ + cells among CD8 + T cells and T-bet + and Foxp3 + among CD4 + T cell ( n = 6). (B and C) Tumor volume, representative tumor images, and tumor weight from CTSD knockdown CT26 tumor cells in BALB/c mice depleted of CD8 + /CD4 + T cells. s.c represents subcutaneous injection. (D) Immunofluorescent staining of CD8 in CT26 tumor and quantification of CD8 signals ( n = 6). Scale bars, 50 μm. (E) Primary tumors from mice were harvested for flow cytometry to determine the percentages of Ki67 + cells among CD8 + T cells ( n = 6). (F and G) BALB/c mice bearing subcutaneous CT26 tumor cell expressing shCTSD, received IgG, or antiPD-1 antibody therapy. Tumor volumes (F) and Kaplan-Meier survival curves (G) on the indicated days after inoculation with CT26 cells ( n = 6). s.c represents subcutaneous injection. (H) Immunofluorescent staining of CD8 in CT26 tumors from (F); quantification of CD8 signals ( n = 6). Scale bars, 50 μm. (I) Primary tumors from mice were harvested for flow cytometry to determine the percentages of GZMB + and IFN-γ + cells among CD8 + T cells ( n = 6). Data are means ± SEM. p values were calculated by two-tailed Student’s t test (A, C, D, and E), one-way ANOVA (H and I), two-way ANOVA for tumor volumes (F), and log rank test for Kaplan-Meier survival curves (G). n.s represents no significance.

Article Snippet: InVivoMAb anti-mouse CD4 , Bio X Cell , Cat# BE0003-1; RRID: AB_1107636.

Techniques: Activation Assay, Flow Cytometry, Knockdown, Injection, Staining, Expressing, Two Tailed Test

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 3. CmAb-(IL10)2-Mediated Antitumor Effects Depend on Host Immunity (A and B) Tumor growth in C57BL/6J (A) or Rag1/ (B) mice (n = 5) bearing B16-cEGFR tumors treated by intratumoral (i.t.) injection of Cetuximab, CmAb-(IL10)2, or control IgG (indicated by arrows). (C) Quantification of OVA tetramer-positive (OVA-specific) CD8+ T cells in tumor tissues collected from B16-cEGFR-OVA tumor-bearing C57BL/6J mice (n = 4–5) treated twice by i.t. injection with control IgG or CmAb-(IL10)2 on days 11 and 14 after tumor cell inoculation. Tumor tissues were collected 7 days after first treatment and analyzed by flow cytometry. (D) IFN-g ELISPOT assay of splenocytes collected from B16-cEGFR-OVA tumor-bearing C57BL/6J mice (n = 5–6) treated three times by i.t. injection of control IgG or CmAb-(IL10)2. The spleens were harvested 9 days after the first treatment. OT1 peptide, OVA-derived SIINFEKL peptide; SIY, a control peptide SIYRYYGL. (E) Tumor growth in C57BL/6J mice (n = 5) bearing B16-cEGFR tumors treated with control IgG or CmAb-(IL10)2 (i.t., indicated by arrows). a-CD8 or a-CD4 antibodies were administered for T cell depletion during the CmAb-(IL10)2 treatment. (F and G) Tumor growth in NSG-SGM3 (F) and NSG-SGM3 humanized (G) mice (n = 5) bearing A431 tumors treated with CmAb-(IL10)2 or Cetuximab on days 11, 14, 17, and 20 after tumor cell inoculation. (A–G) Data are shown as means ± SEM. **p < 0.01, ****p < 0.0001; ns, not significant. See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Injection, Control, Cytometry, Enzyme-linked Immunospot, Derivative Assay

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 4. DCs are Essential for the Antitumor Effects of CmAb-(IL10)2 by Preventing Apoptosis of Antigen-Specific CD8+ T Cells (A and B) Proliferation of CFSE-labeled CD8+ OT1 T cells co-cultured with BMDCs from C57BL/6J mice in the presence of OVA and treated with CmAb-(IL10)2, Cetuximab, or vehicle. The percentage (A) and the number (B) of proliferating CD8+T cells were assessed by flow cytometry at the indicated time points. (C) Apoptosis of proliferating CD8+ T cells at 72 h after co-culture as described in (A and B), assessed by flow cytometry.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Labeling, Cell Culture, Cytometry, Co-Culture Assay

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 5. IL-10R Signaling on DCs Is Required for Preventing Apoptosis of Antigen-Specific CD8+ T Cells (A) Proliferation of CFSE-labeled CD8+ OT1 T cells co-cultured with BMDCs from WT (left) or Il10r/ (right) mice in the presence of OVA treated with CmAb-(IL10)2 or vehicle, assessed by flow cytometry at the indicated time points. (B) Cell number of proliferating CD8+ OT1 T cells co-cultured with BMDCs from Il10r/ mice in the presence of OVA and treated with CmAb-(IL10)2 or vehicle, assessed by flow cytometry at the indicated time points. (C) Apoptosis of proliferating CD8+ T cells at 72 h after co-culture as described in (B), assessed by flow cytometry. (D) Scheme of adoptive transfer of CD8+ T cells (2 3 104 OT1 CD8+ mixed with 2 3 106 WT CD8+ T cells) and CmAb-(IL10)2 treatment of Rag1/ or Il10r/ Rag1/

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Labeling, Cell Culture, Cytometry, Co-Culture Assay, Adoptive Transfer Assay

