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: Oncology Letters

Article Title: Role of programmed death ligands in effective T-cell interactions in extranodal natural killer/T-cell lymphoma

doi: 10.3892/ol.2014.2356

Figure Lengend Snippet: Representative immunohistochemical streptavidin-peroxidase staining in extranodal natural killer/T-cell lymphoma (upper row) and rhinitis tissues (lower row). The positive cases of (A and D) programmed death 1, (B and E) PD-L1 and (C and F) PD-L2 (magnification, ×200). PD-L, programmed death ligand.

Article Snippet: The antigen retrieval was conducted in 0.01 mol/l citrate (pH 6.0) and the slides were incubated overnight with rabbit anti-human PD-L1 polyclonal antibody (1:120; Proteintech, Chicago, IL, USA), rabbit anti-human PD-L2 polyclonal antibody (1:150) and mouse anti-human PD-1 monoclonal antibody (mAb; 1:100) (both ZSGB-BIO, Beijing, China) and phosphate-buffered saline was used as a blank control.

Techniques: Immunohistochemical staining, Staining

Journal: Oncology Letters

Article Title: Role of programmed death ligands in effective T-cell interactions in extranodal natural killer/T-cell lymphoma

doi: 10.3892/ol.2014.2356

Figure Lengend Snippet: PD-1 expression in (A) CD4 + and (B) CD8 + T-cell subsets in 20 ENKL patients was significantly increased compared with that in 10 HVs (P<0.05). Representative PD-1 expression in (C) CD4 + and (D) CD8 + T-cell subsets in six ENKL patients was (E) downregulated with chemotherapy. (F) T-helper cell type 1 cytokine (IL-2 and IFN-γ) mean production levels in the serum of 20 ENKL patients were significantly lower than those in 10 HVs (P<0.05). PD1, programmed death 1; ENKL, extranodal natural killer/T-cell lymphoma; HVs, healthy volunteers; IL-2 interleukin 2; IFN-γ, interferon γ.

Article Snippet: The antigen retrieval was conducted in 0.01 mol/l citrate (pH 6.0) and the slides were incubated overnight with rabbit anti-human PD-L1 polyclonal antibody (1:120; Proteintech, Chicago, IL, USA), rabbit anti-human PD-L2 polyclonal antibody (1:150) and mouse anti-human PD-1 monoclonal antibody (mAb; 1:100) (both ZSGB-BIO, Beijing, China) and phosphate-buffered saline was used as a blank control.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Role of programmed death ligands in effective T-cell interactions in extranodal natural killer/T-cell lymphoma

doi: 10.3892/ol.2014.2356

Figure Lengend Snippet: (A) Purity of CD8 + T cells separated by magnetic-activated cell sorting was 99%. (B) Purity of CD8 + PD-1 + T cells was 96.2% following the stimulation of allogeneic CD8 + T cells with phytohemagglutinin for 48 h. (C) SNK-6 cells were used as the control group and, following the coculture of SNK-6 cells and CD8 + T cells for 72 h, a significant inhibitory effect of PD-L1 on allogeneic CD8 + T-helper type 1 cytokine (IL-2 and IFN-γ) secretion was observed; (A and B) P<0.05. (D) CD8 + T-cell apoptosis in groups A and B was not altered significantly compared with activated CD8 + T cells at 72 h (P>0.05). (E) SNK-6 cells were used as the control group and cells harvested at 0, 24, 48 and 72 h were analyzed by flow cytometry gating CFSE + events. The proliferation index was not significantly different among the groups (P>0.05). PD-1, programme death 1; PD-L. programmed death ligand; IL-2 interleukin 2; IFN-γ, interferon γ; CFSE, carboxy-fluorescein succinimidyl ester.

Article Snippet: The antigen retrieval was conducted in 0.01 mol/l citrate (pH 6.0) and the slides were incubated overnight with rabbit anti-human PD-L1 polyclonal antibody (1:120; Proteintech, Chicago, IL, USA), rabbit anti-human PD-L2 polyclonal antibody (1:150) and mouse anti-human PD-1 monoclonal antibody (mAb; 1:100) (both ZSGB-BIO, Beijing, China) and phosphate-buffered saline was used as a blank control.

Techniques: FACS, Flow Cytometry

Journal: Immunity

Article Title: Single-cell chromatin accessibility landscape identifies tissue repair program in human regulatory T cells

doi: 10.1016/j.immuni.2021.03.007

Figure Lengend Snippet:

Article Snippet: VioBright 515 anti-human PD-1 (CD279) , Miltenyi , AB_2752077.

Techniques: Purification, Control