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: Cell Death & Disease

Article Title: Oncogenic activation of SMYD3-SHCBP1 promotes breast cancer development and is coupled with resistance to immune therapy

doi: 10.1038/s41419-025-07570-8

Figure Lengend Snippet: a The protein levels of Smyd3 and Shcbp1 in normal mouse mammary glands, and tumors initiated with HP5712 cells in 3 months old FVB virgin mice as shown by Western blots. b The protein levels of Smyd3 and Shcbp1 in HP5712 cells expressing sgSmyd3, OE-Smyd3, or OE-Smyd3/sgShcbp1 by Western blots. Representative tumor images ( c ) and tumor weight plots ( d ) at day 32 from FVB virgin mice implanted with HP5712 parental (HP tumors), OE-Smyd3-HP5712, sgSmyd3-HP5712, and OE-Smyd3/sgShcbp1-HP5712 cells at 1X10 6 cells per mammary fat pad (n = 8 mice/group). e The plot of relative spleen weight from the same cohort of mice in c and d . f The protein levels of Smyd3, H3K4me3, and Shcbp1 in tumors initiated with HP5712 parental, OE-Smyd3-HP5712, sgSmyd3-HP5712, and OE-Smyd3/sgShcbp1-HP5712 cells by Western blots. g The protein levels of pMek, pErk, and Kras in tumors initiated with HP5712 parental, OE-Smyd3-HP5712, sgSmyd3-HP5712, and OE-Smyd3/sgShcbp1-HP5712 cells as shown by Western blots. h tSNE visualized immune cells from normal FVB mouse mammary glands (FVB MG, n = 6 mice), tumors implanted with parental HP5712 cells (HP Vector Ctr), HP-OE-Smyd3, HP-OE-Smyd3/sgShcbp1, and HP-sgSmyd3 from FVB mice (n = 6 mice/group) by CyTOF analysis. Quantifications of PMN-MDSCs and M-MDSCs ( i ), CD4+ and CD8 + T cells ( j ) in CD45+ immune cell populations from the same cohorts of mice in h (n = 3 biological independent samples/per group). k , l The WT-T cells were activated with CD3 and CD28 antibody. Co-cultured the activated T cells without or with MDSCs from the mice implanted with HP parental cells, HP cells expressing Smyd3, or sgSmyd3, or OE-Smyd3/sgShcbp1 for 72 hours and examined T cells proliferation status ( k ) and quantifications ( l ) of data in k (n = 3 mice/group). Representative images of CD8+/PD1+ double positive cells in mammary tissues from the mice with the genotypes of HP-Ctr, HP-OE-Smyd3, HP-sgSmyd3, and HP-OE-Smyd3/sgShcbp1 ( m ) and quantifications of CD8+/PD1 double positive cells from mammary tissues ( n ) in these mice (n = 4 mice/group, and at least 20 images for each sample were counted). Scale bar: 20 μm. arrow heads point to PD1/CD8 positive cell. o Expressions of PD1 from the spleen with implantation of parental HP cells, HP cells with OE-Smyd3, HP-sgSmyd3, and HP-OE-Smyd3/sgShcbp1 in FVB mice as determined by qPCR. p Summary of Smyd3-Shcbp1oncogenic signals shape the TIME.

Article Snippet: In Vivo anti-mouse CD8a , BioX Cell , BP0117 , , .

Techniques: Western Blot, Expressing, Plasmid Preparation, Cell Culture

Journal: Cell Death & Disease

Article Title: Oncogenic activation of SMYD3-SHCBP1 promotes breast cancer development and is coupled with resistance to immune therapy