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 6. CmAb-(IL10)2 Prevents Antigen-Specific CD8+ T Cell Apoptosis through Regulating DC-Mediated IFN-g Production (A) Apoptosis assessment of re-stimulated CD8+ T cells by co-culturing antigen-activated CD8+ OT1 T cells with BMDCs from C57BL/6J mice in the presence of OVA and CmAb-(IL10)2 or vehicle, determined at 48 h after re-stimulation by flow cytometry. (B) IFN-g production from the indicated co-cultures of DCs and CD8+ OT1 T cells in the presence of OVA and treated with CmAb-(IL10)2 or vehicle. (C and D) Apoptosis of proliferating CD8+ T cells co-cultured with BMDCs from WT (C) or Il10r/ (D) mice in the presence of OVA and treated with CmAb-(IL10)2, IFN-g, or a-IFN-g (10 mg/mL), assessed by flow cytometry. (E) Apoptosis of CD8+ T cells in B16-cEGFR-OVA tumor tissues from Rag1/ mice (n = 5–6) i.t. treated with 1 3 106 antigen-activated CD8+ OT1 T cells plus control IgG, CmAb-(IL10)2 or anti-IFN-g (150 mg, i.p.) on day 11 after tumor cell inoculation. Tumor tissues were collected 2 days after treatment and analyzed by flow cytometry. (F) Apoptosis of OVA tetramer-positive CD8+ T cells in B16-cEGFR-OVA tumor tissues from Ifng/ mice (n = 7) i.t. treated with control IgG or CmAb-(IL10)2 on days 8 and 11 after tumor cell inoculation. Tumor tissues were collected 7 days after first treatment and analyzed by flow cytometry. (A–F) Data are shown as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Cytometry, Cell Culture, Control

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 7. CmAb-(IL10)2 Can Prevent Antigen-Specific CD8+ TIL Apoptosis and Improve the Antitumor Effects of Immune Checkpoint Blockade in the Treatment of Advanced Tumors (A) Apoptosis of OVA tetramer-positive CD8+ T cells in B16-cEGFR-OVA tumor tissues from C57BL/6J mice (n = 7) treated twice by a-PD-L1 and a-CTLA-4 (immune checkpoint blockade [ICB]) in combination with control IgG or CmAb-(IL10)2. Tumor tissues were collected 7 days after first treatment and analyzed by flow cytometry. (B and C) Scheme (B) (top), tumor growth (B) (bottom), and survival curve (C) of the advanced B16cEGFR tumor-bearing (80–120 mm3) C57BL/6J mice (n = 6–7) treated with CmAb-(IL10)2, ICB, or the combination therapy as indicated. (D) Tumor growth after challenge with B16-cEGFR cells in treatment-naı¨ve mice or mice cured by the combination therapy for ICB and CmAb-(IL10)2. (E) Scheme of treatment (left) and tumor growth (right) of C57BL/6J mice (n = 5–7) bearing advanced B16-cEGFR tumors treated with CmAb-(IL10)2, ICB, or the combination therapy as indicated. (A–E) Data are shown as means ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Control, Cytometry

Journal: American Journal of Cancer Research

Article Title: OX40 agonist combined with irreversible electroporation synergistically eradicates established tumors and drives systemic antitumor immune response in a syngeneic pancreatic cancer model

doi:

Figure Lengend Snippet: The anti-tumor activity of combination therapy is CD8+ T-cell dependent. A. Tumor cell implantation, immune cell depletion, and treatment strategy in unilateral subcutaneous mouse model. B. Successful depletion of CD4+ T and CD8+ T cells is confirmed using flow cytometry, and no cells are depleted after control IgG antibody injection. C and D. Tumor-free rates and survival rates after depletion of CD4+ or CD8+ T cells in unilateral subcutaneous tumor mice treated with combination therapy. N = 6 per group. E. Tumor cell implantation, immune cell depletion, and treatment strategy in bilateral subcutaneous tumor mouse model. F and G. Growth curves of the secondary untreated tumors and survival rates after depletion of CD4+ or CD8+ T cells in bilateral subcutaneous tumor mice treated with combination therapy. N = 6 per group. **P < 0.01, ***P < 0.001.

Article Snippet: For the T cell depletion experiment, anti-mouse CD8 monoclonal antibody (Cat #BE0061, clone 2.43, Bio X Cell), anti-mouse CD4 monoclonal antibody (Cat #BE0003-1, clone GK1.5, Bio X Cell), or isotype control antibody (Cat #BE0094, Bio X Cell) (250 μg in 100 μL PBS) were delivered per mouse (n = 6 in each group) four times by IP injection, two times per week, beginning on day 11 after inoculation of the primary tumor (1 d before initiation of treatment).

Techniques: Activity Assay, Flow Cytometry, Control, Injection

Journal: American Journal of Cancer Research

Article Title: OX40 agonist combined with irreversible electroporation synergistically eradicates established tumors and drives systemic antitumor immune response in a syngeneic pancreatic cancer model

doi:

Figure Lengend Snippet: Immune cell infiltration in the primary tumor 10 days after initiation of treatments. (A-C) Representative images of immunohistochemistry and corresponding quantifications for CD3 (A), CD4 (B), and CD8 (C). (D, E) Representative images of immunofluorescence for MDSCs (D), and T-regs (E) and corresponding quantifications. MDSCs: CD11b+Gr1+; T-regs: CD4+Foxp3+. Scale bars = 50 µm. N = 5 per group. *P < 0.05 vs. control group, **P < 0.01 vs. control group; #P < 0.05 vs. IRE group. IRE, irreversible electroporation.

Article Snippet: For the T cell depletion experiment, anti-mouse CD8 monoclonal antibody (Cat #BE0061, clone 2.43, Bio X Cell), anti-mouse CD4 monoclonal antibody (Cat #BE0003-1, clone GK1.5, Bio X Cell), or isotype control antibody (Cat #BE0094, Bio X Cell) (250 μg in 100 μL PBS) were delivered per mouse (n = 6 in each group) four times by IP injection, two times per week, beginning on day 11 after inoculation of the primary tumor (1 d before initiation of treatment).

