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. Author manuscript; available in PMC: 2011 Mar 29.
Published in final edited form as: J Immunol. 2010 Mar 17;184(8):4228–4235. doi: 10.4049/jimmunol.0901908

Figure 5. Itk(BtkSH3) exhibits enhanced signaling in T cells.

Figure 5

(a–c) Itk−/− primary CD4+ T cells were infected with the retrovirus vector alone (MSCV, solid line), a retrovirus expressing wild-type Itk (Itk, long dashed line) or a retrovirus expressing Itk(BtkSH3) (short dashed line); GFP+ cells were sorted on day 4 post-infection. (a) Histograms of GFP fluorescence at the time of analysis are shown compared to non-infected control cells (filled histogram). Cells were stimulated with anti-CD3 antibody for 5 minutes, fixed, permeabilized, and stained with anti-phospho-ERK antibody. The percentages of phospho-ERK+ cells are indicated on each histogram. The non-stimulated control shown (NS) is MSCV infected cells. Non-stimulated controls for all cell lines are provided in Supplementary Materials. (b) The graph shows data from three experiments described in (a). Bars indicate mean±S.D. of the percentage of phospho-ERK+ T cells. *, p<0.0005; **, p<0.0006; ***, p<0.004 (using Paired t-test). (c) Itk−/− T cells infected with vector alone (lane 3), retrovirus expressing wild-type Itk (lane 4) or retrovirus expressing Itk(BtkSH3) (lane 5) were stimulated with crosslinking anti-CD3 antibody for 5 minutes, lysed, and immunoblotted for Itk (top panel), phospho-ERK (middle panel) and total ERK (bottom panel). Lanes 1 and 2 represent primary Itk+/+ CD4+ T cells stimulated with anti-CD3 antibody for 0 and 5 minutes, respectively. (d and e) Itk+/+ primary CD4+ T cells were infected with the retrovirus vector alone (MSCV, solid line), a retrovirus expressing wild-type Itk (Itk, long dashed line) or a retrovirus expressing Itk(BtkSH3) (short dashed line) and treated in a manner identical to the data experiment shown in (a) and (b). (f) Wild-type or Itk−/− CD4+ T cells were infected with pMX-IRES-hCD8 vector alone or vector containing Itk constructs. Four days after infection, hCD8+ T cells were gated on and analyzed for calcium mobilization in response to anti-CD3 stimulation. Levels of wild-type Itk and Itk(BtkSH3) protein in the transduced Itk−/− T cells were comparable, as assessed by hCD8 staining (See Supplementary Materials). Data are representative of three independent experiments with similar results.