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 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

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

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

Different Ca²⁺ responses of CB and adult CD31⁺ naive T cells.

<p>(A-B) Ca²⁺ mobilization in CD4⁺CD45RA⁺CD31⁺ T cells of one healthy donor (representative of at least eight healthy individuals) of PBMCs (A, adult) or CB (B) were performed in response to 0.05 μg/ml anti-CD3 Ab plus 0.5 μg/ml soluble anti-CD28 Ab (red curve) or only 0.05 μg/ml of anti-CD3 Ab alone (black curve, with anti-CD28 isotype Ab) in combination with GAMIg. The blue dotted line displays the maximum Ca²⁺ response of adult CD31⁺ naive T cells for anti-CD3 Ab stimulation alone. (C) Box plot with scatter plots representing means and SD of Ca²⁺ influx response normalized by maximal Ca²⁺ influx response to ionomycin of adult (gray circle) and CB (black circle) and their dependency on anti-CD28 Ab costimulation (anti-CD3 Ab plus anti-CD28 Ab (red box) or anti-CD3 Ab with anti-CD28 Ab isotype (blue box)). Statistical significance between groups * <i>P<</i>0.05 was determined by two-tailed ANOVA with Tukey-Kramer post-hoc test. n = number of individuals. (D) Comparison of different CD4⁺ T cell subset stimulated with anti-CD3 Ab plus anti-CD28 Ab (red box) or with anti-CD3 Ab alone (blue box). Box plot with scatter plots representing means and standard deviations of Ca²⁺ influx response normalized by maximal Ca²⁺ influx response to ionomycin. Statistical significance of differences between anti-CD3/anti-CD28 Ab or anti-CD3 Ab stimulation at concentration 0.05 μg/ml of anti-CD3 Ab CD31⁺ between groups of different T cell subsets was determined by two-tailed ANOVA <i>P</i> = 0.7745, CD45RA⁺ <i>P</i> = 0.8195, CD4⁺ <i>P</i> = 0.9926. ns = not significant. n = number of individuals. (E) CD4⁺CD45RA⁺CD31⁺ T cells of CB (filled line), and adults (dashed line) treated with 0.5 μg/ml soluble anti-CD3 Ab and anti-CD28 Ab cross-linked with GAMIg in the presence (red) or absence (black) of 2 mM EGTA. One representative experiment out of three comparable experiments is shown. (F) STIM1 protein expression in CD4⁺ T cells of CB (black) and in naive CD31⁺ T cells from adults (gray) after stimulation using anti-CD3/anti-CD28 Ab. The densitometric analyses of the ratio of STIM1/α Tubulin are shown. Results are representative of at least two experiments.</p

T Cells of Infants Are Mature, but Hyporeactive Due to Limited Ca²⁺ Influx

<div><p>CD4 T cells in human infants and adults differ in the initiation and strength of their responses. The molecular basis for these differences is not yet understood. To address this the principle key molecular events of TCR- and CD28-induced signaling in naive CD4 T cells, such as Ca²⁺ influx, NFAT expression, phosphorylation and translocation into the nucleus, ERK activation and IL-2 response, were analyzed over at least the first 3 years of life. We report dramatically reduced IL-2 and TNFα responses in naive CD31⁺ T cells during infancy. Looking at the obligatory Ca²⁺ influx required to induce T cell activation and proliferation, we demonstrate characteristic patterns of impairment for each stage of infancy that are partly due to the differential usage of Ca²⁺ stores. Consistent with those findings, translocation of NFATc2 is limited, but still dependent on Ca²⁺ influx as demonstrated by sensitivity to cyclosporin A (CsA) treatment. Thus weak Ca²⁺ influx functions as a catalyst for the implementation of restricted IL-2 response in T cells during infancy. Our studies also define limited mobilization of Ca²⁺ ions as a characteristic property of T cells during infancy. This work adds to our understanding of infants’ poor T cell responsiveness against pathogens.</p></div

Visualizing the occurrence of age-dependent characteristics of T cell activation.

<p>The requirement of activation by the TCR/CD3 complex is less dependent on costimulation by CD28 in CB naive CD4⁺ T cells compared to that seen in adult cells. The intensity of Ca²⁺ influx, NFATc2 expression and IL-2 response are all age-dependent. A dramatic shift is seen in the naive T cell response at the age of 2 months. The cells’ capacity to produce high amounts of IL-2 is suddenly abrogated. Ca²⁺ influx declines to the lowest values observed in life. At 6 months of age, the IL-2 response starts to improve slowly. This “reprogramming” of T cells takes place as the passively transferred maternal Abs in the infant are beginning to decline. Limited T-cell responses likely contribute to the high risk of infants to suffer from infections and infection-related pathologies such as Sepsis and SIDS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166633#pone.0166633.ref043" target="_blank">43</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166633#pone.0166633.ref044" target="_blank">44</a>] during the first months of life.</p

Ca²⁺ influx is a key event for IL-2 in adult CD31⁺ naive T cells.

