Journal: Cancer immunology research

Article Title: Inhibition of the NKp44-PCNA Immune Checkpoint Using a mAb to PCNA

doi: 10.1158/2326-6066.CIR-19-0023

Figure Lengend Snippet: (A) ELISA demonstrating the binding specificity of 14-25-9 mAb with plate-bound recombinant MBP- and His-tagged hPCNA. His-DJ1 (negative control) and mIgG1 (isotype control). (B) Western blot image demonstrating specific binding of 14-25-9 with His-hPCNA (30kDa) and MBP-hPCNA (71.5kDa) and no binding with His-DJ1. (C) ProteOn array showing the affinity of 14-25-9 at a concentration range of 0–100nM to His-hPCNA. Concentrations represented with lines are, 0nM (Violet, base-line), 6.25nM (Yellow), 25nM (Green), 50nM (Blue), and 100nM (Red). Analysis: bivalent binding model. (D-E) Western blot images showing recognition of endogenous PCNA. Data are representative of three independent experiments. (F) ELISA demonstrating inhibition of NKp44-Ig binding with plate-bound MBP-hPCNA by titrated 14-25-9 and PC10. Percentages of NKp44-Ig binding, were calculated against no-antibody incubation value. Data represents mean±SD of two independent experiments in n=3 biological replicates for each dose. One-way ANOVA was performed among the groups; comparison was done between PC10 and 14-25-9 groups. * p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Small interfering RNA and NKp44-derived peptide-based downregulation of PCNA Small interfering RNA (siRNA) against human PCNA and control siRNA (Santa Cruz Biotechnology, sc-29440, sc-36869) were used to silence PCNA expression in HeLa cells.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant, Negative Control, Control, Western Blot, Concentration Assay, Inhibition, Incubation, Comparison

Journal: Cancer immunology research

Article Title: Inhibition of the NKp44-PCNA Immune Checkpoint Using a mAb to PCNA

doi: 10.1158/2326-6066.CIR-19-0023

Figure Lengend Snippet: Interferon-γ ELISA results showing impact of 14-25-9 on primary human NK and NK92-NKp44-1 cells, while interacting with cancer cell lines. NK cells were co-incubated overnight with different cancer cell lines in the presence of titrated 14-25-9, 5μg/ml of mIgG1 and PBS as controls. IFNγ concentration (pg/ml) was assayed by ELISA. Fold change values of IFNγ with respect to control PBS were determined for (A) 721.221-Cw6 (B) K562 (C) A549 (D) HeLa and (E) Du145. (F) Representative bar graph showing absolute IFNγ concentration (pg/ml) for 721.221-Cw6 cells when interacting with pNK cells. NK92-NKp44-1 cells were co-incubated overnight with different cancer cell lines in presence of titrated 14-25-9, 20μg/ml of mIgG1 and PBS as controls. IFNγ concentration (pg/ml) was assayed by ELISA. Fold change values of IFNγ in respect to control PBS were determined for (G) 721.221-Cw6 (H) K562 (I) A549 (J) HeLa and (K) Du145. (L) Representative bar graph showing absolute IFNγ concentration (pg/ml) for 721.221-Cw6 cells when interacting with NK92-NKp44-1cells. Data represents mean±SD of two to three independent experiments in n = 3 biological replicates for each treatment. One-way ANOVA was performed among the groups, comparison was done between mIgG1 and 14-25-9 treatment groups. *p<0.05, **p<0.01, ***p<0.001, ns=not significant. To determine the effect of 14-25-9 on cytotoxic activity of NK92-NKp44-1 cells, they were incubated with EL4 cells (M) and hPCNA overexpressing EL4 clone1 cells (N) (E: T= 1:1, 2.5:1, 5:1) for 6h in presence of titrated 14-25-9 and 20μg/ml of mouse IgG1 as control. (O) 721.221 and 721.221-Cw6 cells were incubated with NK92-NKp44-1 cells (E: T= 1:1) for 6h in presence of titrated 14-25-9 and 20μg/ml of mIgG1 as control. Specific cytotoxicity was assayed by CFSE/GFP and PI-based FACS analysis. Two-way ANOVA and Bonferroni post tests were performed among the groups. Comparison was done among IgG1 and different treatment groups for EL4 and EL4-PCNA clone1. ‘ns’ represent no significant increase in cytotoxicity with treatment of 14-25-9 and **p<0.01, ***p<0.001 represent significant increase in cytotoxicity. For 721.221 versus 721.221-Cw6, comparisons were done both from control and mIgG1. Significant decrease in killing of 721.221-Cw6 compared to 721.221 control and treated with mouse IgG1 (***p<0.001). No significant increase (ns) is observed in killing of 721.221 treated with three doses of 14-25-9. Significant increase is observed in killing of 721.221-Cw6 cells treated with three doses of 14-25-9 compared to 721.221-Cw6 cells treated with mIgG1 ## (p<0.01). The results are from three experiments.

Article Snippet: Small interfering RNA and NKp44-derived peptide-based downregulation of PCNA Small interfering RNA (siRNA) against human PCNA and control siRNA (Santa Cruz Biotechnology, sc-29440, sc-36869) were used to silence PCNA expression in HeLa cells.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Control, Comparison, Activity Assay

Journal: Cancer immunology research

Article Title: Inhibition of the NKp44-PCNA Immune Checkpoint Using a mAb to PCNA

doi: 10.1158/2326-6066.CIR-19-0023

Figure Lengend Snippet: (A) Staining data showing cell surface PCNA staining by 14-25-9 (brown) and no staining by PC10 (green) compared to secondary anti-mouse IgG alone (black) for cancer cell lines and primary human cells: foreskin fibroblast, NK and human embryonic stem cell (WA09). (B) Primary cancer cells derived from PDX from HNSCC cancer patients (Pat1–3) were stained with 14-25-9 (orange), PC10 (green) and secondary anti-mIgG alone (red). (C) EL4 cells were stained with 14-25-9 (orange), PC10 (sky blue) and mIgG1 (red). (D-E) Human PCNA-transfected EL4 clones (clone 1, 15, 25, 27 and 32, depicted according to their histogram color) were stained with PC10 (D) and 14-25-9 (E). (F) siRNA based intracellular PCNA downregulation was done in HeLa cells to confirm PCNA staining by 14-25-9. (F) SiRNA study: western blot image representing downregulation of PCNA in HeLa by siRNA and quantitation (bar-histogram); FACS histogram: cell-surface staining by 14-25-9 in the same siRNA-based PCNA-silenced HeLa cells. (G) Cell-surface staining by 14-25-9 in HeLa cells treated with R11-NLS-pep8. DMSO and R11-NLS-pep8 short served as controls. Data are representative of three independent experiments.

Article Snippet: Small interfering RNA and NKp44-derived peptide-based downregulation of PCNA Small interfering RNA (siRNA) against human PCNA and control siRNA (Santa Cruz Biotechnology, sc-29440, sc-36869) were used to silence PCNA expression in HeLa cells.

Techniques: Staining, Derivative Assay, Transfection, Clone Assay, Western Blot, Quantitation Assay