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. 2010 Jul;30(14):3596-609.
doi: 10.1128/MCB.01657-09. Epub 2010 May 10.

In Vivo Significance of ITK-SLP-76 Interaction in Cytokine Production

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Free PMC article

In Vivo Significance of ITK-SLP-76 Interaction in Cytokine Production

Juris A Grasis et al. Mol Cell Biol. .
Free PMC article

Abstract

In vitro data have suggested that activation of the inducible T-cell kinase (ITK) requires an interaction with the adaptor protein SLP-76. One means for this interaction involves binding of the ITK SH3 domain to the polyproline-rich (PR) region of SLP-76. However, the biological significance of this association in live cells and the consequences of its disruption have not been demonstrated. Here, we utilized a polyarginine-rich, cell-permeable peptide that represents the portion of the SLP-76 PR region that interacts with the ITK SH3 domain as a competitive inhibitor to disrupt the association between ITK and SLP-76 in live cells. We demonstrate that treatment of cells with this peptide, by either in vitro incubation or intraperitoneal injection of the peptide in mice, inhibits the T-cell receptor (TCR)-induced association between ITK and SLP-76, recruitment and transphosphorylation of ITK, actin polarization at the T-cell contact site, and expression of Th2 cytokines. The inhibition is specific, as indicated by lack of effects by the polyarginine vehicle alone or a scrambled sequence of the cargo peptide. In view of the role of ITK as a regulator of Th2 cytokine expression, the data underscore the significance of ITK as a target for pharmacological intervention.

