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: Cell

Article Title: Generation of tumor-reactive T cells by co-culture of peripheral blood lymphocytes and tumor organoids

doi: 10.1016/j.cell.2018.07.009

Figure Lengend Snippet: Key Resources Table

Article Snippet: Anti-human PD1 , Merus , N/A.

Techniques: Control, Recombinant, Membrane, Cell Culture, Clinical Proteomics, Cell Recovery, Staining, Caspase-3 Assay, Software

Journal: PLoS ONE

Article Title: The role of autophagy in the treatment of BRAF mutant colorectal carcinomas differs based on microsatellite instability status

doi: 10.1371/journal.pone.0207227

Figure Lengend Snippet: ( A ) Cell viability of the mutant BRAF V600E colon cancer cell lines RKO after 72 hours treatments with 1μM anti-EGFR mAbs Cetuximab (C) or panitumumab (P), 0,5 μM checkpoint inhibitors nivolumab (N), Pembrolizumab (PE) or ipilimumab (IPI), 5mM of the autophagic inhibitor 3-MA (A) or 20μΜ of Hydroxychloroquine (HCQ) and in combination with a constant dose of E (C or P) and /or I (E+I, A+E, A+I and A+E+I). ( B ) Western blot analysis after 24 hours exposure of cells alone or in combination with a constant dose of E, I E+I, A+E, A+I and A+E+I. The protein levels of apoptotic cell death were identified by antibody against PARP and cl. caspase-3. The protein levels of p-EGFR, PD-1, LC3 and p62 are also presented. The quantification of LC3 reflects the ratio of LC3II/LC3I in comparison with control in each sample separately. Protein levels were normalized against actin. ( C ) Western blot analysis after treatment of RKO for 24 hours with 0,5 μΜ I, 1μΜ of E and 20μΜ of autophagy inhibitor (A) Hydroxychloroquine (HCQ), alone or in combination of A, A+E, A+I, A+E+I. The detection of p-EGFR, LCE3, p62, PARP and cl. Caspase 3 is tested by specific antibodies against each protein. The quantification of LC3 reflects the ratio of LC3II/LC3I in comparison with control in each sample separately. Protein levels were normalized against actin.

Article Snippet: Anti-EGFR MoAbs Erbitux (Cetuximab) Merck KGaA and Vectibix (Panitumumab) Amgen Europe B.V, checkpoint inhibitors; Nivolumab (a human programmed death receptor-1 (PD-1) blocking antibody) (Bristol Mayer Squibb), Pembrolizumab (a human programmed death receptor-1 (PD-1) blocking antibody) (Merck KGaA), Ipilimumab (Bristol Mayer Squibb).

Techniques: Mutagenesis, Western Blot

Journal: PLoS ONE

Article Title: The role of autophagy in the treatment of BRAF mutant colorectal carcinomas differs based on microsatellite instability status

doi: 10.1371/journal.pone.0207227

Figure Lengend Snippet: Confocal microscope images of three-dimensional culture in RKO and Colo-205 cell lines.( A ) RKO cells treated with 1μM anti-EGFR mAbs Cetuximab (C) or panitumumab (P), 5mM of the autophagic inhibitor 3-MA or 20μΜ of (HCQ), 0,5 μM checkpoint inhibitors nivolumab (N), Pembrolizumab (PE) or ipilimumab (IPI) for 48 hours alone or in 3-Methyladeninein combination with a constant dose of E, I and / or A. ( B ) Colo-205 cells treated with 1μM anti-EGFR mAbs Cetuximab (C) or panitumumab (P), 0,5μM of check point inhibitors nivolumab (NI), pembrolizumab (PE), ipilimumab (IPI), 5mM of the autophagic inhibitor 3-MA or 20μΜ of HCQ and 1μΜ MEK inhibitor PD-0325901 for 48 hours. Colo-205 were treated with [3-Methyladeninee (3-MA)] and in combination with a constant dose of E, I, PD, and / or A respectively for 48 hours. ( C ) HT29 cells treated 1μM anti-EGFR mAbs Cetuximab (C) or panitumumab (P), 0,5μM of check point inhibitors nivolumab (NI), pembrolizumab (PE), ipilimumab (IPI), 20μΜ of the autophagy inhibitor HCQ and 1μΜ MEK inhibitor PD-0325901 for 48 hours alone or in combination with a constant dose of E, I, PD and /or A for 48 hours. Nuclei were detected with DAPI (blue) and cleaved caspase-3 with the specific antibody (red). High concentration of cleaved caspase-3 and apoptotic nuclei are shown with yellow arrows.

