Journal: Molecular Therapy Oncology

Article Title: Exclusion of PD-1 from the immune synapse: A novel strategy to modulate T cell function

doi: 10.1016/j.omton.2024.200839

Figure Lengend Snippet: Full-length anti-PD-1 antibodies removed PD-1 from the IS (A) Coomassie staining of non-reduced full-length nivolumab and nivolumab treated with pepsin. (B) A flow cytometry graph of Jurkat T cells treated with full-length nivolumab and nivolumab F(ab′) 2 at different doses, showing the binding affinity of the different versions of the antibodies to the cells. This experiment was done three times, and representative data are shown. (Ci and Cii) GFP-PD-1-expressing Jurkat T cells co-cultured with SEE-loaded Raji B cells expressing mCherry PD-L1 treated with nivolumab and its F(ab′) 2 (Ci) and quantification of the number of cells where PD-1 was recruited to the IS at different concentrations of both antibodies (Cii). n = 5, p < 0.001 (unpaired t test with Welch’s correction between the non-linear fitting curves). (D) GFP-PD-1-expressing primary human T cells co-cultured with SEE-loaded Raji B cells expressing mCherry PD-L1 treated with nivolumab and its F(ab′) 2 . (E) GFP-PD-1 expressing Jurkat T cells co-cultured with SEE-loaded Raji B cells expressing mCherry PD-L1 treated with an isotype control antibody. (F) Z stack-based three-dimensional reconstruction images of Jurkat T cells expressing GFP-PD-1 co-cultured with SEE-loaded Raji B cells expressing mCherry PD-L1 and treated as indicated.

Article Snippet: Clinical-grade nivolumab fragments, clinical-grade durvalumab fragments, and rat-anti-mouse-PD-1 antibody (catalog number BE0146, InVivoMAb) were prepared by Pierce F(ab′)2 Macro Preparation Kit (catalog number 44988).

Techniques: Staining, Flow Cytometry, Binding Assay, Expressing, Cell Culture, Control

Journal: Molecular Therapy Oncology

Article Title: Exclusion of PD-1 from the immune synapse: A novel strategy to modulate T cell function

doi: 10.1016/j.omton.2024.200839

Figure Lengend Snippet: Full-length anti-PD-L1 antibodies removed PD-1 from the IS (A) Coomassie staining of non-reduced full-length durvalumab and durvalumab treated with pepsin. (B) Flow cytometry of Raji B cells treated with full-length durvalumab and durvalumab F(ab′) 2 , as indicated. (Ci) GFP-PD-1-expressing Jurkat T cells co-cultured with SEE-loaded Raji B cells expressing mCherry PD-L1 treated with durvalumab and its F(ab′) 2 and quantification of the number of cells where PD-1 was recruited to the IS at different concentrations of both antibodies (Cii). n = 3, p < 0.001 (unpaired t test with Welch’s correction between the non-linear fitting curves).

Article Snippet: Clinical-grade nivolumab fragments, clinical-grade durvalumab fragments, and rat-anti-mouse-PD-1 antibody (catalog number BE0146, InVivoMAb) were prepared by Pierce F(ab′)2 Macro Preparation Kit (catalog number 44988).

Techniques: Staining, Flow Cytometry, Expressing, Cell Culture

Journal: Molecular Therapy Oncology

Article Title: Exclusion of PD-1 from the immune synapse: A novel strategy to modulate T cell function

doi: 10.1016/j.omton.2024.200839

Figure Lengend Snippet: Removing PD-1 from the IS is associated with increased T cell activation (A) IL-2 levels of Jurkat T cell and Raji B cell with different concentrations of either full-length nivolumab or its F(ab′) 2 in the presence of SEE. n = 3, p < 0.001 (unpaired t test with Welch’s correction between the non-linear fitting curves). (B) IL-2 and IFN-gamma levels of human PBMC assay of cells treated with SEE and full-length nivolumab or its F(ab′) 2 as indicated and measured by ELISA. n = 3, p < 0.001 (unpaired t test with Welch’s correction between the non-linear fitting curves). (C) A Co-immunoprecipitation (Co-I) experiment of Jurkat T cells treated with SEE or anti-PD-1 antibodies for 5 min, followed by PD-1 precipitation with anti-GFP antibodies and western blotting with anti-4G10 antibodies (Ci). The quantitative analysis of the experiment described in C is shown (Cii). n = 3–4 independent experiments, paired two-tailed t test; ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Clinical-grade nivolumab fragments, clinical-grade durvalumab fragments, and rat-anti-mouse-PD-1 antibody (catalog number BE0146, InVivoMAb) were prepared by Pierce F(ab′)2 Macro Preparation Kit (catalog number 44988).

Techniques: Activation Assay, PBMC Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: Molecular Therapy Oncology

Article Title: Exclusion of PD-1 from the immune synapse: A novel strategy to modulate T cell function

doi: 10.1016/j.omton.2024.200839

Figure Lengend Snippet: Anti-PD-1 F(ab′) 2 cannot inhibit tumor growth in vivo (A) Coomassie staining of non-reduced full-length anti-murine PD-1 treated with pepsin. (B) Flow cytometry of the EL4 murine T cell line treated with the antibodies, as indicated. (C) MC38 syngeneic tumor model showing tumor growth of mice treated with full-length anti-PD-1 antibodies twice a week for four doses or with the same molar amount of anti-murine PD-1 F(ab′) 2 . n = 5, two-way ANOVA. (D) H&E staining of the tumors at the end of the experiment. (E) Flow cytometry of both the full length and the F(ab′) 2 versions of the anti-PD-1 antibodies using T cells isolated from the spleens of these mice. The numbers displayed in the boxes are the percentages of the cells bound with the target full length and the F(ab′) 2 versions of the anti-PD-1 antibodies. (F) Model for antibody-mediated PD-1 removal from the IS.

Article Snippet: Clinical-grade nivolumab fragments, clinical-grade durvalumab fragments, and rat-anti-mouse-PD-1 antibody (catalog number BE0146, InVivoMAb) were prepared by Pierce F(ab′)2 Macro Preparation Kit (catalog number 44988).

Techniques: In Vivo, Staining, Flow Cytometry, Isolation

Journal: Frontiers in Immunology

Article Title: Negative Regulation of Humoral Immunity Due to Interplay between the SLAMF1, SLAMF5, and SLAMF6 Receptors

doi: 10.3389/fimmu.2015.00158

Figure Lengend Snippet: Administration of anti-SLAMF6 (330) F(ab’) 2 antibody has a similar negative effect on GC B cell and Tfh cell development in protein-immunized WT mice . Mice were immunized with 40 μg of NP-OVA in CFA and some were injected with 250 μg of anti-SLAMF6 F(ab’) 2 on day 0 and day 4. The mice were sacrificed on day 9 and serum was collected to measure IgG production. (A) NP-specific IgG titers were determined by ELISA. (B–C) The percentage and number of Germinal Center B cells (B220 + GL7 + Fas + ) were determined by flow cytometry. (D–E) The percentage and number of Tfh cells (CD4 + PD-1 + CXCR5 + ) were determined by flow cytometry. Results are representative of three independent experiments.

Article Snippet: Anti-SLAMF6 F(ab’) 2 fragments were generated from whole SLAMF6 mAb, using the Thermo Scientific Pierce F(ab’) 2 Preparation Kit (PI-44988) according to manufacturer instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Flow Cytometry