Techniques: Immunohistochemistry, Immunofluorescence, Control, Electroporation

Journal: American Journal of Cancer Research

Article Title: OX40 agonist combined with irreversible electroporation synergistically eradicates established tumors and drives systemic antitumor immune response in a syngeneic pancreatic cancer model

doi:

Figure Lengend Snippet: Immune cell infiltration and levels of cytokines or chemokines in the secondary tumor 10 days after initiation of treatments. A. Proportions of tumor-infiltrating CD3+CD8+ T cells, CD3+CD4+ T cells, ki-67+CD8+ cells, ki-67+CD4+ cells, IFN-γ+CD8+ cells, IFN-γ+Foxp3-CD4+ cells, T-regs (CD4+CD25+Foxp3+), and MDSCs (Gr-1+CD11b+) assessed using flow cytometry, n = 5 per group. B and C. Levels of cytokines or chemokines in the secondary tumor were measured using a Mouse Cytokine 23-plex Luminex Assay. Data values (pg/mL) are normalized to total protein content and are presented in pg/mg protein. Cytokines or chemokines that did not show any significant difference in all groups were not included in this figure. n = 5 per group. *P < 0.05 vs. control group.

Article Snippet: For the T cell depletion experiment, anti-mouse CD8 monoclonal antibody (Cat #BE0061, clone 2.43, Bio X Cell), anti-mouse CD4 monoclonal antibody (Cat #BE0003-1, clone GK1.5, Bio X Cell), or isotype control antibody (Cat #BE0094, Bio X Cell) (250 μg in 100 μL PBS) were delivered per mouse (n = 6 in each group) four times by IP injection, two times per week, beginning on day 11 after inoculation of the primary tumor (1 d before initiation of treatment).

Techniques: Flow Cytometry, Luminex, Control

Journal: American Journal of Cancer Research

Article Title: OX40 agonist combined with irreversible electroporation synergistically eradicates established tumors and drives systemic antitumor immune response in a syngeneic pancreatic cancer model

doi:

Figure Lengend Snippet: Percentages of OX40+ T lymphocytes in spleen and TDLN in mice in the IRE and control groups 24 h, 48 h, 7 days, and 10 days post-IRE, analyzed using flow cytometry. A. Representative flow cytometry plots showing OX40+CD8+ T cells in spleen. B and C. Percentages of OX40+CD8+ and OX40+CD4+ T cells in spleen. D. Representative flow cytometry plots showing OX40+CD8+ T cells in TDLN. E and F. Percentages of OX40+CD8+ and OX40+CD4+ T cells in TDLN. n = 5 per group. *P < 0.05, **P < 0.01. IRE, irreversible electroporation; TDLN, tumor-draining lymph nodes.

Article Snippet: For the T cell depletion experiment, anti-mouse CD8 monoclonal antibody (Cat #BE0061, clone 2.43, Bio X Cell), anti-mouse CD4 monoclonal antibody (Cat #BE0003-1, clone GK1.5, Bio X Cell), or isotype control antibody (Cat #BE0094, Bio X Cell) (250 μg in 100 μL PBS) were delivered per mouse (n = 6 in each group) four times by IP injection, two times per week, beginning on day 11 after inoculation of the primary tumor (1 d before initiation of treatment).

Techniques: Control, Flow Cytometry, Electroporation

Journal: Blood Advances

Article Title: AMPK activation induces immunogenic cell death in AML

doi: 10.1182/bloodadvances.2022009444

Figure Lengend Snippet: AML cells pretreated with GSK621 induce a vaccination effect in vivo. (A) Schema of the vaccination assay performed with C1498 cells or MLL-AF9 cells in immunocompetent C57BL/6 mice. (B) Representative interferon gamma enzyme-linked immunospot (ELISpot) assay using single-cell suspensions from the spleens of 1 animal of each arm of the experiment activated by culture with the parental AML cells. Splenocytes were cocultured with a syngeneic C57BL/6 melanoma cell line (B16F10) or the ovalbumin antigen alone (OVA) as negative controls, or anti-CD3 as a nonspecific positive control. Each column represents ELISpots from splenocytes of 1 spleen seeded in triplicates. (C) Kaplan-Meier curves showing the overall survival in each group (5 mice per group) vaccinated with MLL-AF9 cells pretreated as indicated and fixed before injection, then challenged with live MLL-AF9 cells. Log-rank test. (D) Tumor growth on the challenge site of individual animals in panel C. Of note, none of the mice vaccinated with GSK621-treated cells developed tumors (red line on x-axis). (E) Kaplan-Meier curves showing the overall survival in each group (5 mice per group) vaccinated with MLL-AF9 cells pretreated with GSK621 before injection, then challenged with live MLL-AF9 cells. One group was depleted of CD4/CD8 T cells with intraperitoneal injections of neutralizing antibodies starting 2 days before the challenge (dashed line). The mice of the other group received a control IgG (solid line). Log-rank test. (F) Tumor growth on the challenge site of individual animals in panel E.

Article Snippet: For T-cell depletion, animals received intraperitoneal injections of 200 μg each of mouse anti-CD4 and anti-CD8 antibodies (InVivoMAb anti-mouse CD4, Clone: GK1.5, BioXcell no. BE0003-1; InVivoMAb anti-mouse CD8α, Clone 2.43, BioXcell no. BE0061), or immunoglobulin G (IgG) control antibody (InVivoMAb rat IgG2b isotype control, BioXcell no. BE0090).

