Journal: Cell Reports Methods

Article Title: Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV

doi: 10.1016/j.crmeth.2022.100236

Figure Lengend Snippet:

Article Snippet: 155Gd_PD-1 , Fluidigm , Cat# 3155009B, RRID: AB_2811087.

Techniques: Virus, Recombinant, Electroporation, Antibody Labeling, Labeling, Software

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Increased antitumor activities of glypican-3-specific chimeric antigen receptor-modified T cells by coexpression of a soluble PD1–CH3 fusion protein

doi: 10.1007/s00262-018-2221-1

Figure Lengend Snippet: In vitro behavior of CAR-T cells after repetitive stimulation with SK-HEP-1-GPC3 cells. a CAR expression on the T-cell surface after three rounds of antigen-specific stimulation with SK-HEP-1-GPC3 cells. b Each data set is pooled from four independent experiments with individual donors (n = 4, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001); c The ratio of CD8+ versus CD4+ T cells after three rounds of antigen-specific stimulation (n = 3, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01); d Proliferation of both CAR-T cells after three rounds of antigen-specific stimulation. The two groups of CAR-T cells were prestained with CFSE before the third stimulation, and then the stained CAR-T cells were cocultured with SK-HEP-1-GPC3 at a 1:1 effector-to-target ratio for 96 h. The intensity of CFSE in each group was measured by flow cytometry. e After three rounds of antigen-specific stimulation with SK-HEP-1-GPC3 cells, the expression of exhaustion biomarkers on the T-cell surface, including PD-1, TIM-3 and LAG-3, was determined using flow cytometry with the indicated antibodies; f Each data set is pooled from three independent experiments with individual donors (n = 3, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001); g Cytokine release of the engineered T cells after three rounds of antigen-specific stimulation with SK-HEP-1-GPC3 cells. A total of 1 × 106 engineered T cells were cocultured with 1 × 106 tumor cells for 24 h. The levels of IL-2, IFN-γ and TNF-α in the supernatants were evaluated by ELISA (n = 6, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: The level of sPD1 secreted into the culture medium of the transduced T cells was detected using human PD1/PDCD1/CD279 ELISA kits (Sino Biological) according to the manufacturer’s instructions.

Techniques: In Vitro, Expressing, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Discovery of galectin-8 as an LILRB4 ligand driving M-MDSCs defines a class of antibodies to fight solid tumors

doi: 10.1016/j.xcrm.2023.101374

Figure Lengend Snippet:

Article Snippet: The plate was then washed with PBST (PBS, 0.05% Tween 20) (MA0015, Meilunbio) (T8220, Solarbio) and blocked with 3% BSA (97061-420, VWR) at 37°C for 90 min. After repeated washing, hFc-tagged recombinant proteins, including CD3ε (10977-H02H; Sino Biological), CTLA-4 (CT4-H5255; Acro Biosystems), CD28 (CD8-H525a; Acro Biosystems), CD96 (TAE-H5252; Acro Biosystems), LAG-3 (LA3-H5255; Acro Biosystems), TIM-3 (TM3-H5258; Acro Biosystems), CD40 (CD0-5253; Acro Biosystems), ICOS (ICS-H5258; Acro biosystems), OX40 (OX0-H5255; Acro Biosystems), TIGHT (TIT-H5254; Acro Biosystems), LY86 (10242-H02H; Sino Biological), LILRB4 (16742-H02H; Sino Biological), CD27 (CD7-H5254; Acro biosystems), PD-1 (10377-H02H; Sino Biological), and CD8b (11031-HCCH; Sino Biological), were added into each well.

