Journal: Materials Today Bio

Article Title: Developing immune-regulatory materials using immobilized monosaccharides with immune-instructive properties

doi: 10.1016/j.mtbio.2020.100080

Figure Lengend Snippet: Flow cytometry data are shown as a heat map summarizing the expression profile of three surface markers (CD86, CD40, and CD274) on DCs after incubation on immobilized monosaccharide libraries in the presence of a TLR4 ligand (LPS). Unstimulated (immature) DCs were used as a negative control. All changes are expressed as a percentage of expression levels in mature (LPS-stimulated) DCs. A reduction in CD40 expression was observed in most conditions with the highest reduction in fucose-contained libraries compared to LPS alone. CD86 expression was higher in a number of conditions (particularly mixers of Gal1 and Gal2); however, these did not reach statistical significance. CD274 (PD-L1) expression showed a significant increase in Man1 and Gal2 combinations. Data are shown as mean ± SD of three independent donors where ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001, and ∗∗∗∗ p <0.0001.

Article Snippet: DCs were then incubated with labeled antibodies CD274 (APC clone REA1197), CD40 PE (clone HB14), and CD86 FITC (clone FM95) and isotype-matched mouse antibody controls (all from Miltenyi Biotec) for 20 min in the dark at 4 °C.

Techniques: Flow Cytometry, Expressing, Incubation, Negative Control

Journal: Scientific Reports

Article Title: Combining DNMT and HDAC6 inhibitors increases anti-tumor immune signaling and decreases tumor burden in ovarian cancer

doi: 10.1038/s41598-020-60409-4

Figure Lengend Snippet: DNMT1 protein levels are decreased by combination treatment of DNMTi and HDAC6i. ( A ) Ovarian cancer cell lines were treated as in Fig. and protein was extracted at Day 7 after treatment with IFN-gamma (IFN-γ+) (to assess MHC I and PD-L1 expression, in later figures) or control (IFN-γ -). Protein was isolated and immunoblots were run for the DNMT1 protein and α-tubulin as a loading control. Immunoblot membranes were cut and probed separately for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here, and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( B ) The TykNu cell line was treated as in ( A ) and the protein synthesis cycloheximide added to cells on Day 7 for 0, 4, and 8 hours at 10 μM as indicated on the blot. Protein was isolated and immunoblots were run for the DNMT1 protein and α-tubulin as a loading control. Immunoblot membranes were cut and probed separately for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here, and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( C ) Stable knockdowns of the HDAC6 protein were generated in the ID8 Trp53+/+ and Trp53−/− cell lines . Protein was extracted and immunoblots were run for the DNMT1 protein with B-actin as a loading control. Immunoblot membranes were probed for DNMT1 (about 188 kDa) and α-tubulin (50 kDa). Cropped blots are shown here and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( D ) Immunoblot showing knockdown of HDAC6 protein with a-Tubulin as a loading control. Protein was extracted and immunoblots were run for the HDAC6 protein with B-actin as a loading control. Immunoblots were probed for HDAC6 (131 kDa) and tubulin (50 kDa). Cropped blots are shown here and black lines indicate where one part of the blot ends and another begins. Figure shows the entire blot images. ( E ) Ovarian cancer cell lines were treated as in Fig. and RNA was extracted at Day 7. qRT-PCR was run for DNMT1, DNMT3a, and DNMT3b and TBP was used as a reference gene. *p < 0.05 compared to Mock.

Article Snippet: The antibodies used for immunoblotting included: DNMT1 (Sigma, D4692), PD-L1 (ProSci, 4059), HDAC1 (Cell Signaling, 2062), HDAC2 (Cell Signaling, 2540), HDAC6 (Assay Biotech, C0026), acetyl-alpha Tubulin (Cell Signaling, 3971), alpha-Tubulin (Cell Signaling, 3873).

