Spinophilin plays a functional role in antigen presentation in vivo. (a) Protocol: CD4+ T cells were isolated form TCR-transgenic mice (OT-II) and labeled with CFSE. 106 labeled cells were injected i.v. into WT and KO littermates. 1 d later, animals were injected i.v. with 10 μg ovalbumin + 100 ng LPS or LPS alone. On day three, spleen cells from WT and KO mice were isolated and analyzed for proliferation as measured by CFSE dilution or restimulated with ovalbumin (0, 10, and 20 μg/ml) for an additional 3 d. Intracellular cytokine staining was then performed. (b) The CD69hi population of adoptively transferred T cells was smaller in spinophilin KO (red) than in spinophilin WT (black) mice. Representative flow cytometry plots (left) and the data pooled from four independent experiments (right) is shown (n = 14 WT and 15 KO mice total; *, P < 0.05 by Student's t test). (c) The relative abundance of IFNγ-producing effector T cells was significantly greater in WT than in KO, as measured by flow cytometry of intracellular cytokine staining. (left) Representative flow cytometry plots. (right) Data pooled from three independent experiments (n = 8 animals total for WT and KO; ***, P < 0.001 by Student's t test).
