Inhibition of the kinase Csk in thymocytes reveals a requirement for actin remodeling in the initiation of full TCR signaling

Abstract

Signaling via the T cell antigen receptor (TCR) is initiated by Src-family kinases (SFKs). To understand how the kinase Csk, a negative regulator of SFKs, controls the basal state and the initiation of TCR signaling, we generated mice that express a Csk variant sensitive to an analog of the common kinase inhibitor PP1 (Csk(AS)). Inhibition of Csk(AS) in thymocytes, without engagement of the TCR, induced potent activation of SFKs and proximal TCR signaling up to phospholipase C-γ1 (PLC-γ1). Unexpectedly, increases in inositol phosphates, intracellular calcium and phosphorylation of the kinase Erk were impaired. Altering the actin cytoskeleton pharmacologically or providing costimulation via CD28 'rescued' those defects. Thus, Csk has a critical role in preventing TCR signaling. However, our studies also revealed a requirement for actin remodeling, initiated by costimulation, for full TCR signaling.

Figure 1

Inhibition of Csk^AS in primary murine T cells induces hyperactivation of SFKs and phosphorylation of TCR proximal signaling molecules. (a) Wild-type (WT) or Csk^AS (AS) thymocytes or (b) peripheral CD4⁺ T cells were treated for the indicated times with 10 µM 3-IB-PP1 and analyzed by immunoblotting. (c) Wild-type (WT) or Csk^AS (AS) thymocytes were treated for 3 min with vehicle (DMSO), 3-IB-PP1 or anti-CD3ε and analyzed by immunoblotting. (d) Wild-type (WT) or Csk^AS (AS) thymocytes were treated for 3 min with vehicle (DMSO), 10µM 3-IB-PP1 or 20µg/mL anti-CD3ε, then lysed and immunoprecipitated with anti-CD3 ζ antibody. Immunoprecipitates were analyzed by immunoblotting. (e) Wild-type (WT) or Csk^AS (AS) thymocytes were treated for 3 min with vehicle (DMSO), 10µM 3-IB-PP1 or 20µg/mL anti-CD3ε and analyzed by immunoblotting. All data are representative of 3 independent experiments.

Figure 2

Increases in intracellular calcium and Erk phosphorylation are impaired following Csk^AS inhibition. (a) Wild-type (WT) or Csk^AS (AS) thymocytes or purified peripheral CD4⁺ T cells loaded with Indo-1AM dye were stimulated with vehicle (DMSO: shaded gray histogram), 10µM 3-IB-PP1 (black) or 20µg/mL anti-CD3ε (gray). Ratiometric assessment of intracellular calcium of CD4⁺CD8⁺ thymocytes or CD4⁺ T cells over time is shown. (b) Wild-type (WT) or Csk^AS (AS) thymocytes or splenocytes were stimulated for 2 min with vehicle (DMSO: shaded gray histogram), 10µM 3-IB-PP1 (black) or 20µg/mL anti-CD3ε (gray), then analyzed for pErk content. Histograms are gated on CD4⁺CD8⁺ thymocytes or CD4⁺ splenocytes. All data are representative of three independent experiments.

Figure 3

Calcium flux and Erk phosphorylation are restored by altering the actin cytoskeleton following Csk^AS inhibition. (a) Csk^AS(AS) thymocytes were stimulated with vehicle (DMSO), 10µM 3-IB-PP1, 20µg/mL anti-CD3ε, 10µM cytochalasin D (CytoD) alone or with 10µM 3-IB-PP1 for 5 min, and total inositol phosphate content was measured (mean, ± S.E.M., n = 3 (technical replicates), P-values from paired two-tailed student’s t-test). (b) Wild-type (WT) or Csk^AS (AS) thymocytes were stimulated with vehicle (DMSO: gray), 10µM 3-IB-PP1 (magenta), 20µg/mL anti-CD3ε (blue), 10µM cytochalasin D (CytoD: black) alone or with 10µM 3-IB-PP1(green). Ratiometric assessment of intracellular calcium of CD4⁺CD8⁺ thymocytes over time is shown. (c) Wild-type (WT) or Csk^AS (AS) thymocytes were stimulated for 2 min as in b, then analyzed for pErk content. Histograms are gated on CD4⁺CD8⁺ thymocytes. (d) Wild-type (WT) or Csk^AS (AS) thymocytes were treated for 3 min as in a and analyzed by immunoblotting. (e) Wild-type (WT) or Csk^AS (AS) splenocytes were stimulated as in b. Ratiometric assessment of intracellular calcium of CD4+ splenocytes over time is shown. (f) Wild-type (WT) or Csk^AS (AS) splenocytes were stimulated for 2 min as in b, then analyzed for pErk content. Histograms are gated on CD4⁺ splenocytes. Shaded histogram is DMSO control. (g) Wild-type (WT) or Csk^AS (AS) thymocytes or (h) purified T cells were stimulated for 2 min as in b, then analyzed for pErk content. Histograms are gated on CD4⁻ CD8⁻ γδ TCR⁺ cells.. All data are representative of three independent experiments.