doi: 10.1038/s41419-025-07570-8

Figure Lengend Snippet: Representative tumor images ( a ), and tumor weight plots ( b ) at day 22 from FVB mice implanted with HP5712 cells at 1×10 6 cells per mammary fat pad, and treatment with PBS, PD1, trametinib (Tra), and PD1+Tra (n = 8 mice/group). c The plot of tumor volume in the processes of treatment in ( a , b ). d The plot of relative spleen weight from the same cohort of mice in a , b . Quantifications of PMN-MDSCs and M-MDSCs ( e ), CD4+ and CD8 + T cells ( f ) in CD45+ immune cell populations from the same cohorts of mice in a , b by CyTOF analysis at D12 (n = 3 mice/group). Quantifications of PMN-MDSCs and M-MDSCs ( g ), CD4+ and CD8 + T cells ( h ) in CD45+ immune cell populations from the same cohorts of mice in a , b by CyTOF analysis at D22 (n = 3 mice/group). i , j Tumor images from the HP Ctr mice, HP mice treated with αPD1 and Tra, and HP mice treated with αPD1 and Tra with depletion of T cell using CD8 antibody ( i ). The plot of tumor weight ( j ) in the same cohort of mice in ( i ) (n = 8 mice in each group). The protein levels of Smyd3 and Shcbp1 in tumors initiated with HP5712 cells and treatment with PBS, αPD1, Tra, and αPD1+Tra in FVB mice at D12 ( k ) and D22 ( l ) as shown by Western blots. The protein levels of pMek, pErk, Kras, and Grb2 in tumors initiated with HP5712 cells and treatment with PBS, αPD1, Tra, and αPD1+Tra in FVB mice at D12 ( m ) and D22 ( n ) as shown by Western blots. Expressions of Smyd3 ( o ) and Shcbp1 ( p ) in G600 cells with the treatment of E2, Tra, and E2 together with Tra at 1 hour, 2 hours, 4 hours, and 24 hours as determined by qPCR.

Article Snippet: In Vivo anti-mouse CD8a , BioX Cell , BP0117 , , .

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Oncogenic activation of SMYD3-SHCBP1 promotes breast cancer development and is coupled with resistance to immune therapy

doi: 10.1038/s41419-025-07570-8

Figure Lengend Snippet: Antibodies used for ChIP, IHC, IF, WB and in vivo mouse study.

Article Snippet: In Vivo anti-mouse CD8a , BioX Cell , BP0117 , , .

Techniques: Immunohistochemistry-IF, In Vivo

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity.

doi: 10.1186/s13046-023-02831-w

Figure Lengend Snippet: Fig. 6 Suppression of CTLs by CD11b+ MDSCs is responsible for the acceleration of tumor progression by Regnase-1 downregulation. A-D Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1 KO murine pancreatic cancer cells. Representative macro images of pancreatic tumors (A). Relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (B) (N = 6 per group). Representative images of HE (C, left panel) and CD8a immunostaining (C, right panel) and the number of CD8-positive cells (C, right) (N = 6 per group). Dot plots of CD3+CD8+ cells evaluated by flow cytometry (D, left) and the proportion of CD8 + cells among CD45+ cells (D, right) (N = 3 per group). E–H Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1-KO murine pancreatic cancer cells with or without depletion of CD8+ cells upon anti-CD8a antibody or IgG treatment. Experimental schematic (E). Dot plots of CD3+ and CD8.+ cells in WT or Regnase-1-KO syngeneic tumors upon anti-CD8a antibody or IgG treatment evaluated by flow cytometry (F). Tumor weights (G) (N = 6 per group). The relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (H) (N = 6 per group). Student’s t test was used to evaluate differences between 2 groups. One-way ANOVA with Tukey’s post hoc test was used to compare differences among 4 groups. *P < 0.05, scale bars: 100 μm (insets)

Article Snippet: BE0061, a fully neutralizing mAb recognizing CD8a, and control IgG were obtained from Bioxcell.

