Journal: Frontiers in Immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: Pre-engagement of Ipilimumab and Tremelimumab with soluble CTLA-4-Ig prevents CTLA4 binding to cell bound CD80 or CD86 ligand. (A) Schematic of experimental set-up. A fixed dose (2µg/ml) of soluble APC conjugated Abatacept was incubated on ice with a titration of soluble anti-CTLA-4 Abs to pre-engage CTLA-4 with anti-CTLA-4. This was added to DG75 B cells expressing either CD80 or CD86-GFP and incubated on ice for 30 minutes. Cells were washed and analysed for Abatacept-ligand interaction by flow cytometry. All reagents were chilled on ice before use. (B) Representative concatenated FACS plots show impact of Abatacept-ligand engagement as the dose of anti-CTLA-4 Abs increased. (C) Anti-CTLA-4 doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC 50 for ipilimumab(Ipi) (Black line) and tremelimumab(Treme) (Blue line). (D) Graphs show the mean IC 50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates and data presented as mean +/- SD from 3 independent experiments.

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Binding Assay, Incubation, Titration, Expressing, Flow Cytometry, Transformation Assay

Journal: Frontiers in Immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: Ipilimumab and Tremelimumab outcompete cell bound CD80 or CD86 for engagement with soluble CTLA-4-Ig. (A) Schematic of experimental set-up. DG75 B cells expressing either CD80 or CD86-GFP were mixed with a titration of soluble anti-CTLA-4 Abs. A fixed dose (2µg/ml) of soluble APC conjugated Abatacept was added to assess direct competition between soluble anti-CTLA-4 and cell bound ligand for Abatacept binding. Cells were incubated on ice, washed and Abatacept-ligand engagement analysed by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show reduced Abatacept-ligand engagement as the dose of anti-CTLA-4 Abs increased. (C) Anti-CTLA-4 doses were Log(x) transformed and dose response curves fitted using Prism v6 to obtain Log IC 50 for ipilimumab (Black line) and tremelimumab (Blue line). (D) Graphs show the mean IC 50 with 95% confidence interval calculated using Prism v6. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates and data presented as mean +/- SD from 3 independent experiments.

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Expressing, Titration, Binding Assay, Incubation, Flow Cytometry, Transformation Assay

Journal: Frontiers in Immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: Ipilimumab and Tremelimumab are unable to displace pre-engaged CTLA-4-Ig from cell bound CD80 or CD86. (A) Schematic of experimental set-up. DG75 B cells expressing either CD80 or CD86-GFP were pre-incubated on ice with a fixed dose (2µg/ml) of soluble APC conjugated Abatacept to pre-engage soluble CTLA-4 with ligand. Cells were washed, treated with a titration of soluble anti-CTLA-4 Abs and incubated on ice or at 37°C. Cells were washed and analysed for Abatacept-ligand engagement by flow cytometry. All reagents/cells were chilled on ice before use. (B) Concatenated FACS plots show Abatacept-ligand binding at different doses of anti-CTLA-4 Abs when incubated on ice or when incubated at 37°C. (C) Dose response curves for ipilimumab (Black line) and tremelimumab (Blue line) presented as MFI CTLA-4-Ig binding from data shown in (B) Data presented are mean +/- SD from 3-6 independent experiments. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=3-6).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Expressing, Incubation, Titration, Flow Cytometry, Ligand Binding Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: Ipilimumab and Tremelimumab block CD80 and CD86 ligand loss for APC by transendocytosis. (A) CTV stained CHO cells expressing either CD80 or CD86-GFP were cultured in the presence of CHO cells expressing CTLA-4 at a 1:1 ratio for 5 hours at 37°C. A titration of soluble ipilimumab (Black line) or tremelimumab (Blue line) were added at 0h. Percentage of CD80 or CD86-GFP ligand loss from CTV+ CHO cells by transendocytosis was determined by making the GFP MFI of CTV+ CHO cells relative to a negative control where no CTLA-4 was present. (B) Anti-CTLA-4 doses were Log(x) transformed and dose response curves fit using Prism v6 to obtain Log EC 50 for Iipilimumab (Black line) and tremelimumab (Blue line). (C) Graphs show the mean EC 50 with 95% confidence interval calculated using Prism v6. (D-F) As in A, B & C except with CTLA-4 expressing Jurkat and CTV stained DG75 B cells expressing CD80 or CD86-GFP. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates, data presented as mean +/- SD from 3 independent experiments.

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Blocking Assay, Staining, Expressing, Cell Culture, Titration, Negative Control, Transformation Assay

Journal: Frontiers in Immunology

Article Title: Impact of CTLA-4 checkpoint antibodies on ligand binding and Transendocytosis

doi: 10.3389/fimmu.2022.871802

Figure Lengend Snippet: Ipilimumab and Tremelimumab interfere with pre-established transendocytosis of CD80 and CD86 to prevent further ligand loss from APC. (A) CTV stained CHO cells expressing either CD80 or CD86-GFP were cultured in the presence of CHO cells expressing CTLA-4 at a 1:1 ratio for 0, 3 or 6 hours at 37°C. Transendocytosis was established for 3 hours in the absence of anti-CTLA-4 Abs at which point soluble ipilimumab (dotted black line) or tremelimumab (dotted blue line) were spiked into culture. Transendocytosis was continued for a further 3 hours to observe the effect of anti-CTLA4 spike. Soluble anti-CTLA-4 antibodies were also added at timepoint 0h to block transendocytosis from the start of the assay (solid black – ipilimumab or blue – tremelimumab lines). Transendocytosis was also performed in the absence of anti-CTLA-4 treatment (Purple line). (B) % of CD80 or CD86-GFP ligand loss from CTV+ CHO cells by transendocytosis was determined by making the GFP MFI CTV+ CHO cells relative to a negative control where CTLA4 was not expressed (n=3). ( C , D ) As in A & B except with CTLA-4 expressing Jurkat and CTV stained DG75 B cells expressing CD80 or CD86-GFP cultured at a Jurkat:DG75 ratio of 2:1. Also an additional spike was performed at 6h and the assay was run for a total of 20h. Data for ipilimumab and tremelimumab were acquired in separate 96-well plates (n=2).

Article Snippet: CD80 (Cat# 10133-B1) and CD86 (Cat# 141-B2) -Ig expressing human IgG1 Fc were purchased from R&D Systems.

Techniques: Staining, Expressing, Cell Culture, Blocking Assay, Negative Control