Figure 4

Mature DCs restore calcium flux and Erk phosphorylation following Csk^AS inhibition. (a) DCs enriched from wild-type splenocytes were activated by overnight culture. Thymocytes from wild-type (WT) or Csk^AS(AS) mice were pelleted with or without activated DCs at a 1:1 ratio and stimulated with vehicle (DMSO), 10µM 3-IB-PP1, 10µM cytochalasin D (CytoD) alone or with 10µM 3-IB-PP1 or 50ng/mL phorbol myristate acetate (PMA) for 3 min. Cells were then analyzed for pErk content. Histograms shown are gated on CD4⁺CD8⁺ thymocytes. Numbers within histograms indicate percentage of pErk⁺ cells. (b) Wild-type (WT) or Csk^AS (AS) thymocytes were loaded with Indo1-AM dye and surface stained for CD4 and CD8. Activated DCs were prepared as in a, and surface stained for CD11c. Thymocytes and activated DCs were mixed at a 1:1 ratio and stimulated for 5 min with 10µM 3-IB-PP1. Cells collected were first gated for CD4⁺CD8⁺ multiplets, then further separated into thymocyte-thymocyte conjugates (CD11c⁻) and thymocyte-DC conjugates (CD11c⁺) before ratiometric assessment of intracellular calcium over time (far right). All data are representative of three independent experiments.

Figure 5

Restoration of full TCR signaling upon Csk^AS inhibition requires both MHC and B7 on DCs. DCs enriched from wild-type or (a) CD80-CD86 double knockout or (b) MHCI and MHCII deficient splenocytes were activated by overnight culture. Wild-type (WT) or Csk^AS (AS) thymocytes were pelleted with or without activated DCs at a 1:1 ratio and stimulated with vehicle (DMSO) or 10µM 3-IB-PP1for 3 min. Cells were then analyzed for pErk content. Histograms shown are gated on CD4⁺CD8⁺ thymocytes. Numbers within histograms indicate percentage of pErk⁺ cells. (c, d) Wild-type (WT) or Csk^AS (AS) thymocytes were loaded with Indo1-AM dye and surface stained for CD4 and CD8. Activated DCs from (c) CD80-CD86 double knockout or (d) MHCI- and MHCII-deficient splenocytes were surface stained for CD11c. Thymocytes and activated DCs were mixed at a 1:1 ratio and stimulated with 10µM 3-IB-PP1. Cells collected were first gated for CD4⁺CD8⁺ multiplets, then further separated into thymocyte-thymocyte conjugates (CD11c⁻) and thymocyte-DC conjugates (CD11c⁺) before ratiometric assessment of intracellular calcium over time. All data are representative of three independent experiments.

Figure 6

CD28 signaling induces actin remodeling and restores robust Erk phosphorylation following Csk^AS inhibition. (a) Csk^AS thymocytes were pelleted with styrene beads coated with 5 µg/ml CD86-Ig (CD86) and/or 5 µg/ml I-A^b tetramer (MHCII) and treated for 3 min with vehicle (DMSO) or 10 µM 3-IB-PP1 and then analyzed for pErk content. Histograms shown are gated on CD4⁺CD8⁺ thymocytes. Data shown are representative of three independent experiments. (b) Csk^AS thymocytes were pretreated for 15 min with DMSO or 10 µM LY294002, then pelleted with or without activated WT DCs at a 1:1 ratio and stimulated as indicated for 3 min. Cells were then analyzed for pErk and pAkt content. Histograms shown are gated on CD4⁺CD8⁺ thymocytes. Numbers within histograms indicate percentage of pErk⁺ or pAkt⁺ cells. Data shown are representative of three independent experiments. (c, d) Csk^AS thymocytes were pelleted with styrene beads coated with 5 µg/ml CD86-Ig or 5 µg/ml anti-CD3ε and treated for 3 min with vehicle (DMSO) or 10µM 3-IB-PP1. Bead-thymocyte conjugates were imaged for F-actin distribution. (c) Representative images show the different F-actin distribution patterns observed. (d) Graph shows the percentage of conjugates with moderate and strong F-actin polarization at the bead-thymocyte interface (means, -S.E.M.(moderate), +S.E.M.(strong), n = 3 (biological replicates)). Total number of conjugates analyzed for each condition is indicated on top of the bars.