Chemokine receptor signaling affects signaling molecules involved in cytoskeleton rearrangements.

<p><b>A</b> Activated Rac was detected by G-LISA in Th1 lymphocytes from TCR^tgCD152-deficient and TCR^tgCD152-competent mice on day 5 of a recall response. Rac activation was induced in serum-starved lymphocytes by treatment with 20 nM CCL4 for 5 or 20 min. <b>B</b> Th1 lymphocytes from TCR^tgCD152-deficient and TCR^tgCD152-competent mice on day 5 of a recall response were incubated with or without 20 nM CCL4 for 5 or 20 min. Fixed and permeabilized lymphocytes were analyzed by flow cytometry for Akt activation using antibodies specific for phosphorylated Akt or total Akt. A protion of the lymphocyteswere incubated for 20 h with the PI3K inhibitor Ly294.002 (filled histograms). Histograms show the expression of Akt on T lymphocytes. <b>C</b> Th1 lymphocytes from TCR^tgCD152-deficient and TCR^tgCD152-competent mice were analyzed to determine their migration affinities for the inflammatory chemokine CCL4 in a transwell system on day 5 of a recall response. A portion of the Th1 lymphocytes were incubated for 20 h with a PI3K inhibitor or Akt inhibitor II, as indicated. The dotted line indicates basal migration toward medium (ns, not significant). Representative data from at least two experiments are shown.</p

Effect of induction with the chemokine CCL4 on signaling molecules.

<p><b>A</b> Th1 lymphocytes from TCR^tgCD152-deficient and TCR^tgCD152-competent mice were analyzed for ERK phosphorylation on day 2 and day 5 of a recall response. Signaling via CCR5 was induced in serum-starved lymphocytes by treatment with 20 nM CCL4 for 5 (heavy black line) or 20 min (heavy grey line). Filled histograms indicate ERK phosphorylation before CCL4 application. <b>B</b> Migration assay in Th1 lymphocytes from TCR^tgCD152-deficient and TCR^tgCD152-competent mice on day 5 of a recall response. A portion of the lymphocytes was incubated with the PKCθ-specific inhibitor Rottlerin for 20 h. <b>C</b> Western blot detection of signaling proteins in lysates of TCR^tgCD152-deficient and TCR^tgCD152-competent Th1 lymphocytes on day 5 of a recall response. The cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031391#pone-0031391-g002" target="_blank">Fig. 2 A</a>. <b>D</b> The band intensity of pGRK2 relative to GRK2 was quantified using Image Gauge 4.0. Representative data from at least two experiments are shown.</p

Chemokine receptor expression in activated T lymphocytes does not reflect the chemotactic capacities of corresponding chemokines.

<p>CD4⁺ T lymphocytes from TCR^tgCD152-deficient (CD152^−/−) and TCR^tgCD152-competent (CD152^+/+) mice were analyzed to detect chemokine receptor expression by flow cytometry (upper panels) and in chemotaxis assays (lower panels) on day 3 after the initiation of recall responses. <b>A</b> CCR7 expression and migration toward medium or CCL19 were detected in T lymphocytes. <b>B</b> Th1-differentiated CD4⁺ T lymphocytes were analyzed for CCR7 expression and migration behavior toward medium or CCL19. <b>C</b> Th1 lymphocytes exhibited similar CCR5 expression levels (M1: TCR^tgCD152-deficient, 60%; TCR^tgCD152-competent, 60%) but different migration rates in transwell systems. Representative data from two experiments are shown.</p

Role of CD28 and CD152 signals in signal transduction via the chemokine receptor CCR5 in Th1 lymphocytes.

<p>Upon binding of its ligand, CCL4, the CCR5 receptor is phosphorylated by CD28-induced GRK2. β-arrestins can now bind to CCR5 and initiate desensitization, which contributes to the degradation or recycling of CCR5. CD152 engagement leads to the inactivation of GRK2, and the phosphorylation of CCR5 is prevented. CD28 and CD152 signal-induced activation of integrins by the Gβγ subunit and via the GTPases Rac1 and Cdc42 or Rap1 ultimately leads to lymphocyte adhesion. Chemokine-induced activation of PI3K and the subsequent phosphorylation and activation of Akt are only initiated in the presence of CD152 signaling, and it is only under these conditions that specific migration occurs along chemokine gradients (orange arrows indicate signal transduction under CD28 signaling, and blue arrows indicate signal transduction under CD152 signaling). The figure shows only those signaling pathways controlled by CD152.</p

Additional file 1: Figure S1. of Early changes in the metabolic profile of activated CD8+ T cells

Purified CD8+ T cells were treated with OT-I-streptamers for the indicated time periods. Samples were analyzed by Western blotting using the indicated Abs to determine the inhibition of mTOR (A) and AKT (B). Purified CD8+ T cells were treated with OT-I-streptamers for 24 h in presence or absence of aIL-2. Samples were analyzed for pSTAT5 activation to determine the function of aIL-2 antibody (C) Toxicity was assessed for the inhibitors rotenone and oxamate and the aIL-2 antibody by AnnexinV PI staining 24 h after stimulation (D). (TIF 189 kb