Techniques: In Vivo, Enzyme-linked Immunospot, Positive Control, Injection, Control

Journal: Nature Communications

Article Title: In vivo armed macrophages curb liver metastasis through tumor-reactive T-cell rejuvenation

doi: 10.1038/s41467-025-58369-2

Figure Lengend Snippet: A UMAP projection of scRNA-seq of liver T and NK cells subclustered. B Gene set enrichment analysis (GSEA) of scRNA-seq data showing normalized enriched score (NES) for selected gene ontology (GO) terms calculated based on genes differentially expressed in T and NK cells in the indicated comparisons ( n = 2, 3, 3, 3 mice/group for IiOVA, OVA.Ifna, OVA.Il12 and OVA.Combo). C Combined gene expression score for genes belonging to the indicated categories in liver CD8 + T cells. D Fraction of positive cells and scaled averaged expression of selected genes belonging to the indicated biological processes in the liver CD8 + T cells (number of mice as in ( B ). E UMAP projection of liver scRNA-seq indicating cells bearing OVA-specific TCRs. F GSEA of scRNA-seq data showing NES for selected GO terms calculated based on genes differentially expressed in OVA-specific CD8 + T cells in the indicated comparisons (number of mice as in B ). G Combined gene expression score of genes belonging to the indicated categories in OVA-specific CD8 + T cells. H Clonotype sharing between liver and tumor CD4 + T cells, grouped by TCR clonotype. I UMAP projection of liver scRNA sequencing indicating CD4 + T cells clonotypes shared between liver and tumor tissue. J Percentage of cells within the CD4 + T cells populations in the liver ( n = 2, 3, 3, 3 mice/group for IiOVA, OVA.Ifna, OVA.Il12, and OVA.Combo, horizontal line represents mean, error bars represent the SEM). K Number of CD4 + T cells divided by TCR clonotype frequency.

Article Snippet: The antibodies used were In-vivoMAB anti-mouse PD-1 (BioXCell, catalog number:BE0146), In-vivoMAB anti-mouse CD4 (BioXCell, catalog number:BE0003-1), In-vivoMAB anti-mouse CD8a (BioXCell, catalog number:BE0004-1), In-vivoMAB anti-mouse NK 1.1 (BioXCell, catalog number:BE0036).

Techniques: Gene Expression, Expressing, Sequencing

Journal: Nature Communications

Article Title: In vivo armed macrophages curb liver metastasis through tumor-reactive T-cell rejuvenation

doi: 10.1038/s41467-025-58369-2

Figure Lengend Snippet: A – D Delivery of dOVA, SIINFEKL or SIINFEKL.TB LV to mice before tumor implantation (10 7 TU/mouse). A s.c. tumor growth ( n = 8 mice/group, mean ± SEM; statistical analysis by two-sided Mann–Whitney test on the area under the curve, p ≤ 0.05 are shown). B LV copies per genome of the tumor by ddPCR analysis (number of mice as in A ; horizontal line represents median; statistical analysis by Kruskal–Wallis with Dunn’s tests, p ≤ 0.05 are shown). C , D FC analysis of the liver (number of mice and statistic as in B , values below the dark gray area are excluded from further analyses). E – H Treatment with, OVA.Combo or SIINFEKL.Combo 7 days after tumor challenge (total dose 1.2 × 10 8 TU/mouse; Control mice are untreated). E Tumor weight ( n = 8, 9, 7 mice/group for Control, OVA.Combo, or SIINFEKL.Combo respectively, horizontal line represents median, statistical analysis by Kruskal–Wallis with Dunn’s tests, p ≤ 0.05 are shown). F OVA gene expression analysis on residual LM (number of mice and statistics as in E ). G , H FC analysis of the liver. Values below the dark gray are excluded from further analysis (number of mice and statistics as in E ). I Expression of selected genes in liver OVA-specific CD8 + T cells ( n = 1 mouse/group). J Combined gene expression score in liver CD4 + T cells ( n = 1 mouse/group). K GSEA of scRNA sequencing data showing NES for selected GO terms in liver CD4 + T cells ( n = 1 mouse/group). L Number of CD4 + T cells divided by TCR clonotype frequency ( n = 1 mouse/group). M – P Mice were injected intrahepatically with MC38.OVA cells and, after 6 days, injected IP with a-CD4, a-CD8, or a-NK. On day 7 mice were injected with OVA.Combo (total dose 1.2 × 10 8 TU/mouse) and then with the respective mabs IP twice weekly. M Tumor weight ( n = 12, 9, 9, 9 mice/group for OVA.Combo, OVA.Combo + a-CD4, OVA.Combo + a-CD8, OVA.Combo + a-NK, horizontal line represents median, statistical analysis by Kruskal–Wallis with Dunn’s tests comparing all groups vs OVA.Combo, p ≤ 0.05 are shown). N OVA gene expression analysis of residual LM (number of mice and statistics as in M ). O , P FC analysis of the liver (number of mice and statistics as in M ).

Article Snippet: The antibodies used were In-vivoMAB anti-mouse PD-1 (BioXCell, catalog number:BE0146), In-vivoMAB anti-mouse CD4 (BioXCell, catalog number:BE0003-1), In-vivoMAB anti-mouse CD8a (BioXCell, catalog number:BE0004-1), In-vivoMAB anti-mouse NK 1.1 (BioXCell, catalog number:BE0036).

