Figure 4

Enhanced Src kinase activity results in phosphorylation of CTLA-4. (A) Primary human T cells transfected with Renilla (ctrl) or PAG (PAGsi) siRNA were stimulated for 72 hours on an anti-CD3+anti-CD28 coated plastic plate. The surface expression of CTLA-4 was measured by extracellular FACS staining. (B) T cells transfected as in (A) were stimulated overnight on anti-CD3+anti-CD28 coated plastic plate, lysed and CTLA-4 was immunoprecipitated. Samples were immunoblotted for phospho-tyrosine (4G10), Shp1, Shp2, Fyn, and CTLA-4. Antibody control (Ab) was included in the IP’s. Panel left shows whole lysates. Data are representative of three independent experiments. Quantification of the blots can be found in the supplemental material (Additional file 7: Figure S7). (C) To exclude an off-target effect of our siRNA, a pool of three siRNAs (all differ from our sequence) was used for the same experiment as in (B). (D) Murine T cells from wildtype and Fyn knockout mice were cultured for three days on anti-CD3+anti-CD28 coated plastic plate, lysed and CTLA-4 was immunoprecipitated. Western blot of a phospho-tyrosine (4G10) and total CTLA-4 staining is shown. Panel left shows whole lysates. Data are representative of two independent experiments. (E). The CTLA-4 mediated reduction of IFNγ production is abbrogated in Fyn^−/− mice. Naïve (CD62L^high) CD8⁺ T cells from WT or Fyn^−/− mice were stimulated with anti-CD3/CD28/CTLA-4 or anti-CD3/CD28/Isotype-coupled microspheres. Surface expression of the activation markers CD25 and CD44, as well as intracellular IFNγ expression were detected by flow cytometry after 2 days of activation.