Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A IOD of ACAT2 expression in CC tissues and adjacent tissues was examined using immunohistochemical staining ( n = 47 biologically independent samples). IOD of DHCR7 B and MSMO1 C expression in CC patients with high ( n = 27 biologically independent samples) or low ( n = 20 biologically independent samples) expression of ACAT2 was examined using immunohistochemical staining. The number of activated CD8 T cells (CD8A + GZMB + ) D or activated NK cells (CD56 + GZMB + ) E infiltrated in the tumor tissues of patients with high ( n = 27 biologically independent samples) and low ACAT2 ( n = 20 biologically independent samples) expression was detected. Data represent mean ± SEM. Statistical analysis was performed using the paired A or unpaired ( B – E ) t-test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: ACAT2 expression in HCeEpiC and CC cell lines was examined using RT-qPCR A and Western blot analysis B ( n = 5 independent experiments). C ACAT2, DHCR7, and MSMO1 expression in CC cells after infection with Scramble-sh, ACAT2-sh #1, and ACAT2-sh #2 was examined using Western blot analysis ( n = 5 independent experiments). D Detection of total cholesterol, free cholesterol, and cholesteryl ester levels in CC cells ( n = 5 independent experiments). The proliferation of CC cells was examined using CCK8 ( E ) and colony formation assays F (n = 5 independent experiments). G CC cells were co-cultured with (E: T = 3:1) with NK cells or CD8 T cells for 6 h, respectively, and the death of CC cells was detected ( n = 5 independent experiments). H IFN-γ and GZMB released from immune cells in a co-culture system with CC cells were examined using ELISA ( n = 5 independent experiments). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( A , B ) or two-way ( C - H ) ANOVA, followed by Tukey’s multiple comparisons test ( A – H ).

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Infection, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A ACAT2 knockdown efficiency in U14 cells was examined using western blot analysis ( n = 10 independent experiments). B Volume changes of transplanted tumors in mice subcutaneously inoculated with U14 cells (n = 10 animals). C The images and weight of the tumors harvested on day 21 ( n = 10 animals). D Protein expression of ACAT2, MSMO1, DHCR7, and PCNA in transplanted tumors was examined using western blot analysis ( n = 10 animals). E Detection of total cholesterol, free cholesterol, and cholesteryl ester levels in transplanted tumors ( n = 10 animals). The gating strategy for GZMB + NK cells and CD8 + T cells F and quantification G were analyzed using flow cytometry ( n = 10 animals). H Survival of mice over 60 days after subcutaneous inoculation of U14 cells was analyzed using the log-rank test ( n = 20 animals). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( A , C , E , G ) or two-way ( B , D ) ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Knockdown, Western Blot, Expressing, Flow Cytometry

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: The proliferation of CC cells was examined using CCK8 A and colony formation assays B ( n = 5 independent experiments). C CC cells were co-cultured with (E: T = 3:1) with NK cells or CD8 + T cells, and the death of CC cells was detected ( n = 5 independent experiments). D IFN-γ and GZMB released from immune cells in a co-culture system with CC cells were examined using ELISA ( n = 5 independent experiments). E TGF-β1 released by CC cells was examined using ELISA ( n = 5 independent experiments). F PD-L1 expression levels in CC cells were observed using immunofluorescence staining ( n = 5 independent experiments). Data represent mean ± SEM. Statistical analysis was performed using the two-way ( A – F ) ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Staining

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A Volume changes of transplanted tumors in mice subcutaneously inoculated with U14 cells ( n = 5 animals). B The images and weight of the tumors harvested on day 21 ( n = 5 animals). The gating strategy for GZMB + NK cells and CD8 + T cells C and quantification D were analyzed using flow cytometry ( n = 5 animals). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( B , D ) or two-way A ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Flow Cytometry

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:

Article Title: Sulfolipid Deficiency Does Not Affect the Virulence of Mycobacterium tuberculosis H37Rv in Mice and Guinea Pigs

doi: 10.1128/IAI.71.8.4684-4690.2003

Figure Lengend Snippet: Representative photomicrographs of immunohistochemical staining for CD4+ (A and C) and CD8+ (B and D) lymphocytes in pulmonary granulomas of guinea pigs 100 days post-aerosol infection with M. tuberculosis H37Rv (A and B) or msl2 (C and D). Positive cells are indicated by red staining of the plasma membrane. Images are 5-μm serial sections. Note the similar numbers and arrangements of positive cells in panels A and B and in panels C and D. Bar, 10 μm.

Article Snippet: Mouse anti-guinea pig monoclonal antibodies specific for guinea pig CD4 (clone CT7), and CD8 (clone CT6) were purchased from Serotec (Oxford, England).

