Journal:

Article Title: Inducing CTLA-4-dependent Immune Regulation by Selective CD28 blockade Promotes Regulatory T cells in Organ Transplantation

doi: 10.1126/scitranslmed.3000116

Figure Lengend Snippet: (A) Mixed-lymphocyte reaction using human (n=10), baboon (n=15) or macaque (n=9) PBMC. Black bars: mean ±SD in control conditions (mouse irrelevant IgG1). White bars: mean ± SD with 10 µg/ml sc28AT. (B) IL-2 secretion by Jurkat T cells stimulated with bacterial superantigen (staphylococcal enterotoxin E, SEE) and Raji B cells in the presence of sc28AT (n=8) or CTLA4-Ig (n=3). Results are expressed as percentage of IL-2 secretion observed in the absence of Ab (100%). (C) Suppressive activity of human Treg is not impeded by CD28 blockade. Tregs were added to CD4+CD25− T cells stimulated with allogeneic irradiated PBMC at the indicated ratio in the presence of 10µg/ml of CD28 or CTLA-4 blocking antibodies. Results are mean cpm ± SD of one representative assay out of 3. (D) Suppressive activity of human Treg pre-treated with CD28 or CTLA-4 blocking Ab. Treg were first cultured with allogeneic mature DC in the presence of sc28AT or anti-CTLA-4 Fab fragments (10µg/ml) for 18h, washed and assessed in a suppression assay. Results are mean cpm ± SD of a representative assay out of 3. *, ** and *** indicate a significant difference at p<0.05, 0.01 and 0.001, respectively.

Article Snippet: Baboon Treg suppression assays CD25 + and CD25 − cells were prepared from PBMC during the 3 rd month post transplantation using FITC anti-human CD25 mAb and specific anti-FITC microbeads (Miltenyi) and positive or negative selection, respectively.

Techniques: Control, Activity Assay, Irradiation, Blocking Assay, Cell Culture, Suppression Assay

Journal:

Article Title: Inducing CTLA-4-dependent Immune Regulation by Selective CD28 blockade Promotes Regulatory T cells in Organ Transplantation

doi: 10.1126/scitranslmed.3000116

Figure Lengend Snippet: (A) CD25+ CD127 lo Treg cells analyzed in blood by flow cytometry after gating on CD3+ CD4+ cells. These cells also expressed CD28, and intra-cellular CTLA-4 and Foxp3. (B-C) Kinetics of CD4+CD25+Foxp3+CD127lo Treg levels in blood, percentage of CD4+ T cells (B) or absolute number (C), in control untreated animals (n=3), sc28AT monotherapy (n=4), Tacrolimus monotherapy (n=4 until week 1 and then n=2), sc28AT + Tacrolimus (n=5 up to 2 weeks, 4 at 1 month and then n=3). D: days, W: weeks, M: month. # and ##, significant difference with the untreated group (P=0.02 and P=0.0011, respectively). ***, ** and * significant difference with the Tacrolimus monotherapy group (P<0.05, 0.01 and 0.001, respectively). (D) Suppressive activity of CD25+ PBMC from sc28AT + Tacrolimus recipients at day 90 after transplantation (black bars, n=3) or from control ungrafted baboons treated with Tacrolimus (white bars, n=4).

Article Snippet: Baboon Treg suppression assays CD25 + and CD25 − cells were prepared from PBMC during the 3 rd month post transplantation using FITC anti-human CD25 mAb and specific anti-FITC microbeads (Miltenyi) and positive or negative selection, respectively.

Techniques: Flow Cytometry, Control, Activity Assay, Transplantation Assay