Techniques: Tumor Implantation, MANN-WHITNEY, Control, Gene Expression, Expressing, Sequencing, Injection

Journal: bioRxiv

Article Title: Preeclampsia-specific immune cell network in placenta revealed by Cytometry by time of flight and single-cell RNA-seq

doi: 10.1101/2024.06.05.597577

Figure Lengend Snippet: (A) Schematic of the experimental workflow in CyTOF experiment. The placentas were obtained from individuals with normal pregnancy (NP, n=9), preeclampsia (PE, n=8), gestational diabetes mellitus (GDM, n=8) or GDM&PE (n=7). (B) t-SNE maps showing 7×10 5 CD45 + cells from the placenta overlaid with color-coded clusters and the distributions of B cells, CD4 + T cells, CD8 + T cells, DC, γδT cells, monocytes, granulocytes, MDSC, and NK cells. (C) Percentages of each cell type of CD45 + cells in placentas. (D) t-SNE maps showing the expression of CD3, CD8, CD4, γδTCR, CD14, CD15, CD56. (E) Heatmap showing the expression levels of markers in CD45 + cell subsets. Data were compared between NP and PE, NP and GDM, NP and GDM&PE using the Shapiro-wilk test and represented as mean±SEM (*P < 0.05, ** P < 0.01, *** P < 0.001; NS, not significant).

Article Snippet: Endogenous macrophages or CD4 + T cells were depleted by injecting PLX3397 (S7818, Selleck, USA), clodronate liposomes (40337ES08, YEASEN, China) or anti-CD4 antibody (BE0003-1, BioXcell, USA) intraperitoneally every three days starting from day 10.5 of gestation.

Techniques: Expressing

Journal: bioRxiv

Article Title: Preeclampsia-specific immune cell network in placenta revealed by Cytometry by time of flight and single-cell RNA-seq

doi: 10.1101/2024.06.05.597577

Figure Lengend Snippet: (A) Distribution of the CD4 + T cells analyzed using t-SNE. (B) Scatter dot plots showing the frequencies of cluster 8 of CD4 + T cells in the placentas of individuals with NP, PE, GDM and GDM&PE. (C) Expression of IL-17A and Foxp3 in CD45RO + CCR7 + CD4 + T cells in placentas of individuals with NP and PE using flow cytometry. (IL-17A: n=10 in NP group, n=15 in PE group; Foxp3: n=7 in NP group, n=9 in PE group). (D) Immunofluorescence co-staining of CD4 (red), CD45RO (green) and DAPI (blue) in frozen placental sections. The right panels show the enlargement of the dotted box in the left panel. Scale bar, 20 µm. (E) Scatter dot plots showing significantly altered markers of in cluster 8 of CD4 + T cells. (F) Distribution of the CD8 + T cells analyzed using t-SNE. (G) Scatter dot plots showing the frequencies of cluster 2 of CD8 + T cells in the placentas of individuals with NP, PE, GDM and GDM&PE. (H) Scatter dot plots showing significantly altered markers in cluster 2 of CD8 + T cells. (I) Distribution of the γδT cells analyzed using t-SNE. (J) Scatter dot plots showing the frequencies of cluster 15 of γδT cells in the placentas of individuals with NP, PE, GDM and GDM&PE. (K) Scatter dot plots showing significantly altered markers in cluster 15 of γδT cells. Data were compared between NP and PE, NP and GDM, NP and GDM&PE using the Shapiro-wilk test and represented as mean±SEM (*P < 0.05, ** P < 0.01, *** P < 0.001; NS, not significant).

Article Snippet: Endogenous macrophages or CD4 + T cells were depleted by injecting PLX3397 (S7818, Selleck, USA), clodronate liposomes (40337ES08, YEASEN, China) or anti-CD4 antibody (BE0003-1, BioXcell, USA) intraperitoneally every three days starting from day 10.5 of gestation.

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Preeclampsia-specific immune cell network in placenta revealed by Cytometry by time of flight and single-cell RNA-seq

doi: 10.1101/2024.06.05.597577

Figure Lengend Snippet: (A) Heatmap showing the expression levels of markers in the CD4 + T subsets. (B) Heatmap showing the expression levels of markers in the CD8 + T cells. (C) Heatmap showing the expression levels of markers in the γδT cells.

Article Snippet: Endogenous macrophages or CD4 + T cells were depleted by injecting PLX3397 (S7818, Selleck, USA), clodronate liposomes (40337ES08, YEASEN, China) or anti-CD4 antibody (BE0003-1, BioXcell, USA) intraperitoneally every three days starting from day 10.5 of gestation.

Techniques: Expressing

Journal: bioRxiv

Article Title: Preeclampsia-specific immune cell network in placenta revealed by Cytometry by time of flight and single-cell RNA-seq

doi: 10.1101/2024.06.05.597577

Figure Lengend Snippet: (A) Schematic of the mice model adoptively transferred CD45 + F4/80 + CD206 - pro-inflam Macs or CD45 + F4/80 + CD206 + anti-inflam Macs. (B) Principal component analysis (PCA) reflected the differences between the two groups of macrophages (n=3). (C) The volcano map shows a comparison of the content and P value of gene expression between pro-inflam Macs and anti-inflam Macs. Differential expression genes were screened out when P < 0.05. Red dots indicate genes with increased expression in pro-inflam Macs. Blue dots indicate genes with decreased expression. (D) The volcano map shows differential expression genes between pro-inflam Macs and anti-inflam Macs. (E) Representative pathways enriched in the identified genes as determined by GSEA (p value<0.05). (F) Embryo abortion rate of the pregnant mice, body weight and crown-rump length of pups measured on day 18.5 of gestation. Black represents mice treated with DMSO (n=8); gray represents mice treated with PLX3397 (n=8); blue represents mice injected with CD45 + F4/80 + CD206 + anti-inflammatory macrophages (n=8); red represents mice injected with CD45 + F4/80 + CD206 - pro-inflammatory macrophages (n=8). (G) SBP and UACR of pregnant mice in the four groups. (H) Frequencies of CD44 + CD4 + IL-17A + cells, CD44 + CD8 + T cells and CD11b + Ly6G + granulocytes analyzed by flow cytometry.