Techniques: Recombinant, Derivative Assay, Microarray, Enzyme-linked Immunosorbent Assay, Lysis, Blocking Assay, Staining, Transfection, Marker, Flow Cytometry, shRNA, Plasmid Preparation, Negative Control, Silver Staining, Bicinchoninic Acid Protein Assay, Selection, Extraction, Labeling, RNA Expression, Cell Culture, Software

Journal: Cancer cell

Article Title: Non-conventional inhibitory CD4 + Foxp3 − PD-1 hi T cells as a biomarker of immune checkpoint blockade activity

doi: 10.1016/j.ccell.2018.05.009

Figure Lengend Snippet: (A,B) Mice were injected with 0.25×105 (n=5, tumor onset in 4 of 5 injected mice), 0.5×105 (n=5), 1×105 (n=5, one mouse died before FACS analysis), 2×105 B16 cells (n=5, one mouse died before FACS analysis), and 2 weeks later 4PD1hi and Tregs (percentage of total CD4+) were analyzed in spleen (SP), tumor-draining lymph nodes (DLNs) and tumor (TM) in comparison with spleens from naive mice (SP naive) (mean ± SEM; unpaired t test) (A). Pearson correlation analyses of tumor burden and intra-tumor 4PD1hi %, Tregs % and the indicated intra-tumor T-cell ratios (B). (C) Percentage of 4PD1hi among CD4+ T cells in healthy donors’ PB (n=7), advanced melanoma patients’ PB (n=47) and tumors (TM, n=10); NSCLC patients’ PB (n=51) and tumors (TM, n=10) (mean ± SEM; unpaired t test), and representative plots of Foxp3 and PD-1 expression in CD4+CD45+ T cells, and CD25 expression in 4PD1hi, Tregs and 4PD1− from the indicated samples. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. See also Figure S1.

Article Snippet: Sections were blocked for 30 min with Background Buster solution (Innovex) followed by avidin/biotin blocking for 8 min. Stainings were performed sequentially starting with an anti-CD4 Ab (polyclonal, R&D Systems, 2 μg/ml) followed by an anti-Foxp3 Ab (clone FJK-16s, eBioscience, 0.5 μg/ml), and finally an anti-PD-1 Ab (polyclonal, Sino Biological, 1 μg/ml).

Techniques: Injection, Expressing

Journal: Cancer cell

Article Title: Non-conventional inhibitory CD4 + Foxp3 − PD-1 hi T cells as a biomarker of immune checkpoint blockade activity

doi: 10.1016/j.ccell.2018.05.009

Figure Lengend Snippet: (A) Schema of in vitro suppression assay of CD45.1+ T cells (target) with 4PD1hi, 4PD1− or PD-1−Tregs FACS-sorted from spleens of naive Foxp3-GFP mice (CD45.1−, effectors). (B) CTV dilution and frequency of CD44+CD25+ in total CD45.1+CD4+ target T cells in the indicated co-cultures at the indicated effector:target ratios after 48-hr incubation (mean ± SD; n=2; 2-way ANOVA, 4PD1hi and Tregs vs 4PD1−). (C) Quantification of IFN-γ, TNF-α and IL-2 by FACS-based bead immunoassay in culture supernatants of CD4+ cells alone or co-cultured with the indicated cells for 48 hr (ratio 1:1; mean ± SD; n=2; unpaired t test). (D) Foxp3, CD25 and PD-1 MFI in effector CD45.1−CD4+ T-cell subsets co-cultured with CD45.1+CD4+ target T cells (ratio 1:1) for 48 hr (mean ± SD; n=2; unpaired t test). (E) In vivo suppression assay with 4PD1hi or Tregs FACS-sorted from B16-bearing Foxp3-GFP mice and co-transferred with CFSE-labeled Pmel/gp100-TCR-specific CD8+ T cells (Pmels) (1:1 ratio) into irradiated CD45.1+ recipients and stimulated in vivo with irradiated B16 cells (schema). Proliferation (CFSE dilution) and activation (CD44 and CD25 expression) of CD45.1−Thy1.1+CD8+ Pmels in recipient spleens (mean ± SEM; n=4–5; unpaired t test). * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. See also Figure S2.

Article Snippet: Sections were blocked for 30 min with Background Buster solution (Innovex) followed by avidin/biotin blocking for 8 min. Stainings were performed sequentially starting with an anti-CD4 Ab (polyclonal, R&D Systems, 2 μg/ml) followed by an anti-Foxp3 Ab (clone FJK-16s, eBioscience, 0.5 μg/ml), and finally an anti-PD-1 Ab (polyclonal, Sino Biological, 1 μg/ml).