Techniques: Immunohistochemical staining, Staining, Aerosol, Infection, Clinical Proteomics, Membrane

Journal:

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8??

doi: 10.1128/JVI.79.17.11422-11433.2005

Figure Lengend Snippet: Flow cytometric analysis of Nef-mediated receptor down-regulation and internalization in retrovirally transduced SupT1 cells. SupT1 cells were transduced with an inducible NA-7.ER construct. At time zero, 4-hydroxytamoxifen (1 μM) was added to the culture medium. (A) Percent down-regulation was calculated, as described in the Materials and Methods. CD4-allophycocyanin (solid line) and CD8β-phycoerythrin (dashed line) were measured as a function of time. (B) The figure shows the percentage of CD4, CD8αβ, and CD28 molecules internalized by HIV-1 NA-7.ER, calculated as described in Materials and Methods. In each graph, Nef-positive (EGFP expressing Nef, solid line) and Nef-negative (EGFP not expressing Nef, dashed line) cells are depicted. The EGFP ranges used for calculation are indicated in Fig. ​Fig.1A1A.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8β primary antibody (5F2 [ 15 ], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Transduction, Construct, Expressing

Journal:

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8??

doi: 10.1128/JVI.79.17.11422-11433.2005

Figure Lengend Snippet: Mutations in the CD8 β-chain and their effect on endocytosis and down-regulation. Daudi cells were cotransduced with CD8α, wild-type or mutant CD8β, and control or wild-type Nef. (A and C) Alignment of amino acid sequences of wild-type (210*) and mutant CD8 β-chain cytoplasmic tails. An asterisk indicates a stop codon. (A) The bar chart shows the percent down-regulation of (mutant) CD8αβ by HIV-1 Nef alleles NA-7, LAI, and NL4-3. In both B and D the percentage of (mutant) CD8αβ molecules internalized by wild-type HIV-1 NA-7 is shown, including in both the same data for a Nef-negative construct as a control (wild-type Nef−). (C) The bar chart represents the percentage of (mutant) CD8αβ down-regulation after transduction with either control virus or wild-type Nef NL4-3. Each bar represents a (mutant) CD8 β-chain, as indicated by the changed amino acid sequence compared with CD8αβ. Percent down-regulation and internalization were calculated as described in Materials and Methods, using the ranges indicated in Fig. ​Fig.1A.1A. In A, B, and D, mean values are shown and standard deviations are calculated from the data generated from three independent experiments. In B the results for NL4-3 are representative of the results with HIV-1 alleles NA-7 and LAI.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8β primary antibody (5F2 [ 15 ], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Mutagenesis, Construct, Transduction, Sequencing, Generated

Journal:

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8??

doi: 10.1128/JVI.79.17.11422-11433.2005

Figure Lengend Snippet: Chimeric constructs. Daudi cells were retrovirally transduced with the CD8α(EC-TM)-CD8α(IC) chimera (cyt tail) or a CD8α(EC-TM)-CD8β(IC) chimera (cyt tail), using bicistronic constructs with ΔNGFR as the reporter. Bivariate dot plots are gated on ΔNGFR-positive cells, at day 2 after transduction of these cells with control virus, HIV-1 Nef (NA-7 allele), and SIV Nef (mac239), using bicistronic constructs with EGFP as the reporter. CD8α-phycoerythrin versus EGFP expression is shown.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8β primary antibody (5F2 [ 15 ], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Construct, Transduction, Expressing

Journal:

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8??

doi: 10.1128/JVI.79.17.11422-11433.2005

Figure Lengend Snippet: Confocal images of 293T cells. Nef.EGFP was detected by direct fluorescence (green, left panels) and CD8β.HA or CD8β by monoclonal antibodies as indicated in Materials and Methods (red, middle panels). Nuclei were visualized by DAPI staining (blue). Right panels show the merged images from Nef.EGFP and CD8β. Areas of colocalization of Nef.EGFP/CD8β are shown in yellow. As indicated, the upper panels show cells expressing wild-type LAI, the middle panels show the LLAA mutant, and alower panels show the PPAA mutant. Scale bars represent 5 μm.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8β primary antibody (5F2 [ 15 ], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Fluorescence, Staining, Expressing, Mutagenesis

Journal: Discovery Immunology

Article Title: Early disruption of the innate-adaptive immune axis in vivo after infection with virulent Georgia 2007/1 ASFV

doi: 10.1093/discim/kyaf014

Figure Lengend Snippet: Dynamics, proliferation and perforin expression of CD2 + CD8α + γδ-TCR + cells in selected lymphoid tissues. (tSNE cluster 8) high dimensional analysis and (Conventional) conventional gating of CD2 + CD8α + γδ-TCR + cell dynamics. (Proliferation) Proliferation and (Perforin) perforin expression of CD2 + CD8α + γδ-TCR + cells. Each datapoint denotes a single animal. Median (centreline), the first and third quartiles (box boundaries), maximum and minimum values within 1.5× the interquartile range (whiskers). SMLN, submandibular lymph node; SPTonsil, soft palate tonsil. Statistical significance determined with one way ANOVA except (d, CLN tSNE Cluster 8) where Kruskal–Wallis and Dunn test were used. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: CD8α , MIL12 , Mouse IgG2a , SBV515 , 1:25 , Bio-Rad Laboratories , MCA1223SBV515.