Article Snippet: Endogenous macrophages or CD4 + T cells were depleted by injecting PLX3397 (S7818, Selleck, USA), clodronate liposomes (40337ES08, YEASEN, China) or anti-CD4 antibody (BE0003-1, BioXcell, USA) intraperitoneally every three days starting from day 10.5 of gestation.

Techniques: Comparison, Gene Expression, Quantitative Proteomics, Expressing, Injection, Flow Cytometry

Journal: bioRxiv

Article Title: Preeclampsia-specific immune cell network in placenta revealed by Cytometry by time of flight and single-cell RNA-seq

doi: 10.1101/2024.06.05.597577

Figure Lengend Snippet: (A) Embryo abortion rate of the pregnant mice, body weight and crown-rump length of pups measured on day 18.5 of gestation. Black represents mice treated with control liposomes (n=6); gray represents mice treated with clodronate liposomes (n=6); blue represents mice injected with CD45 + F4/80 + CD206 + anti-inflam Macs (n=6); red represents mice injected with CD45 + F4/80 + CD206 - pro-inflam Macs (n=6). (B) SBP and UACR of pregnant mice in the four groups. (C) Frequencies of CD44 + CD4 + IL-17A + cells, CD44 + CD8 + T cells and CD11b + Gr1 + granulocytes analyzed by flow cytometry.

Article Snippet: Endogenous macrophages or CD4 + T cells were depleted by injecting PLX3397 (S7818, Selleck, USA), clodronate liposomes (40337ES08, YEASEN, China) or anti-CD4 antibody (BE0003-1, BioXcell, USA) intraperitoneally every three days starting from day 10.5 of gestation.

Techniques: Control, Liposomes, Injection, Flow Cytometry

Journal: bioRxiv

Article Title: Preeclampsia-specific immune cell network in placenta revealed by Cytometry by time of flight and single-cell RNA-seq

doi: 10.1101/2024.06.05.597577

Figure Lengend Snippet: (A) UMAP maps showing the 12 clusters of mouse T/NK cells was listed in the up panel. Bar graph showing the frequencies of clusters of T/NK cells in the two groups of mice was listed in the down panel. (B) Heatmap showing clustering analysis for markers distinguished 12 different clusters of T/NK cells. (C) UMAP maps showing the distribution of specific markers of cluster 0, 1 and 2. (D) Dot plot depicting GO enrichment terms that were significantly enriched in the differentially expressed genes in cluster 0 from the pro-inflam Macs group and the control group. (E) Violin plot of specific differential gene expression in cluster 0 between the pro-inflam Macs group and the control group. (F) Frequencies of CD4 + CD44 + T cells and the percentages of IL-17A + cells in CD4 + CD44 + T cells at the maternal-fetal interface in Sham and RUPP group analyzed by flow cytometry. (G) The embryo abortion rate of pregnant mice, body weight, and crown-rump length of pups measured on day 18.5 of gestation in mice injected PBS, Sham mouse-derived or RUPP mouse-derived CD4 + CD44 + T cells. Black represents mice injected with PBS (n=6); blue represents mice injected with Sham mouse-derived CD4 + CD44 + T cells (n=6); red represents mice injected with RUPP mouse-derived CD4 + CD44 + T cells (n=6). (H) SBP and UACR of pregnant mice injected with PBS, Sham mouse-derived or RUPP mouse-derived CD4 + CD44 + T cells.

Article Snippet: Endogenous macrophages or CD4 + T cells were depleted by injecting PLX3397 (S7818, Selleck, USA), clodronate liposomes (40337ES08, YEASEN, China) or anti-CD4 antibody (BE0003-1, BioXcell, USA) intraperitoneally every three days starting from day 10.5 of gestation.

Techniques: Control, Gene Expression, Flow Cytometry, Injection, Derivative Assay

Journal: bioRxiv

Article Title: Preeclampsia-specific immune cell network in placenta revealed by Cytometry by time of flight and single-cell RNA-seq

doi: 10.1101/2024.06.05.597577

Figure Lengend Snippet: (A) Experimental design of mice model with PE by reducing uterine perfusion pressure (RUPP) on day 12.5 of gestation. Mice with sham operation were considered as controls. Systolic blood pressure (SBP) and urine albumin creatine ratio (UACR) were measured on day 12.5 and 16.5 of gestation respectively. Mice were sacrificed on day 18.5 of gestation. (B) The embryo abortion rate of pregnant mice, body weight, and crown-rump length of pups measured on day 18.5 of gestation in the Sham and RUPP group. Embryo abortion rate = number of absorbed embryos/total number of embryos. Blue represents mice in the Sham group (n=9); Red represents mice in the RUPP group (n=9). (C) SBP and UACR of pregnant mice in Sham and RUPP group. (D) Embryo abortion rate of pregnant mice, body weight and crown-rump length of pups measured on day 18.5 of gestation. Black represents mice with previous normal pregnancy (n=9); gray represents mice with previous pregnancy with PE (n=9). (E) SBP and UACR of second pregnant mice with a history of PE or NP in the first pregnancy measured on day 16.5 of gestation. (F) Frequencies of CD4 + CD44 + T cells and the levels of IL-17A in CD4 + CD44 + T cells in mice with a pregnancy history with NP and PE analyzed by flow cytometry. Data were compared using the Student’s t-test and represented as mean±SEM (*P < 0.05, ** P < 0.01, *** P < 0.001; NS, not significant).