Techniques: In Vitro, Suppression Assay, Incubation, Cell Culture, In Vivo, Labeling, Irradiation, Activation Assay, Expressing

Journal: Cancer cell

Article Title: Non-conventional inhibitory CD4 + Foxp3 − PD-1 hi T cells as a biomarker of immune checkpoint blockade activity

doi: 10.1016/j.ccell.2018.05.009

Figure Lengend Snippet: (A) FACS gating strategy to sort human 4PD1hi, 4PD1− and Tregs based on PD-1 and CD25 expression in CD4+ T cells and related Foxp3 expression (left). Proliferation (CTVlow) and activation (CD25 MFI) of autologous target CD4+ T cells co-cultured with donor-derived 4PD1hi, 4PD1− and Tregs at 1:1 ratio for 72 hr (middle) (mean ± SD; n=3; unpaired t test, 4PD1hi and Tregs vs 4PD1−). Heatmap with unsupervised hierarchical clustering of the indicated cytokines in co-culture supernatants as assessed by Luminex-based bead immunoassay (right). (B,C) Proliferation (CTVlow) of target autologous (auto) CD4+ TILs (B) or donor-derived allogeneic circulating CD8+ T cells (C) co-cultured with human tumor-infiltrating 4PD1hi, 4PD1− or Tregs (1:1 ratio) for 72 (B) or 96 hr (C), and cytokine production by Luminex-based bead immunoassay in the same cultures. Mean ± SD (B melanoma, n=2 with Tregs and 4PD1hi, n=6 with 4PD1−; B NSCLC, n=2 with 4PD1hi, n=3 with 4PD1− and Tregs; C, n=3); unpaired t test, 4PD1hi and Tregs vs 4PD1−. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. See also Figure S3.

Article Snippet: Sections were blocked for 30 min with Background Buster solution (Innovex) followed by avidin/biotin blocking for 8 min. Stainings were performed sequentially starting with an anti-CD4 Ab (polyclonal, R&D Systems, 2 μg/ml) followed by an anti-Foxp3 Ab (clone FJK-16s, eBioscience, 0.5 μg/ml), and finally an anti-PD-1 Ab (polyclonal, Sino Biological, 1 μg/ml).

Techniques: Expressing, Activation Assay, Cell Culture, Derivative Assay, Co-Culture Assay, Luminex

Journal: Cancer cell

Article Title: Non-conventional inhibitory CD4 + Foxp3 − PD-1 hi T cells as a biomarker of immune checkpoint blockade activity

doi: 10.1016/j.ccell.2018.05.009

Figure Lengend Snippet: (A) Fold changes in circulating 4PD1hi (percentage of total CD4+) in advanced NSCLC patients during treatment with nivo3 (nivolumab 3 mg/kg, once every 2 weeks (q2wks), n=10), nivo3+ipi1 (nivolumab 3 mg/kg + ipilimumab 1 mg/kg, q3wks, q6wks+q2wks, or q12wks+q2wks, n=21), nivo1+ipi1 (nivolumab 1 mg/kg + ipilimumab 1mg/kg, q3wks, or q6wks, n=11) or nivo1+ipi3 (nivolumab 1mg/kg + ipilimumab 3mg/kg, q3wks, n=8) (average ± SEM; 2-way ANOVA with Bonferroni correction, nivo3 vs nivo1+ipi1 and nivo3 vs nivo1+ipi3). Representative FACS plots of Foxp3 and PD-1 expression in CD4+ T cells from NSCLC patients treated with nivo1+ipi3 (red) or nivo3 (blue) at the indicated time points. (B) Circulating 4PD1hi (percentage of CD4+) in B16-bearing mice treated with αCTLA-4 monotherapy (100 μg or 300 μg/cycle; average ± SEM, n=7–10) relative to naive mice (n=5) (2-way ANOVA with Bonferroni correction, treated vs naive mice). (C) Pairwise comparison of 4PD1hi (percentage of CD4+) at the indicated time points relative to baseline in advanced melanoma patients during ipilimumab (ipi, 3 mg/kg, q3wks; n=47) or pembrolizumab treatment (pembro, 2 or 10 mg/kg, q3wks; n=52) (average ± SEM) (left). Representative FACS plots of Foxp3 and PD-1 expression in CD4+ T cells from melanoma patients treated with ipi (red) or pembro (blue) at the indicated time points (middle). Pairwise comparison of circulating 4PD1hi/CD4+ % at baseline and 3 weeks after pembro (right). (D) Average ± SEM tumor diameter (left; n=10; 2-way ANOVA with Bonferroni correction) and Kaplan-Meier tumor-free survival curves (right; pooled data from 3 independent experiments, n=30; log-rank test; number of tumor-free mice approximately 100 days after tumor implantation is reported for each group) of B16-bearing mice treated with VRP-TRP2 and αCTLA-4 and/or αPD-1 as indicated. (E) Intra-tumor 4PD1hi and Tregs frequencies one day after treatment completion in B16-bearing mice treated as in D (average ± SEM; n=9–10; unpaired t test). * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. See also Figure S4.