Article Snippet: Anti-EGFR MoAbs Erbitux (Cetuximab) Merck KGaA and Vectibix (Panitumumab) Amgen Europe B.V, checkpoint inhibitors; Nivolumab (a human programmed death receptor-1 (PD-1) blocking antibody) (Bristol Mayer Squibb), Pembrolizumab (a human programmed death receptor-1 (PD-1) blocking antibody) (Merck KGaA), Ipilimumab (Bristol Mayer Squibb).

Techniques: Microscopy, Concentration Assay

Journal: PLoS ONE

Article Title: The role of autophagy in the treatment of BRAF mutant colorectal carcinomas differs based on microsatellite instability status

doi: 10.1371/journal.pone.0207227

Figure Lengend Snippet: ( A ) Western blot analysis of protein levels of pEGFR, pERKs, LC3, p62, PD-L1 and actin after 24 hours treatment with 1μM anti-EGFR mAbs Cetuximab or panitumumab and 0,5 μM checkpoint inhibitors nivolumab, pembrolizumab or ipilimumab and the combination of E+I in mutant BRAF V600E cell line RKO (upper panel) and in PD, PD+E (PD+C, PD+P), PD+I (PD+NI, PD+PE, PD+IPI) (lower panel). The quantification of LC3 reflects the ratio of LC3II/LC3I in comparison with control in each sample separately. ( B ) Western blot analysis of protein levels of pEGFR, pERKs, LC3, p62, PD-L1 and actin after treatment for 24 hours treatment with 1μM anti-EGFR mAbs Cetuximab or panitumumab and 0,5 μM checkpoint inhibitors nivolumab, pembrolizumab or ipilimumab, 1μM of MEK inhibitor PD 0325901 and in PD+E (PD+C, PD+P), PD+I (PD+NI, PD+PE, PD+IPI) in colo-205 cell line for 24 hours. The quantification of LC3 reflects the ratio of LC3II/LC3I in comparison with control in each sample separately.

Article Snippet: Anti-EGFR MoAbs Erbitux (Cetuximab) Merck KGaA and Vectibix (Panitumumab) Amgen Europe B.V, checkpoint inhibitors; Nivolumab (a human programmed death receptor-1 (PD-1) blocking antibody) (Bristol Mayer Squibb), Pembrolizumab (a human programmed death receptor-1 (PD-1) blocking antibody) (Merck KGaA), Ipilimumab (Bristol Mayer Squibb).

Techniques: Western Blot, Mutagenesis

Journal: PLoS ONE

Article Title: The role of autophagy in the treatment of BRAF mutant colorectal carcinomas differs based on microsatellite instability status

doi: 10.1371/journal.pone.0207227

Figure Lengend Snippet: Anti-EGFR mAbs (Cetuximab, panitumumab) and check point inhibitors (nivolumab, pembrolizumab, ipilimumab) trigger autophagy in MSI-H and MSS CRC cells in ERKs dependent and independent pathways, respectively. Moreover, inhibition of autophagy decreases the protein levels of PD-L1 in MSI-H cells. In MSS cells, the ERKs independent initiation of autophagy requires both autophagy and MEK inhibition. The triple inhibition A+E+I and A+PD+E/I initiate apoptotic cell death based on the microsatellite instability status.

Article Snippet: Anti-EGFR MoAbs Erbitux (Cetuximab) Merck KGaA and Vectibix (Panitumumab) Amgen Europe B.V, checkpoint inhibitors; Nivolumab (a human programmed death receptor-1 (PD-1) blocking antibody) (Bristol Mayer Squibb), Pembrolizumab (a human programmed death receptor-1 (PD-1) blocking antibody) (Merck KGaA), Ipilimumab (Bristol Mayer Squibb).

Techniques: Inhibition