Article Snippet: Endogenous macrophages or CD4 + T cells were depleted by injecting PLX3397 (S7818, Selleck, USA), clodronate liposomes (40337ES08, YEASEN, China) or anti-CD4 antibody (BE0003-1, BioXcell, USA) intraperitoneally every three days starting from day 10.5 of gestation.

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Preeclampsia-specific immune cell network in placenta revealed by Cytometry by time of flight and single-cell RNA-seq

doi: 10.1101/2024.06.05.597577

Figure Lengend Snippet: (A) Signaling modules indicated by ligand-receptor pairing between macrophages and other types of immune cells at the maternal-fetal interface using CellPhoneDB. (B) Frequencies of IGF1 + CD14 + cells and IGFIR + CD4 + cells in placentas of individuals with NP and PE (n=12 in NP group, n=4 in PE group). (C) Schematic of the experimental workflow to induce memory-like T cells in vitro. Macrophages, after incubating with PBS, NP-EVs or PE-EVs, were co-cultured with CD4 + naïve T cells treated with DMSO or BMS-754807. Cells were isolated from human peripheral blood. (D) Frequencies of CD45RO + CCR7 + Th17 cells. Black represents CD4 + naïve T cells treated with DMSO; gray represents CD4 + naïve T cells cocultured with NP-EVs-treated macrophages; blue represents CD4 + naïve T cells co-cultured with PE-EVs-treated macrophages; red represents CD4 + naïve T cells treated with BMS-754807 before co-cultured with PE-EVs-treated macrophages (n=10 in each group). (E) Schematic of mice transferred CD4 + T cells treated with BMS-754807 or PBS. Anti-CD4 antibody was used to deplete CD4 + T cells in mice on day 10.5 of gestation. CD4 + T cells were transferred into mice on day 11.5 of gestation. 20 µg/kg lipopolysaccharide (LPS) was intraperitoneally injected on day 12.5 and 15.5 of gestation to induce a PE-like pregnant mice model. Mice were sacrificed on day 18.5 of gestation. (F) Embryo abortion rate of pregnant mice, body weight and crown-rump length of pups were measured on day 18.5 of gestation. Black represents the control group mice (n=6); gray represents mice treated with LPS (20μg/kg) to construct an animal model of PE (n=6); blue represents anti-CD4 antibody treated PE mice (n=6); red represents anti-CD4 antibody treated PE mice injected with CD4 + T cells with DMSO treatment (n=7); orange represents anti-CD4 antibody treated PE mice injected with CD4 + T cells with BMS754807 treatment (n=7). (G) SBP and UACR of pregnant mice in the five groups. (H) The frequencies of CD4 + CD44 + IL-17A + cells analyzed by flow cytometry. The results were compared using one-way ANOVA and represented as mean±SEM (*P < 0.05, ** P < 0.01, *** P < 0.001; NS, not significant).

Article Snippet: Endogenous macrophages or CD4 + T cells were depleted by injecting PLX3397 (S7818, Selleck, USA), clodronate liposomes (40337ES08, YEASEN, China) or anti-CD4 antibody (BE0003-1, BioXcell, USA) intraperitoneally every three days starting from day 10.5 of gestation.

Techniques: In Vitro, Cell Culture, Isolation, Injection, Control, Construct, Animal Model, Flow Cytometry

Journal: bioRxiv

Article Title: Lactose blocks intercellular spreading of Galectin-1 from cancer cells to T-cells and activates tumor immunological control

doi: 10.1101/2023.12.19.572484

Figure Lengend Snippet: B4GALT1 knockout and control OT-1 cells were incubated with biotin-recombinant Gal-1 (rGal-1) and then stained with streptavidin-PE. Blue and red curves indicate control and B4GALT1 knockout OT-1 cells, respectively; gray and green dotted curves indicate control and B4GALT1 knockout OT-1 cells, respectively, stained with streptavidin-PE only. OT-1 cells with (red) or without (blue) TCR activation by anti-CD3/28 antibodies and OT-1 cells cocultured with MC38 cells with (orange) or without (green) prior Ova-peptide pulsing were stained with anti-Gal-1 antibody. The gray dotted curve indicates OT-1 cells stained with isotype control antibody. OT-1 cells show no significantly increased surface Gal-1 after coculture with Gal-1 knockout MC38 cells (red curve) compared with OT-1 cells without coculture (gray dotted curve). The blue curve indicates OT-1 cells cocultured with control wild-type MC38 cells (blue curve). Schematic of surface Gal-1 transfer from cancer cells to T-cells. rGal-1 treatment (2.5 µg/ml) decreases the mRNA expression levels of Tnfα and Ifnγ after TCR activation by anti-CD3/CD28 antibodies in control wild-type OT-1 cells (1×10 5 cells/ml) but shows no significant effect on B4GALT1 knockout OT-1 cells. The p value was calculated by a two-tailed Student’s t test. Compared with wild-type MC38 cells, coculture with Gal-1 knockout MC38 cells increased the mRNA expression levels of Tnfα and Ifnγ in control wild-type OT-1 cells after activation by anti-CD3/CD28 antibodies. Such an effect is absent for B4GALT1 knockout OT-1 cells. The p value was calculated by a two-tailed Student’s t test. B4GALT1 KO and control wild-type OT-1 cells were used to kill wild-type control (left panel) and Gal-1 knockout (right panel) MC38 cells pulsed with Ova-peptide. The p value was calculated by a two-tailed Student’s t test. The fold change in the killing ability of B4GALT1 knockout OT-1 cells versus wild-type control OT-1 cells was calculated when wild-type or Gal-1 knockout MC38 cells were used. Data for a 2:1 ratio (T-cells: MC38 cells) were used. The p value was calculated by a two-tailed Student’s t test. MC38 cells infected with exogenous expression of Gal-1-EGFP fusion or control EGFPs were inoculated subcutaneously into wild-type C57BL/6J mice. After 3 weeks, tumor-infiltrated CD8 + T-cells showed a GFP signal only in Gal-1-EGFP tumors. The p value was calculated by a two-tailed Student’s t test. Tumor-infiltrated CD8 + T-cells showed a surface GFP signal by anti-GFP antibody staining only for Gal-1-EGFP tumors. The p value was calculated by a two-tailed Student’s t test. Gal-1 knockout (sgGalectin-1) MC38 and wild-type control MC38 cells were inoculated subcutaneously into wild-type C57BL/6J (left panel) and NPG (NOD-Prkdc scid IL2rg null ) mice (right panel). The p value was calculated by two-way ANOVA. Gal-1 knockout MC38 cells were inoculated subcutaneously into wild-type mice depleted of CD8 + T-cells, CD4 + T-cells, and NK cells. The p value was calculated by two-way ANOVA. B2m knockout and Gal-1/B2m double knockout MC38 cells were inoculated subcutaneously into wild-type C57BL/6J mice. After 3 weeks, tumor-infiltrated CD8 + T-cells were stained with anti-Gal-1 antibody. The p value was calculated by a two-tailed Student’s t test. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. NS, not significant.