Article Snippet: Sections were blocked for 30 min with Background Buster solution (Innovex) followed by avidin/biotin blocking for 8 min. Stainings were performed sequentially starting with an anti-CD4 Ab (polyclonal, R&D Systems, 2 μg/ml) followed by an anti-Foxp3 Ab (clone FJK-16s, eBioscience, 0.5 μg/ml), and finally an anti-PD-1 Ab (polyclonal, Sino Biological, 1 μg/ml).

Techniques: Expressing, Tumor Implantation

Journal: Cancer cell

Article Title: Non-conventional inhibitory CD4 + Foxp3 − PD-1 hi T cells as a biomarker of immune checkpoint blockade activity

doi: 10.1016/j.ccell.2018.05.009

Figure Lengend Snippet: (A) In vitro suppression assays with 4PD1hi, Tregs and 4PD1− from spleens of sRBC-immunized or control B16-bearing Foxp3-GFP mice at the indicated ratios. Mean ± SD proliferation (CTVlow) and activation (CD25+CD44+) of target CD4+ T cells, and Foxp3 and PD-1 MFI in CD45.1− 4PD1hi, 4PD1− or Tregs from the same co-cultures after 48-hr incubation (n=2–3; 2-way ANOVA, NT-4PD1hi vs sRBC-4PD1hi). (B) In vitro suppression assays with CXCR5+ and CXCR5− 4PD1hi, 4PD1− and Tregs from spleens (SP) and tumors (TM) of naive and B16-bearing (TB) Foxp3-GFP mice immunized with sRBC. Mean ± SD proliferation (CTVlow) of target CD45.1+CD4+ T cells and quantification of IL-2 by Luminex-based bead immunoassay in culture supernatants after 72-hr incubation with effector cells (1:1 ratio; 4×104 cells from SP; 1×104 cells from TM; n=2–4; unpaired t test). (C) In vitro suppression assays with 4PD1hi, CD44hiPD-1−Foxp3−CD4+ Tmem and Foxp3+ Tregs from spleens of sRBC-immunized Foxp3-GFP mice at the indicated effector:target ratios. Mean ± SD proliferation (CTVlow) and activation (CD25+CD44+) of target CD8+ and CD4+ T cells from 48- and 72-hr co-cultures respectively (n=2–3; 2-way ANOVA, 4PD1hi and Tregs vs Tmem). (D) Average ± SEM tumor diameter of B16-bearing Sh2d1a (SAP) KO and WT C57BL/6J mice treated with αCTLA-4 or control isotype IgG (100 μg x4) starting on day 7 after tumor implantation (suboptimal treatment) (n=4–5; 2-way ANOVA with Bonferroni correction). * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. See also Figure S7.

Article Snippet: Sections were blocked for 30 min with Background Buster solution (Innovex) followed by avidin/biotin blocking for 8 min. Stainings were performed sequentially starting with an anti-CD4 Ab (polyclonal, R&D Systems, 2 μg/ml) followed by an anti-Foxp3 Ab (clone FJK-16s, eBioscience, 0.5 μg/ml), and finally an anti-PD-1 Ab (polyclonal, Sino Biological, 1 μg/ml).

