Journal: bioRxiv

Article Title: PTPN22 interacts with EB1 to regulate T cell receptor signaling

doi: 10.1101/481507

Figure Lengend Snippet: A Compared with the PTPN22 WT, PTPN22 ΔP1 can increase remarkably the phosphorylation levels of ZAP-70, Lck and Erk. B, C, D Western blot and qPCR assays were used to test the protein and mRNA expression levels of CD25, CD69 and IL-2, and the results shown that the protein and mRNA expression levels of CD25, CD69 and IL-2 increased significantly in PTPN22 ΔP1 transfected cells. Western blot assays were used to test the protein expression levels of CD25 and CD69 (B-C) or qPCR assays were used to test the mRNA expression levels of CD25, CD69 and IL-2 (D). E, F, G Deletion of P1 domain of PTPN22 will not affect its enzyme activity. pNPP hydrolysis with PTPN22 catalytic domain and PTPN22 ΔP1 (E) or Kinetics parameters for the wild-type and the mutants of PTPN22 toward pNPP (F,G). Data information: All Experiments were carried three times (A-E) with similar results. In (A-C), Significant differences were calculated with t-test and are indicated with *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: CD25 ELISA was performed using the pre-packaged CD25 ELISA kit (DY2438, R & D system), while CD69 ELISA was performed using the pre-packaged CD69 ELISA kit (TWp022633, www.tw-reagent.com ) following the manufacturer’s protocols.

Techniques: Western Blot, Expressing, Transfection, Activity Assay

Journal: bioRxiv

Article Title: PTPN22 interacts with EB1 to regulate T cell receptor signaling

doi: 10.1101/481507

Figure Lengend Snippet: A, B Test the protein expression levels of CD25 and CD69 by use of ELISA. ELISA assays were used to test the expression levels of CD25 (A) or Same assays were used to test the expression levels of CD69 (B). C, D Test the mRNA transcription levels of CD25 and CD69 by use of qPCR. qPCR assays were used to test the expression levels of CD25 (C) or Same assays were used to test the expression levels of CD69 (D). Data information: Significant differences were calculated with t-test and are indicated with *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: CD25 ELISA was performed using the pre-packaged CD25 ELISA kit (DY2438, R & D system), while CD69 ELISA was performed using the pre-packaged CD69 ELISA kit (TWp022633, www.tw-reagent.com ) following the manufacturer’s protocols.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Multiple intravenous injections of allogeneic equine mesenchymal stem cells do not induce a systemic inflammatory response but do alter lymphocyte subsets in healthy horses

doi: 10.1186/s13287-015-0050-0

Figure Lengend Snippet: Multiple allogeneic mesenchymal stem cell (MSC) injections result in changes in splenic regulatory T cell percentages. (A-D) There were no significant changes in splenic CD21 + B-cell (A) , CD4 + T-cell (B) , or CD8 + T-cell percentages (C) or CD4/CD8 ratios (D) following multiple MSC injections. (E) There were no significant changes in activated (CD25 + ) lymphocyte proportions. (F) There were significantly higher percentages of splenic FoxP3 + regulatory T cells in the horses injected with bone marrow (BM)-derived MSCs compared with horses injected with adipose tissue (AT)-derived MSCs. Data are presented as mean ± standard error of the mean. * P <0.05.

Article Snippet: The following antibodies were used: mouse-anti-equine CD3 (clone UC F6G 1:250; Jeffery Stott, University of California, Davis, CA, USA) [ ], mouse-anti-human CD21 (clone B-ly4 1:20; BD Pharmingen, San Jose, CA, USA) [ , ], polyclonal goat-anti-human CD25 (clone AF-223; R&D Systems) [ ], rat-anti-mouse/human FoxP3 (clone PCH101; ebioscience, San Diego, CA, USA) [ ], mouse-anti-CD4 (clone HB61A 1:133; VMRD, Pullman, WA, USA) [ ], mouse-anti-equine CD8 (clone F18P 1:500; J. Stott) [ ], and a donkey-anti-mouse secondary when necessary (1:50; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA).

