Journal: PLoS ONE

Article Title: Expression of the Receptor Tyrosine Kinase EphB2 on Dendritic Cells Is Modulated by Toll-Like Receptor Ligation but Is Not Required for T Cell Activation

doi: 10.1371/journal.pone.0138835

Figure Lengend Snippet: BMDCs were matured from mouse bone marrow (A) and incubated with the MF2.2D9 T cell hybridoma and chicken ovalbumin (OVA) (B) or OVA 257-278 peptide (C) for 18 hours. The amount of IL-2 secreted by the MF2.2D9 T cell hybridoma was measured using HT-2 cells and the cell number enumerated using an ATP-luminescence assay. The graphs represent the mean value ±SD of 3 replicate wells for each dilution of EphB2-/- BMDCs (gray lines) or littermate control BMDCs (black lines). Circles represent no addition of a stimulus and stimulated BMDCs are represented by squares. (D) BMDCs derived from bone marrow of EphB2-/- or EphB2+/+ mice were incubated with splenic CD4+ T cells purified from naïve OT-II T cell receptor transgenic mice (n = 5) and OVA protein for 5 days. The development and expansion of Th1 and Th2 cells were measured by intracellular staining of interferon (IFN)-γ and interleukin (IL)-4 respectively (E). All graphs are representative of at least 2 experiments, with the exception of (E) which is the median value of pooled data ±SD from 3 separate preparations of BMDCs.

Article Snippet: T cells were purified from single cell splenocyte preparations from 5 naïve OT-II mice using CD4+ Miltenyi MACs beads according to the manufacturer’s instructions or sorted into CD4+Vα2TCR+ cells on a FACS Aria cell sorter (Becton Dickinson).

Techniques: Incubation, Luminescence Assay, Derivative Assay, Purification, Transgenic Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: The structural features that distinguish PD-L2 from PD-L1 emerged in placental mammals

doi: 10.1074/jbc.AC119.011747

Figure Lengend Snippet: Effect of PD-ligand affinity for PD-1 on T-cell inhibition. A–G, proliferation of primary human CD4+ T-cell blasts in response to anti-CD3 and anti CD-28 antibodies was measured by CFSE dilution without (A) or with the indicated ligand absorbed at the indicated concentration. H, cumulative data from multiple independent experiments with the indicated absorbed concentration. I, correlation between T-cell inhibition and affinity for PD-ligand variants. A–I, data are from a single healthy donor. J, IL-2 release from primary human T cells in response to SEE bound to Raji B cells with or without ectopic and equal expression of PD-ligands (24 h co-culture). Data are from three healthy donors assessed independently. K, effect of 20 μg/ml Nivolumab or the indicated PD-ligand Fc fusion protein on IL-2 secretion from human PBMCs in response to increasing concentrations of SEB superantigen. Data shown represent independent experiments from four healthy donors. Bar graphs: paired t tests: *, p < 0.05; **, p < 0.01. Dose-response curves: two-way analysis of variance; ****, p < 0.0001; ns, not significant.

Article Snippet: Primary human CD4 + T cells were isolated from peripheral blood of a healthy volunteer using the CD4 MicroBeads kit (MACS Miltenyi Biotec).

Techniques: Inhibition, Concentration Assay, Expressing, Co-Culture Assay