Journal: Clinical Cancer Research

Article Title: IFNα Augments Clinical Efficacy of Regulatory T-cell Depletion with Denileukin Diftitox in Ovarian Cancer

doi: 10.1158/1078-0432.ccr-20-4594

Figure Lengend Snippet: Figure 5. DCs mediate IFNa effects on Treg suppression through IL6. A, Flow cytometric analysis of suppression of T-cell proliferation by Tregs from the PEC of ID8- challenged IFNR/ mice adoptively transferred with WT or IFNR/ DCs and then treated with DD plus IFNa (samples pooled for each experiment, all groups n ¼ 3). B, Luminex analysis of IL6 and IL10 in supernatant with Tregs, DCs and/or IFNa (one of three experiments with similar results). Suppression of T-cell proliferation by Tregs in the presence of DCs, IFNa, and/or anti-IL6 (n ¼ 3 for all groups; C) or in the presence of DCs, IFNa, and/or recombinant IL6 (n ¼ 3 for all groups; D). E, Suppression of T-cell proliferation by Tregs in the presence of DCs, recombinant IL6, anti-CD80 and/or anti-CD86 (n ¼ 3 for all groups). F, Treg function in mLNs of mice challenged with ID8, treated with DDþIFNa PBS or anti-IL6 in vivo (n ¼ 5 for both groups).

Article Snippet: Recombinant mouse IL6 (R&D Systems) at 0.1 or 1.0 mg/mL was added withoutDCs as indicated.

Techniques: Luminex, Recombinant, In Vivo

Journal: Clinical Cancer Research

Article Title: IFNα Augments Clinical Efficacy of Regulatory T-cell Depletion with Denileukin Diftitox in Ovarian Cancer

doi: 10.1158/1078-0432.ccr-20-4594

Figure Lengend Snippet: Figure 7. Pegylated IFNa has activity in the clinical response to failed DD in patients with ovarian cancer. A, CA-125 serum levels in patient 103. IFNa given as weekly, subcutaneous pegylated IFNa2a. B, CA-125 serum levels in patient 105. C, Serum IL6 in patient 103 after treatment with DD alone and after addition of pegylated IFNa2a. Mean of triplicate replicates. D, Flow cytometric analysis of Treg prevalence (CD4þCD25hiCD127lo cells among all blood CD3þCD4þ

Article Snippet: Recombinant mouse IL6 (R&D Systems) at 0.1 or 1.0 mg/mL was added withoutDCs as indicated.

Techniques: Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Loading of the Nonhomologous End Joining Factor, Ku, on Protein-occluded DNA Ends

doi: 10.1074/jbc.m611125200

Figure Lengend Snippet: FIGURE 7. The DNA binding activity of Ku at RAG bound signal ends. A, a signal end complex is defined here as RAG1, RAG2, and HMG1 bound to two- oligonucleotide DNA duplexes, one containing the 12-RS and the other con- taining the 23-RS. A streptavidin-biotin complex was used to block the ends of the DNA duplexes distal to the site of cleavage. B, approximately 10 ng/l of purified RAG1 and RAG2 and 425 nM HMG1 were incubated with 0.4 nM radiolabeled23-RS-containingand10nM12-RS-containingDNAfragmentsat 37 °C for 10 min. 5 M streptavidin was added at 25 °C for 5 min prior to addition of 25 nM Ku. Antibody supershifts required an additional 10-min room temperature incubation step with either 0.2 g of the monoclonal anti- body to Ku or 1 l of a polyclonal antisera recognizing the maltose binding domain fused to recombinant RAG proteins. The inferred composition of each of the generated species is indicated to the side of the panel: species I, 23-RS; species II, HMG1-bound 23-RS; species III, RAGs and HMG1 bound to the 23-RS; species IV, SEC; species V, streptavidin-blocked SEC (upper arrow indi- cates -MBP supershift); species VI, Ku bound to incompletely formed SEC (upper arrow indicates -Ku supershift).

Article Snippet: The relative amounts of Ku and histoneH3 in each of the excised complexes were determined by semi- quantitative Western analysis probing with a polyclonal rabbit antibody raised against native, recombinant human Ku and a polyclonal antibody against histone H3 (Ab1791; Abcam) using fluorescent detection and a Typhoon imager (GE Healthcare).

Techniques: Binding Assay, Activity Assay, Blocking Assay, Purification, Incubation, Recombinant, Generated