Techniques: Immunostaining, Flow Cytometry

Journal: BJC reports

Article Title: A detoxified TLR4 agonist inhibits tumour growth and lung metastasis of osteosarcoma by promoting CD8+ cytotoxic lymphocyte infiltration.

doi: 10.1038/s44276-024-00120-3

Figure Lengend Snippet: Fig. 3 Lipo-MP-LPS induces immunological changes in both primary tumours and lung metastases. a, c Representative immunohis- tochemistry images of xenograft tumours and lung metastases stained with mouse specific anti-CD8a antibodies. Scale bar = 50 μm. b, d Quantification of CD8a positive cells in xenograft tumours and lung metastases. Data are represented as means ± SD; *p < 0.05, using Mann–Whitney U test. e, g Representative immunohistochemistry images of xenograft tumours and lung metastases stained with mouse specific anti-F4/80 antibodies. Scale bar = 50 μm. f, h Quantification of F4/80 positive cells in xenograft tumours and lung tissues. Data are represented as means ± SD; n = 6 mice per group, *p < 0.05, using Mann–Whitney U test. (i) iNOS, MHC II, TNF-α, CD206, and IL10 gene expression in xenograft tumours and lung metastases determined by RT-qPCR. Data are presented as means ± SD; n = 6 mice per group, *p < 0.05, **p < 0.01, using two-tailed t-test. NS not significant.

Article Snippet: In vivo depletion of CD8+ T cells Mice were depleted of CD8+ T cells by the intraperitoneal administration of an anti-CD8a antibody (YTS169.4, rat IgG2b; BioXCell, West Lebanon, NH, USA).

Techniques: Staining, MANN-WHITNEY, Immunohistochemistry, Gene Expression, Quantitative RT-PCR, Two Tailed Test

Journal: BJC reports

Article Title: A detoxified TLR4 agonist inhibits tumour growth and lung metastasis of osteosarcoma by promoting CD8+ cytotoxic lymphocyte infiltration.

doi: 10.1038/s44276-024-00120-3

Figure Lengend Snippet: Fig. 4 The tumour suppressive effect of Lipo-MP-LPS depends on CD8+ T cells. a Representative images of IHC staining for CD8a positive cells of xenograft tumours from mice treated with anti-CD8a antibody or IgG2b isotype. Scale bar = 100 μm. b CD8a expression in spleen tissues determined by RT-qPCR. Data are represented as means ± SD; n = 4. ***p < 0.001, using two-tailed t-test. c Tumour growth curve in C3H/HeN mice treated with empty liposome or Lipo-MP-LPS in addition to injection with anti-CD8a or isotype IgG2b antibodies (n = 6 mice per group). Data are shown as mean tumour volume ± SD. *p < 0.05, using Mann–Whitney U test. d Kaplan–Meier survival curves. p < 0.05 was considered significant using the log-rank test. Representative images of H&E staining of xenograft tumours (e) and lungs (g) for each group. Scale bar = 200 μm. f Quantification of necrotic areas in tumour tissues. h Quantification of lung metastasis areas. Data are presented as means ± SD; *p < 0.05, **p < 0.01; NS not significant, using Mann–Whitney U test.

Article Snippet: In vivo depletion of CD8+ T cells Mice were depleted of CD8+ T cells by the intraperitoneal administration of an anti-CD8a antibody (YTS169.4, rat IgG2b; BioXCell, West Lebanon, NH, USA).

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, Two Tailed Test, Injection, MANN-WHITNEY, Staining

Journal: BJC reports

Article Title: A detoxified TLR4 agonist inhibits tumour growth and lung metastasis of osteosarcoma by promoting CD8+ cytotoxic lymphocyte infiltration.

doi: 10.1038/s44276-024-00120-3

Figure Lengend Snippet: Fig. 6 Survival analysis of patients with osteosarcoma according to immune cell infiltration levels estimated by consensus TME. Of the 84 patients with osteosarcoma, the top third (28 patients) and bottom third (28 patients) were classified into high and low score groups, respectively. Kaplan–Meier curves of (a) CD8+ T cells (b) macrophage, (c) M1 macrophage, and (d) M2 macrophage infiltration for OS (left) and PFS (right) are presented. p < 0.05 was considered significant using the log-rank test.

Article Snippet: In vivo depletion of CD8+ T cells Mice were depleted of CD8+ T cells by the intraperitoneal administration of an anti-CD8a antibody (YTS169.4, rat IgG2b; BioXCell, West Lebanon, NH, USA).

