Journal: The Journal of Immunology Author Choice

Article Title: A Small Molecule Inhibitor of ITK and RLK Impairs Th1 Differentiation and Prevents Colitis Disease Progression

doi: 10.4049/jimmunol.1501828

Figure Lengend Snippet: PRN694 leads to impaired P-selectin binding and inhibits chemokine-induced CD4+ T cell migration. (A–E) Isolated naive CD4+ T cells were cultured in the presence or absence of PRN694 (25 or 50 nM) for 72 h in each Th polarization condition. (A) Histograms show polarized CD4+ T cells stained with recombinant P-selectin–human IgG fusion protein as an assay for P-selectin glycoprotein ligand 1 binding. Bold numbers above region indicate the percentages of cells staining positively for P-selectin–Ig based on comparison with an isotype control stain; numbers at the left of each histogram indicate the mean fluorescence intensity of P-selectin–Ig staining in each panel. (B) Graph shows a compilation of data from two independent experiments. The data were normalized to the percentage of P-selectin–binding cells among each Th-polarizing condition in the absence of inhibitor. (C–E) Cultured CD4+ T cells (5.0 × 105 cells) were plated in the upper chamber of HTS Transwell-96 well permeable supports (3.0-μm pore); bottom chambers were loaded with diluted CXCL11 (100 ng/ml) (C), CCL20 (100 ng/ml) (D), or CCL25 (300 ng/ml) (E). After 3 h at 37°C, the numbers of migrated cells were counted by the flow cytometer. The chemotaxis index was calculated by dividing the number of cells that migrated in response to each chemokine by the number of spontaneously migrated cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Recombinant P-selectin–human IgG fusion protein (BD Biosciences) was added to the cells (1:200 dilution) at 4°C for 30 min.

Techniques: Binding Assay, Migration, Isolation, Cell Culture, Staining, Recombinant, Fluorescence, Flow Cytometry, Chemotaxis Assay