Journal: PLoS ONE

Article Title: Regulatory B Cells Inhibit Cytotoxic T Lymphocyte (CTL) Activity and Elimination of Infected CD4 T Cells after In Vitro Reactivation of HIV Latent Reservoirs

doi: 10.1371/journal.pone.0092934

Figure Lengend Snippet: Bregs or non-Bregs B cells were co-cultured for 72 hours with VPD450-labeled FACS-purified CD4 + T cells, activated using Anti-Biotin MACSiBead (Miltenyi, T Cell Activation/Expansion Kit) and IL-2 (2 U/ml) and proliferation of CD4+ T cells determined by flow cytometry. ( a ) Right panel, shows a representative overlay and right panel depicts compilation of results, whereby the reduction in proliferation is normalized to CD4 + T cell proliferation alone (n = 4). ( b ) In antibody blocking experiments, labeled and activated CD4+ T cells were co-cultured with Bregs and under conditions shown above the representative histograms (n = 4) (α-IL-10 = IL-10R blocking antibody (20 ug/ml, Biolegend), α-PD-1 = PD-1 blocking antibody (10 ug/ml, Biolegend).

Article Snippet: To determine the effect of PD-L1/PD-1 and IL-10/IL-10R interactions in Breg-mediated inhibition of CD4+ T cell proliferation, VPD450-labeled FACS-purified CD4 + T cells were activated using Anti-Biotin MACSiBead loaded CD2, CD3, and CD28 antibodies (Miltenyi, T Cell Activation/Expansion Kit) and IL-2 (20U, NIH AIDS repository), cultured with medium alone, with Bregs, with Bregs and IL-10R blocking antibody (20 ug/ml, BD Pharmingen), with Bregs and PD-1 blocking antibody (10 ug/ml, BD Pharmingen) or with Bregs with both IL-10R and PD-1 blocking antibodies.

Techniques: Cell Culture, Labeling, Purification, Activation Assay, Flow Cytometry, Blocking 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 ) 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