Techniques: In Vitro, Activation Assay, Incubation, Luminex, Tumor Implantation

Journal: Cancer cell

Article Title: Non-conventional inhibitory CD4 + Foxp3 − PD-1 hi T cells as a biomarker of immune checkpoint blockade activity

doi: 10.1016/j.ccell.2018.05.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sections were blocked for 30 min with Background Buster solution (Innovex) followed by avidin/biotin blocking for 8 min. Stainings were performed sequentially starting with an anti-CD4 Ab (polyclonal, R&D Systems, 2 μg/ml) followed by an anti-Foxp3 Ab (clone FJK-16s, eBioscience, 0.5 μg/ml), and finally an anti-PD-1 Ab (polyclonal, Sino Biological, 1 μg/ml).

Techniques: Purification, Blocking Assay, Recombinant, Staining, Expressing, Knock-Out, Transgenic Assay, Software

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE blockade of PD-1/PD-L1 interaction in coculture cell-based luciferase assay. (A, B) Cytotoxicity assay performed using Cell Counting Kit-8 (CCK) assay. The hPD-1/NFAT Jurkat T cells (A) and hPD-L1/TCR CHO-K1 cells (B) after treatment with SRE for 24 hours. (C, D) The PD-1/PD-L1 blockade bioassay was performed using the Bio-Glo™ luciferase assay system. After addition of hPD-1/NFAT Jurkat T cells and SRE (C) and anti-PD-1 antibodies (αPD-1) (D) , hPD-L1/TCR CHO-K1 cells were seeded for 20 hours. Data are presented as the mean ± SD. * p < 0.05 and *** p < 0.001 compared to the control.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Luciferase, Cytotoxicity Assay, Cell Counting

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE-induced activation of T cells and cytotoxic effect of T cell-mediated cancer cells. (A, B) The cell viability was performed using the CCK-8 assay. Splenocytes were isolated from hPD-L1 MC38 cell-bearing hPD-1 knockin mice. Murine CRC hPD-L1 MC38 cells (A) and hPD-1 mice splenocytes (B) were treated with SRE for 72 hours. (C) Cocultured hPD-L1 MC38 cell viability tested by crystal violet staining; (D) Lactate dehydrogenase (LDH) released by damaged cells, detected via LDH cytotoxicity assay; (E) Relative interleukin-2 (IL-2) level, determined using the mouse IL-2 ELISA set. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Activation Assay, CCK-8 Assay, Isolation, Knock-In, Staining, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: SRE elevated the activation of hPD-1 + CD8 + T cells and the CD8 + T cell-mediated killing effect on hPD-L1 MC38 cancer. (A) Cocultured hPD-L1 MC38 cell viability, tested by crystal violet staining. Cocultured hPD-L1 MC38 cells detected with fluorescence microscopy (× 200) (B) and determined by fluorescent-activated cell sorting analysis (C) . (D) LDH released from damaged cells; (E) Relative perforin 1 (PRF1) level, determined with use of the mouse PRF1 ELISA kit. Data are presented as the mean ± SD. ** p < 0.01 and *** p < 0.001 compared to the vehicle group.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Activation Assay, Staining, Fluorescence, Microscopy, FACS, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Sanguisorbae Radix Suppresses Colorectal Tumor Growth Through PD-1/PD-L1 Blockade and Synergistic Effect With Pembrolizumab in a Humanized PD-L1-Expressing Colorectal Cancer Mouse Model

doi: 10.3389/fimmu.2021.737076

Figure Lengend Snippet: Sanguisorbae Radix extract reduced tumor growth in the hPD-L1 MC38 cell allograft hPD-1 mouse model. (A) Body weight (grams); (B) Tumor volume after 18 days; (C) Tumor weight after 18 days; (D) Images of tumor tissues (bar indicates 5 mm); (E) hPD-L1 MC38 tumor-bearing mice 18 days after treatment; (F) Representative microscopic images (×400) of CD8 and PRF1-positive area of tumor tissues calculated using immunohistochemical analysis. Data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the vehicle group.

Article Snippet: The biotinylated hPD-1 (#71109, BPS Bioscience) of 0.5 μg/mL was added to each well and incubated for 2 hours at RT.

Techniques: Immunohistochemical staining, Standard Deviation