Techniques: Expressing

Journal: Journal of Virology

Article Title: Recombinant duck enteritis virus harboring the hemagglutinin genes of influenza virus rapidly induces specific cellular immunity in ducks

doi: 10.1128/jvi.02014-25

Figure Lengend Snippet: Expansion of T cells induced by rDEV-dH5/H7 and vDEV. Four-week-old ducks were inoculated i.m. with two doses of 10 5 TCID 50 of rDEV-dH5/H7 or vDEV at a 3-week interval. The percentages of ( A ) CD3 + T cells, ( B ) CD3 + CD8 + T cells, and ( C ) CD3 + CD4 + T cells among the PBMCs were analyzed by flow cytometry. Data are presented as means and plotted with connecting lines. Each data point represents one sample value. The red triangles represent the time points of inoculation.

Article Snippet: The PBMCs were plated in 1.5 mL centrifuge tubes (10 6 cells/tube) and fixed with 100 μL of 0.3% paraformaldehyde for 20 min at room temperature and subsequently stained for 30 min at room temperature with either mouse anti-duck CD4 mAb (Du CD4-2; Bio-Rad, MCA2478) or mouse anti-duck CD8 mAb (Du CD8-1; Bio-Rad, MCA2479) diluted in PBS containing 0.05% Tween-20.

Techniques: Flow Cytometry

Journal: Journal of Virology

Article Title: Recombinant duck enteritis virus harboring the hemagglutinin genes of influenza virus rapidly induces specific cellular immunity in ducks

doi: 10.1128/jvi.02014-25

Figure Lengend Snippet: Specific CD8 + T-cell responses induced by rDEV-dH5/H7 and vDEV. (A) Timeline of inoculation and sample testing. Arrows indicate inoculation, and red circles indicate bleeds, tests, and post-vaccination time points. ( B ) Frequency of specific CD3 + CD8 + T cells expressing IFN-γ + induced by DEV virion. ( C–F ) Frequency of HA-specific CD3 + CD8 + T cells expressing IFN-γ + . PBMCs were stimulated with single inactivated virus antigens: GZ/S4184 (H5N6), LN/SD007 (H5N1), GX/SD098 (H7N9), or a mixture of antigens of these three influenza virus antigens; CD3 + CD8 + IFN-γ + T cells were then quantified. ( G and H ) Uncorrelated control. CD3 + CD8 + IFN-γ + T cells were quantified after PBMCs were stimulated with WSN (H1N1) or La Sota (NDV). Data are presented as means in histograms. Each data point represents one sample value. Statistical significance: * P < 0.05, ** P <0.01, and *** P < 0.001. P values were determined using a two-tailed unpaired Student’s t test. ( I ) Representative gating strategy for HA-specific CD3 + CD8 + T cells expressing IFN-γ + detected at 31 days post-prime vaccination, following stimulation with the influenza virus antigens mixture. RPMI 1640 and PMA plus ionomycin served as negative and positive controls, respectively.

Article Snippet: The PBMCs were plated in 1.5 mL centrifuge tubes (10 6 cells/tube) and fixed with 100 μL of 0.3% paraformaldehyde for 20 min at room temperature and subsequently stained for 30 min at room temperature with either mouse anti-duck CD4 mAb (Du CD4-2; Bio-Rad, MCA2478) or mouse anti-duck CD8 mAb (Du CD8-1; Bio-Rad, MCA2479) diluted in PBS containing 0.05% Tween-20.

Techniques: Expressing, Virus, Control, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Effect of mucosal adjuvant IL-1β on heterotypic immunity in a pig influenza model

doi: 10.3389/fimmu.2023.1181716

Figure Lengend Snippet: Antibodies used:

Article Snippet: CD8β , PPT23 , Mouse IgG1 , FITC , Bio-Rad , , .

Techniques:

Journal: Frontiers in Immunology

Article Title: Effect of mucosal adjuvant IL-1β on heterotypic immunity in a pig influenza model

doi: 10.3389/fimmu.2023.1181716

Figure Lengend Snippet: Tetramer recognition and functional profile of porcine T cells in different tissues. (A) Proportions of AAV- (left) and DFE- (right) specific CD8β + T cells in different tissues of pH1N1 infected or Ad-HA/NP ± Ad-IL-1β immunized pigs at day 21. IFNγ, IL-2, and TNFα cytokine secretion were measured by intracellular staining in (B) CD8 + and (C) CD4 + T cells from the indicated tissues at day 21 post-immunization or infection. Statistical significances were analyzed by two-way ANOVA followed by Tukey’s Multiple Comparison Test. Asterisks denote significance between indicated groups (*, p <0.05; **, p <0.01; ***, p <0.001 and **** p <0.0001).

Article Snippet: CD8β , PPT23 , Mouse IgG1 , FITC , Bio-Rad , , .

Techniques: Functional Assay, Infection, Staining, Comparison