Article Snippet: For CD8 + T-, CD4 + T-, NK-cell depletion, 100 μg NK depletion antibody in 200 μl PBS was injected intraperitoneally at day −3 and day −1 prior to tumor inoculation; 250 μg CD8 depletion antibody (Clone 2.43, BioXCell, BE0061) in 200 μl PBS was injected intraperitoneally at day −2 and day −1 prior to tumor inoculation; 400 μg CD4 depletion antibody (Clone GK1.5, BioXCell, BE0003-1) in 200 μl PBS was injected intraperitoneally at day −2 and day −1 prior to tumor inoculation, respectively.

Techniques: Knock-Out, Control, Incubation, Recombinant, Staining, Activation Assay, Expressing, Two Tailed Test, Infection, Double Knockout

Journal: bioRxiv

Article Title: Lactose blocks intercellular spreading of Galectin-1 from cancer cells to T-cells and activates tumor immunological control

doi: 10.1101/2023.12.19.572484

Figure Lengend Snippet: Lactose treatment increases in vitro specific killing activities of OT-1 CD8 + T-cells on OVA-peptide pulsed MC38 cells. The p values were calculated by a two-tailed Student’s t test. Lactose treatment increases the expression of Tnfα in OT-1 CD8 + T-cells after coculture with OVA peptide-pulsed MC38 cells. The relative mRNA expression levels of Tnfα were measured by quantitative RT‒qPCR. The p values were calculated by a two-tailed Student’s t test. rGal-1 treatment reduces the expression of Tnfα in OT-1 CD8 + T-cells after anti-CD3/CD28 antibody stimulation. Such an inhibitory effect of rGal-1 could be reversed by lactose treatment. The relative mRNA expression levels of Tnfα were measured by quantitative RT‒qPCR. The p values were calculated by a two-tailed Student’s t test. The effect of intravenous injection of lactose on growth of subcutaneous MC38 tumors in syngeneic C57BL/6J wild-type mice. The p value was calculated by two-way ANOVA. Intravenous lactose injection increases percentages of MC38 tumor-infiltrated IFNγ-positive CD8 + T-cells. The p value was calculated by a two-tailed Student’s t test. The effect of intravenous lactose injection on the growth of subcutaneous MC38 tumors in immunodeficient NPG mice. The p value was calculated by two-way ANOVA. The effect of intravenous lactose injection on the growth of MC38 tumors in syngeneic C57BL/6J wild-type mice depleted of CD8 + T-cells, CD4 + T-cells, and NK cells. The p values were calculated by two-way ANOVA. The effect of intravenous lactose injection on the growth of B2m knockout MC38 tumors in syngeneic C57BL/6J wild-type mice. The p value was calculated by two-way ANOVA. The effect of intravenous lactose injection on the growth of 4T1 tumors in syngeneic BALB/c wild-type mice. The p value was calculated by two-way ANOVA. The effect of intravenous lactose injection on the growth of CT26 tumors in syngeneic BALB/c wild-type mice. The p value was calculated by two-way ANOVA. The effect of intravenous lactose injection on the growth of B16F10-puro tumors in syngeneic C57BL/6J wild-type mice. The p value was calculated by two-way ANOVA. The effect of intravenous lactose injection on the growth of B16F10-OVA tumors in syngeneic C57BL/6J wild-type mice. The p value was calculated by two-way ANOVA. Data are shown as the mean ± SEM. *P < 0.05; ***P < 0.001.

Article Snippet: For CD8 + T-, CD4 + T-, NK-cell depletion, 100 μg NK depletion antibody in 200 μl PBS was injected intraperitoneally at day −3 and day −1 prior to tumor inoculation; 250 μg CD8 depletion antibody (Clone 2.43, BioXCell, BE0061) in 200 μl PBS was injected intraperitoneally at day −2 and day −1 prior to tumor inoculation; 400 μg CD4 depletion antibody (Clone GK1.5, BioXCell, BE0003-1) in 200 μl PBS was injected intraperitoneally at day −2 and day −1 prior to tumor inoculation, respectively.

Techniques: In Vitro, Two Tailed Test, Expressing, Injection, Knock-Out