Figure 2. RGS16 inhibits T_H2 chemotaxis.

(A) Naïve T cells were isolated from peripheral LNs and differentiated into T_H1, T_H2, or T_H17 cells as outlined in the Methods. Relative Rgs16 expression was measured by real-time PCR (**P = 0.005, *P < 0.01, unpaired t-test). (B) Whole splenocytes or splenic CD4 T cells C57/Bl6 and Rgs16^–/– mice were exposed to chemokine gradients in transwell plates for 3 h at 37°C, followed by enumeration of migrated cells by flow cytometry. (C) Migration of T_H1 or T_H2 cells towards CXCL9 or CCL17 gradients, respectively, at the indicated concentrations was measured as in (B) (*P = 0.04, **P < 0.003, unpaired t-test). (D) Chemokinesis was measured by incubating cells with equimolar concentrations of CCL17 (50 nM) in the upper and lower chambers of transwell plates followed by enumeration of cells by flow cytometry. (E) Rgs16 expression in cells migrated to the lower chamber of CCL17-containing transwell plates (“migratory”) was compared to cells retained in the upper chamber (“non-migratory”) by real-time PCR (*P = 0.04, paired t-test). (F) T_H2 cells from Rgs16^–/– mice were left untreated or pre-incubated with TAT-GFP or TAT-RGS16 (60-500 nM) for 1 h prior to exposure to CCL17 gradients in transwell assays (***P < 0.001, 1 way ANOVA, TAT-RGS16 compared to untreated or TAT-GFP). All data are mean ± S.E.M of 3-4 independent experiments using cells from 1 mouse/group in each.