Techniques: Expressing, Control, Isolation, Western Blot, Generated, Knockdown, Quantitative RT-PCR

Journal: Cancers

Article Title: Chemotherapeutics Used for High-Risk Neuroblastoma Therapy Improve the Efficacy of Anti-GD2 Antibody Dinutuximab Beta in Preclinical Spheroid Models

doi: 10.3390/cancers15030904

Figure Lengend Snippet: Chemotherapy-induced immune-checkpoint ligand-cell surface abundance on tumor cells. 1 × 10 6 tumor cells treated for 24 h under cell culture conditions with either carboplatin (2 µg/mL, open circles), cisplatin (1 µg/mL, open triangles), etoposide (0.5 µg/mL, open squares), vincristine (0.05 µg/mL, open diamonds), or the cyclophosphamide metabolite 4-HPC (1 µg/mL, open hexagons). After 72 h of culturing, LAN-1 (left panel), CHLA-20 (center), and CHLA-136 (right panel) were analyzed for surface abundance of ( A ) PD-L1, ( B ) CD86, ( C ) CD155, and ( D ) Gal-9, using flow cytometry. Data represent at least five biological replicates. Means and SEM are indicated as black lines and error bars, respectively. For statistical analysis, ANOVA with appropriate post hoc test was used; * p < 0.05 vs., ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus untreated control (medium).

Article Snippet: Immune checkpoint ligand expression analysis by tumor cells was performed in analogy to the stress ligand expression analysis detailed above, using the following antibodies: anti-human CD80-BV421 (Biolegend, mouse IgG1,κ, clone 2D10, 1:20), CD86-PerCP-Vio 700 (Miltenyi Biotec, Bergisch Gladbach, REA968, 1:50), CD112-APC (Miltenyi Biotec, REA1195, 1:50), CD155-PE-Vio 770 (Miltenyi Biotec, REA1081, 1:50), Galectin-9-PE (Miltenyi Biotec, REA435, 1:50), and CD274 (PD-L1)-Vio Bright B515 (Miltenyi Biotec, REA1197, 1:50).

Techniques: Cell Culture, Flow Cytometry, Control

Journal: Stem Cells and Development

Article Title: Suppression of Canine Dendritic Cell Activation/Maturation and Inflammatory Cytokine Release by Mesenchymal Stem Cells Occurs Through Multiple Distinct Biochemical Pathways

doi: 10.1089/scd.2016.0199

Figure Lengend Snippet: MSC use multiple immune suppressive pathways to abrogate DC activation. IFNγ-treated MSC and DC were cocultured (1:10 ratio) for 24 hr and activated with LPS (50 ng/mL) for 24 hr. The MSC:DC cocultures were also incubated with the following immune pathway inhibitors: aminoguanidine (0.5 nM), indomethacin (100 μM), CSC-A2 inhibitor (0.2 μM), SB-43154 (10 μM), 1-methyltryptophan (MT) (500 μM), or programmed death ligand-1 (PD-L1) blocking antibody (10 μg/mL). Controls included medium with DMSO or methanol vehicle or with an isotype matched antibody for PD-L1 (data not shown). (i) DC:MSC cocultures or (ii) DC alone were incubated at 37°C for 24 hr in the presence of inhibitors and LPS. DC were subsequently harvested and immunostained for flow cytometric quantitation of expression of: (A) MCHII, (B) CD86, and (C) CD40 on CD11c+ events. Horizontal dashed lines denote the average maximal level of suppression of MHCII, CD86, and CD40 expression that is observed in DC cultures treated with IFNγ+MSC+LPS. Data are representative of four separate experiments. P values calculated for statistical variance were determined using a paired two-tailed Mann–Whitney test. *P < 0.05, **P < 0.01, and ***P < 0.005.

Article Snippet: Anti-canine-programmed death ligand-1 (PD-L1) and programmed death protein-1 (PD-1) monoclonal antibodies were from Merck Research Products, Kenilworth, NJ.

Techniques: Activation Assay, Incubation, Blocking Assay, Quantitation Assay, Expressing, Two Tailed Test, MANN-WHITNEY