<p>(A) No Ca²⁺ influx signal with GAMIg alone (black curve; anti-CD28 isotype Ab were substituted for anti-CD28 Ab) or anti-CD28 Ab plus GAMIg alone (red curve) in T cells stained for anti-CD4, anti-CD45, and anti-CD31. Results representative of at least 3 experiments are shown. (B-C) Ca²⁺ influx into adult CD31⁺ naive T cells stimulated using anti CD3 Ab and anti CD28 Ab. (B) A representative Ca²⁺ influx experiment and (C) box plots for the different T cell subsets showing scatter plots of mean value and SD of Ca²⁺ influx experiments by normalizing maximal Ca²⁺ signals to the maximal Ca²⁺ influx ionomycin are shown (* <i>P<</i>0.05; two-tailed ANOVA of differences). (D) Increased Ca²⁺ influx in adult CD31⁺ naive T cells compared with anti-CD3/anti-CD28 Ab (red) in response to anti-CD3 Ab alone (blue, with anti-CD28 isotype Ab) (* <i>P<</i>0.05; two-tailed ANOVA of differences) (E) Increased levels of NFATc2 protein expression in response to anti-CD3/anti-CD28 Ab stimulation in naive T cells. The immunoblot detection of the relative protein expression level by ratio of NFATc2 or pNFATc2 to αTubulin is shown for naive CD31⁺ or CD31^- T cells of adults stimulated with anti-CD3 Ab in combination with soluble anti-CD28 Ab (dark gray) or anti-CD28 Ab isotype (light gray). Laminin and αTubulin were used as loading controls. Results are representative of at least two experiments. unstim. = unstimulated. (F) IL-2 cytokine in supernatants does not differ in cultures under TCR/CD3 stimulation between subtypes of naive CD4 T cells (* <i>P<</i>0.05; two-tailed ANOVA of differences). ns = not significant.</p

Key signaling pathways for IL-2 transcription are activated in TCR/CD3 stimulated naive CD4⁺ T cells of CB.

<p>(A) Protein expression by Western blot of ERK1/2 and phosphorylated ERK1/2Tyr202/ Tyr204 (pERK1/2) in naive CD4⁺ T cells of CB as well as for naive CD31⁺ adult T cells. The ratios of relative protein expression levels are indicated below the respective bands. Data are representative of two independent experiments. (B) Whole cell protein extract of NFATc2 and phosphorylated NFATc2 (pNFATc2) in CB naive CD4⁺ T cells and adult naive CD31⁺ T cells under different stimulation conditions. The densitometric analyses of the immunoblot detection for the relative protein expression level are shown as ratio of NFATc2 or pNFATc2 to αTubulin. Lysates from three different donors were pooled. Data are representative of at least three independent experiments. αTubulin was used as a loading control. (C-D). The NFATc2 protein expression in cytoplasm or nucleoplasm (separated through a dashed line) in (C) naive CD4⁺ T cells of CB or (D) CD31⁺ naive T cells of adult were detected and the phosphorylated (pNFATc2) and dephosphorylated (NFATc2) forms quantified. Cells were stimulated as indicated in the presence or absence of cyclosporin A (CsA). One representative experiment out of two comparable experiments is shown. unstim. = unstimulated.</p

Age-dependent signatory Ca²⁺ influx and cytokine concentrations in supernatants of naive CD4⁺ T cells.

<p>(A-B) CD4⁺CD45RA⁺CD31⁺ T cells stimulated with anti-CD3 Ab as indicated either with costimulation by 0.5 μg/ml soluble anti-CD28 Ab (A, dark gray) or with the CD28 isotype control (B, light gray). Compiled data of box plots with scatter plots represent the Ca²⁺ influx response normalized to the maximal Ca²⁺ influx ionomycin response. Statistical significance between groups was determined by two tailed ANOVA Tukey-Kramer post-hoc test * <i>P<</i>0.05. n = number of individuals. (C) NFATc2 expression after anti-CD3 Ab plus anti-CD28 Ab engagement. The densitometric analyses of the immunoblots for the relative protein expression levels are shown as ratios of NFATc2 or pNFATc2 to αTubulin. Lysates from three different donors were pooled. Results are representative of at least two independent experiments. IFNγ (D), IL-2 (E), and TNFα (F) concentrations in the supernatants of unstimulated (white), of soluble anti-CD3 Ab plus anti-CD28 Ab stimulated (dark gray), and of TCR/CD3 stimulated alone (light gray, with anti-CD28 Ab isotype) of naive T cells using a Bio-Plex cytokine assay (Bio-Rad). The mean value and SD are indicated for five independent experiments (two tailed ANOVA Tukey-Kramer post-hoc test * <i>P<</i>0.05). n = number of individuals.</p