Techniques:

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: Cytokine

Article Title: Self-assembling sequentially administered tumor targeted Split IL-12p35 and p40 subunits to improve the therapeutic index of systemically delivered IL-12 therapy for cancer.

doi: 10.1016/j.cyto.2025.156912

Figure Lengend Snippet: Fig. 3. Split subunits are functional and active in vitro. A. Cartoon schematic of antigen independent (left) and antigen dependent (right) STAT4 assay formats. B–C. The potency of the IL-12 split subunits was assessed using the antigen independent STAT4 assay in CD8+ T cells derived from human PBMCs alone (B), or antigen dependent with MCF7-uPAR cells (C). EC50 values are displayed in (D). Technical replicates from single chain and subunit dose-response curves were used to generate EC50 values, error bars represent means ± SD.

Article Snippet: Cells were further incubated with human Fc receptor blocking antibodies (ThermoFisher #14–9161-73), and then stained with fluorophore-conjugated antibodies for the following surface markers: CD45RA BUV395 (BD Biosciences #740315), CD16 BUV496 (BD Biosciences #612944), CD69 BUV737 (BD Biosciences #612817), CD3 BV421 (BD Biosciences #562877), CD14 Pacific Blue (BioLegend #301828), CD20 BV650 (BioLegend #302336), CD8 cFluorB548 (Cytek #R7–20097), CD56 PE (BioLegend #318306), CCR7 PE-Cy7 (BioLegend #353226).

Techniques: Functional Assay, In Vitro, Derivative Assay

Journal: Cytokine

Article Title: Self-assembling sequentially administered tumor targeted Split IL-12p35 and p40 subunits to improve the therapeutic index of systemically delivered IL-12 therapy for cancer.

doi: 10.1016/j.cyto.2025.156912

Figure Lengend Snippet: Fig. 5. Split subunits assemble and form functional IL-12p70 that stimulates target biology in non-human primates. A. Schematic representation of the dosing schedule for targeted split IL-12 subunits. B. Quantitative MSD (meso scale discovery) assay specific for IL-12p70 was used to detect complexed IL-12p35/p40 subunits in serum. Drug levels are shown for both cycles as mean ± SEM for complexed IL-12p70 following sequential injection of IL-12 subunits with a 4- or 24-h interval. C-E. Flow cytometry was used to analyze cell subsets in the peripheral blood of treated NHP. C and D. Cytotoxic NK cells (CD56dim CD16+) and effector memory CD8+ T cells (CCR7−CD45RA−) respond to treatment with split IL-12 subunits demonstrated by both activation (CD69) and dose dependent proliferation (Ki-67). E. Fold change of total monocyte and lymphocyte populations in NHP peripheral blood. F. Data was analyzed with FlowJo v10.8 (BD Biosciences). F. Sera from treated NHP was assessed for cytokines and biomarkers of activation by multi-spot MSD. Neopterin, IL-18, IP-10, MIG, and IL-15 demonstrated an exposure- response relationship, with greater pharmacodynamic response observed in groups with elevated IL-12p70 exposure in Cycle 1. Cycle 2 showed a reduced cytokine/ biomarker response across all treatment groups.

Article Snippet: Cells were further incubated with human Fc receptor blocking antibodies (ThermoFisher #14–9161-73), and then stained with fluorophore-conjugated antibodies for the following surface markers: CD45RA BUV395 (BD Biosciences #740315), CD16 BUV496 (BD Biosciences #612944), CD69 BUV737 (BD Biosciences #612817), CD3 BV421 (BD Biosciences #562877), CD14 Pacific Blue (BioLegend #301828), CD20 BV650 (BioLegend #302336), CD8 cFluorB548 (Cytek #R7–20097), CD56 PE (BioLegend #318306), CCR7 PE-Cy7 (BioLegend #353226).

Techniques: Functional Assay, Injection, Flow Cytometry, Activation Assay, Biomarker Discovery