Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activation-induced cell death of self-reactive regulatory T cells drives autoimmunity

doi: 10.1073/pnas.1910281116

Figure Lengend Snippet: TAZ10 CD4+CD25+Foxp3+ Treg suppress Teff proliferation in vitro. (A) Depletion of CD25+ T cells was done by incubating TAZ10 LNs and splenocytes (from pooled mice, n = 6) with the anti-CD25 monoclonal antibody clone 7D4 in conjunction with rabbit complement. Complement only was used as control. Depletion’s efficiency was verified by costaining with CD4 and CD25 (clone PC61). Numbers in quadrants indicate cell percentages. Untreated (C, red) or CD25 depleted (7D4, blue) TAZ10 splenocytes were challenged with: plate-bound CD3 antibody alone or in conjunction with CD28 for 18 h (B), and the levels of IFNγ, IL-2, and IL-10 were assessed by ELISA (C); CD3 in the presence of increasing concentrations of blocking anti-mouse DTA-1 mAb [0–100 mg/mL] (D); increasing concentrations of TPO peptide (TPO536–547) [0.1–10 μM] (E); TPO536–547 [10 μM] in the presence or absence of sorted TAZ10 CD4+CD25+ Treg (green) (F). (B–F) Data show 1 representative of 3 (B and D–F) or 4 (C) independent experiments, each done in triplicate with 3 independent wells (mean + SEM). Proliferation was assessed by 72-h thymidine incorporation assay adding Tritiated Thymidine in the last 18 h of the assay. (B–F) Statistical analysis was performed by using 2-tailed unpaired Student’s t test (nonsignificant [n.s.], P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001).

Article Snippet: In vitro depletion of CD25 + T cells was achieved with anti-mouse CD25 clone 7D4 and rabbit complement (Harlan Seralab).

Techniques: In Vitro, Control, Enzyme-linked Immunosorbent Assay, Blocking Assay, Thymidine Incorporation Assay