Techniques: Injection, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: Structure and in vitro characterization of CD25-ADC. (A) Structure and (B) in vitro characterization of CD25-ADC. (i) ELISA showing binding of anti-CD25 antibody PC61 to mouse recombinant CD25. (ii–iv) Flow cytometry measurement of PC61 and isotype-control antibody binding to Yac-1, MC38 and CT26 cells. (v–vii) Yac-1, MC38 and CT26 cells’ viability after exposure to CD25-ADC and isotype-ADC (and the naked pyrrolobenzodiazepine-dimer SG3199 in MC38 and CT26 cell lines). MFI, median fluorescence intensity; PABA, para amino benzoic acid.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Binding Assay, Recombinant, Flow Cytometry, Control, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: In vivo antitumor activity of CD25-ADC in the s.c. MC38 syngeneic model. Treatment with (i) vehicle, (ii) anti-PD-1 antibody (5 mg/kg, on days 2, 5, and 8), (iii) non-binding ADC (1 mg/kg, single dose on day 1) alone or (iv) in combination with anti-PD-1 antibody, (v–vii) CD25-ADC (0.1, 0.5, and 1 mg/kg single dose on day 1) alone or (viii–x) in combination with anti-PD-1 antibody, started at a group mean tumor volume of 103 mm 3 . Data are shown as tumor volumes (mm 3 ) over time for each individual mouse (10 mice/group). (xi) Survival of mice shown in i–x. Lines for G4, G5, G8 and G9 are overlapping.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: In Vivo, Activity Assay, Binding Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: In vivo antitumor activity of CD25-ADC in the s.c. CT26 syngeneic model. Treatment with (i) vehicle, (ii) anti-PD-1 antibody (5 mg/kg, on days 2, 5, and 8), (iii) isotype-ADC (1 mg/kg, single dose on day 1) alone or (iv) in combination with anti-PD-1 antibody, (v–vii) CD25-ADC (0.1, 0.5, and 1 mg/kg single dose on day 1) alone or (viii–x) in combination with anti-PD-1 antibody, started at a group mean tumor volume of 110 mm 3 . Data are shown as tumor volumes (mm 3 ) over time for each individual mouse (10 mice/group). (xi) Survival of mice shown in i–x.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: In Vivo, Activity Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: Role of CD8+ T eff cells in CD25-ADC antitumor activity in the MC38 syngeneic model. Depletion of CD8+ T eff cells significantly reduces the antitumor activity of CD25-ADC. CD25-ADC was administered intraperitoneally (i.p.) at a group mean tumor volume of 89 mm 3 as a single dose on day 1 at 0.5 mg/kg alone or in combination with anti-PD-1 antibody (5 mg/kg, on days 2, 5, and 8). Anti-CD8 T-cell depleting antibody (10 mg/kg) was injected i.p. on days 0, 5, 8, and 13. Data are shown as mean tumor volumes (mm 3 ) ± SEM over time (n=10/group).

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: Activity Assay, Injection

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: Intratumoral T-cell immunophenotype analysis in MC38-bearing mice. (A) Absolute quantification of intratumoral T regs , CD8+ T cells and CD8+/T reg ratio following i.p. treatment with anti-PD-1 antibody or CD25-ADC or the combination of CD25-ADC and anti-PD-1. (B) Percentage of CD69+, Ki67+ and IFNγ+ tumor-infiltrating CD8+ T cells. Tumors were processed at the indicated times (days post CD25-ADC dose). Horizontal bars represent median value. Statistical differences between treatment groups were calculated using JMP 15 by the Dunn method for joint ranking. Results were considered significant when p<0.05. *, p≤0.05; **, p≤0.01. IFN, interferon.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: Quantitative Proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: Circulating and thymic T-cell immunophenotype analysis in MC38-bearing mice. (A) Absolute quantification of circulating T regs , CD8+ T cells and CD8+/T reg ratio following i.p. treatment with anti-PD-1 antibody or CD25-ADC or the combination of CD25-ADC and anti-PD-1. Blood was processed at the indicated times (days post CD25-ADC dose). (B) Absolute quantification of thymic T reg cells and CD8+/T reg ratio following i.p. treatment with anti-PD-1 antibody or CD25-ADC or the combination of CD25-ADC and anti-PD-1. Thymus was processed at the indicated times (days post CD25-ADC dose). Statistical differences between treatment groups were calculated using JMP 15 by the Dunn method for joint ranking. Results were considered significant when p<0.05. *, p≤0.05; **, p≤0.01.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: Quantitative Proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: CD25-targeted antibody–drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity

doi: 10.1136/jitc-2020-000860

Figure Lengend Snippet: T-cell dynamic study in non-tumor-bearing mice. Effect of CD25-ADC on the percentage of T regs and T eff levels in non-tumor-bearing mice. Female C57BL/6 mice were injected i.p. with vehicle, CD25-ADC (0.5 mg/kg), or isotype control ADC (0.5 mg/kg) on day 0. (A) Spleen, (B) lymph node, and (C) thymus were collected 4 hours post dose, and 6, 13, and 20 days post dose for T-cell immune profiling. Levels of T regs , CD8+ T, and conventional CD4+ T cells in spleen, lymph nodes, and thymus are presented as % of CD45 cells±SEM over time.

Article Snippet: Binding of PC61 to mouse recombinant CD25 (R&D Systems) was determined by ELISA, using a mouse CD25/human Fc chimeric antigen (R&D Systems) and a secondary goat antirat HRP (Jackson Immunoresearch Laboratories).

Techniques: Injection, Control