Figures

FIG. 1.
FIG. 1.
FITC-R9-QQP entry into Jurkat cells and murine splenocytes. Jurkat cells (A) or murine splenocytes (B) were incubated with various concentrations of FITC-R9-QQP, as indicated in the tables, and analyzed by flow cytometry. The results are displayed as percentages of maximum events (linear) versus fluorescence intensity (logarithmic). The tables display quantification of MFIs under various conditions from the histograms. The images at the bottom are representative midsection confocal and differential interference contrast images of individual Jurkat cells and splenocytes, respectively, treated with 10 μM peptide. (C) Jurkat cells treated with FITC-R9-QQP and then incubated with trypsin (0.05% for 10 min at 37°C), as indicated in the table, before flow cytometric analysis. (Bottom) Midsection confocal images of cells treated with FITC-R9-QQP (20 μM), where the outline of the cell membrane has been accentuated by treatment with Cy5 succinimidyl ester. (D) Murine splenocytes were incubated with 10 μM FITC-R9-QQP for various periods, as indicated, treated with trypsin as for panel C, washed, and stained with Alexa Fluor 647-conjugated anti-CD4 antibody, and the CD4-positive cells were analyzed by flow cytometry for the percentage of FITC-positive cells and MFI. (E) Murine splenocytes treated with the indicated amounts of R9-QQP were stimulated with anti-CD3ɛ and anti-CD28 antibodies, as described in Materials and Methods. The cells were then stained with Alexa Fluor 647-conjugated anti-CD4 antibody and loaded with 7-AAD. The CD4-positive cells were analyzed for the percentage of 7-AAD-positive (dead) cells. The data shown in panels A and B are representative of two replicate experiments. The data in panels C to E are from single experiments.
FIG. 2.
FIG. 2.
R9-QQP specifically disrupts the association between ITK and SLP-76. (A) Jurkat cells treated with various peptide concentrations, as indicated, were stimulated with anti-CD3ɛ (+) or isotype control (−) antibodies, the cells were lysed, the lysates were precleared with rabbit IgG and immunoprecipitated (IP) with anti-SLP-76 antibody, and immune complexes were resolved by SDS-PAGE. The membranes were sequentially immunoblotted (WB) with anti-ITK, anti-LCK, anti-HPK-1, anti-GADS, and anti-SLP-76 antibodies. (B) Graphic representation of SLP-76 association with the indicated signaling partners displayed as percent association with SLP-76. The results are the averages (± standard errors of the mean [SEM]) of three replicate experiments performed as in panel A. The percent association with SLP-76 was calculated by densitometric analysis of immunoblotted bands as detailed in Materials and Methods.
FIG. 3.
FIG. 3.
R9-QQP specifically inhibits the TCR-induced recruitment of ITK, but not SLP-76. Shown are representative confocal images of conjugates between GFP-ITK-nucleofected Jurkat (E6.1) cells (treated or not with 20 μM R9-QQP) and control (nonstimulated) (A) or anti-CD3ɛ antibody-coated (stimulated) (B and C) beads. (Left) Confocal fluorescence images. The asterisks indicate the positions of the beads. (Right) Differential interference contrast images. (D) Wild-type (E6.1) or SLP-76-deficient (J14) Jurkat cells that had been transfected with GFP-ITK by nucleofection were incubated with control (− stimulation) or anti-CD3ɛ (+ stimulation) antibody-coated beads, and the ITK localization index was determined as described in Materials and Methods. The results are displayed as the average ITK localization index (±SEM) of 20 to 40 conjugates from two replicate experiments. (E) Wild-type Jurkat cells (E6.1) transfected with GFP-ITK as described above were pretreated with R9-QQP or R9-SCR peptide and incubated with either control (− stimulation) or anti-CD3ɛ antibody-coated (+ stimulation) beads, as indicated. The ITK localization index was determined as described in Materials and Methods and is expressed as the average (±SEM) of 25 to 46 conjugates from two replicate experiments. (F) SLP-76-deficient Jurkat cells (J14) transfected with GFP-SLP-76 were treated as for panel E, and the SLP-76 localization index was determined as described above. The results are the average SLP-76 localization index (±SEM) of 17 to 32 conjugates from two replicate experiments.
FIG. 4.
FIG. 4.
R9-QQP specifically disrupts TCR-induced actin polarization. Representative midsection confocal images of Jurkat (treated or not with R9-QQP) cell-bead conjugates formed with control (A) or anti-CD3ɛ antibody-coated (B and C) beads. For each panel, the Texas Red fluorescence (left) and differential interference contrast (right) images are shown. The asterisks in the fluorescence images indicate the positions of the beads. (D) Jurkat cells treated with medium, R9-QQP, or R9-SCR were incubated with control (− stimulation) or anti-CD3ɛ antibody-coated (+ stimulation) beads, as indicated. The cell-bead conjugates were imaged, and actin polarization was determined as described in Materials and Methods. The results are displayed as the average percentage (±SEM) of cells with polarized actin from 2 to 6 replicate experiments. Each experiment included 18 to 23 conjugates.
FIG. 5.
FIG. 5.
R9-QQP specifically inhibits the TCR-mediated phosphorylation of ITK. (A) Jurkat cells pretreated with 27 μM R9-QQP, R9-SCR, or no peptide were incubated with anti-CD3ɛ (stimulated) or isotype control antibodies (nonstimulated) and analyzed by flow cytometry using anti-ITK phosphotyrosine 511 antibodies. The data are from one representative experiment presented as the total cell count versus the Itk-pY511 fluorescence intensity (log scale). (B) Averages (±SEM) of four replicate experiments performed as for panel A using Jurkat cells pretreated with the indicated amounts of R9-QQP or control peptides. The data are presented as the percentage of cells positive for ITK Y511 phosphorylation calculated as described in Materials and Methods. (C) Mouse splenocytes pretreated with 30 μM R9-QQP, R9-SCR, or no peptide were stimulated as for panel A, and samples were analyzed for CD4 versus Itk-pY511 fluorescence intensity (log scale). The data are from one representative experiment. (D) Averages (±SEM) of four replicate experiments performed as for panel C using splenic cells pretreated with the indicated amounts of R9-QQP or control peptides. The data were derived and presented as for panel B. (E) Lysates of mouse splenocytes that had been stimulated as for Fig. 2 were immunoprecipitated with anti-ITK antibody and immunoblotted serially with anti-ITK pY511 and anti-ITK antibodies. The results are from one experiment. (F) Averages (±SEM) of three replicate experiments performed as for panel E using splenic cells pretreated with the indicated amounts of R9-QQP or control peptides. The data were derived and calculated as described in Materials and Methods. (G) Lysates of Jurkat cells treated as for panel E and immunoblotted with anti-pY antibody. The results are from a single experiment.
FIG. 6.
FIG. 6.
R9-QQP inhibits the TCR-induced expression of Th2 cytokines. (A) Murine splenocytes were pretreated with R9-QQP (10 μM) or buffer, stimulated with anti-CD3ɛ and anti-CD28 antibodies, and analyzed for intracytoplasmic cytokines by flow cytometry as described in Materials and Methods. The results are presented as CD4 versus intracytoplasmic cytokine fluorescence intensity (log scale). The data are from one representative experiment. (B to D) Averages (±SEM) of three replicate experiments performed as for panel A using splenic cells pretreated with the indicated amounts of R9-QQP or control peptides. The data are presented as percentages of CD4+ cells expressing the indicated cytokines, calculated as described in Materials and Methods. (E to G) Analysis of secreted cytokines from cultures established similarly to those described above (without brefeldin A). The culture supernatants were analyzed using commercial cytokine kits. P/I denotes stimulation with PMA and ionomycin performed as described in Materials and Methods. The results are presented as picograms of the indicated cytokines per milliliter of culture supernatant determined by standard curves (see Materials and Methods). Each determination was performed in triplicate. The results are the averages (±SEM) of four replicate experiments.
FIG. 7.
FIG. 7.
R9-QQP specifically inhibits ITK activation and function when delivered in vivo. Mice were injected intraperitoneally with 20 mg of the indicated peptides per kg weight or received no peptide treatment, as described in Materials and Methods. (A) Harvested splenocytes were stimulated with anti-CD3ɛ or control antibodies, and the association of SLP-76 with the indicated signaling partners was assessed as for Fig. 2. (B) Graphic representation of SLP-76 association with various signaling partners: ITK (three replicate experiments) and LCK, HPK-1, and GADS (one experiment each, performed as for panel A). The results were derived and displayed as for Fig. 2B. (C) Splenocytes were stimulated with anti-CD3ɛ or control antibodies, and ITK pY511 phosphorylation was assessed and presented as for Fig. 5C. (D) Averages (±SEM) of four replicate experiments performed and presented as for Fig. 5D. LCK phospho-flow cytometry was performed using anti-LCK pTyr 394 antibody. (E) Lysates of mouse splenocytes that had been stimulated as for Fig. 2 were immunoprecipitated with anti-ITK antibody and Western blotted serially with anti-ITK pY511, anti-LCK pY394, anti-ZAP-70 pY493, and anti-ITK antibodies. The results are from a single experiment. (F) Splenocytes were stimulated with anti-CD3ɛ plus anti-CD28 antibodies, and the intracellular expression of cytokines was assessed and presented as for Fig. 6A. The data are from one representative experiment. (G) Averages (±SEM) of three replicate experiments performed and presented as for Fig. 6B to D. (H) Analysis of secreted cytokines from cultures established and tested as described for Fig. 6E to G. The results are from a single experiment, with each sample tested in triplicate.

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