cAMP production and PKA activation in T cells treated with high and low concentrations of CyaA and ET.

<p>(A) Time course analysis of cAMP production in purified peripheral blood T lymphocytes treated with high (CyaA hi, 45 nM ; ET hi, 110 nM) (<i>top left</i>) or low (CyaA lo, 0.28 nM; ET lo, 0.11 nM) (<i>top right</i>) concentrations of CyaA or ET, or activated by TCR/CD3 cross-linking (<i>bottom right</i>). The histogram on the bottom left panel also includes the quantification of cAMP in lysates of T cells treated with the adenylase cyclase deficient CyaA and ET mutants (45 nM CyaA-E5, 110 nM EL1) for 2 h or 6 h, respectively. The results, which show the levels of cAMP measured in T cell lysates, are expressed as fmoles/10⁶ cells. Representative experiments, each carried out on duplicate samples from individual healthy donors, are shown (<i>n</i>≥4). (B) <i>Top</i>, Immunoblot analysis of the phosphorylation state of PKA substrates in post-nuclear supernatants of T cells treated with 45 nM CyaA (CyaA hi) or 0.28 nM CyaA (CyaA lo), or 110 nM ET (ET hi) or 0.11 nM ET (ET lo), for 2 h (CyaA) or 6 h (ET), and then lysed as such or after stimulation for 1 min with anti-CD3 mAb (CD3). A sample stimulated with anti-CD3 mAb alone was also included. The immunoblot was carried out using an antibody which recognizes a phosphorylated PKA consensus sequence (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000325#s4" target="_blank">Materials and Methods</a>). The stripped filter was reprobed with a phosphospecific antibody which recognizes the active form of CREB <i>(middle)</i>. The fold activation of CREB in CyaA/ET treated samples <i>vs</i> untreated control in the experiment shown was the following: CyaA low, 8.3; CyaA high, 19.0; ET low, 8.7; ET high, 79.9. The levels of phospho-CREB in the samples treated with CyaA or ET in combination with anti-CD3 mAb <i>vs</i> samples treated with anti-CD3 mAb alone (taken as 100%) were the following: CyaA low+CD3, 98.1±4.8%; CyaA high+CD3, 103.4±8.7%; ET low+CD3, 102.1±3.2%; ET high+CD3, 112.1±8.1% (<i>n</i> = 3). A control anti-actin blot is shown below. None of the treatments modified the expression levels of CREB (data not shown). Representative experiments are presented (<i>n</i>≥3). The migration of molecular mass markers is indicated. (C) Quantification of CREB phosphorylation in post-nuclear supernatants of T cells treated with 45 nM CyaA (CyaA hi)/CyaA-E5 or 0.28 nM CyaA (CyaA lo), or 110 nM ET (ET hi) /EL1 or 0.11 nM ET (ET lo), for 2 h (CyaA) or 6 h (ET). Where indicated, cells were pretreated for 1 h with 20 µM H89. A sample stimulated for 30 min with 100 µM 8-CPT was included as positive control. The data were obtained by laser densitometry of anti-phospho-CREB immunoblots. The results are expressed as relative CREB phosphorylation (fold activation <i>vs</i> untreated controls) (<i>n</i> = 2). (D) Quantification of cAMP in lysates of T cells treated as in B. The results, which show the levels of cAMP measured in T cell lysates, are expressed as fmoles/10⁶ cells. A representative experiment, carried out on duplicate samples from an individual healthy donor, is shown (<i>n</i> = 3). ***<i>P</i>≤0.001; **<i>P</i>≤0.01; *<i>P</i>≤0.05. Error bars, SD.</p

CyaA and ET promote c-maf and GATA-3 expression in primed T cells.

<p>Enriched CD4⁺ T cells from 3 healthy donors were primed with anti-CD3 mAb, as such or following pretreatment for 2 h with 0.28 nM CyaA or 0.11 nM ET. Cells primed in Th2- (IL-4) or Th1- (IL-12) inducing conditions were included as controls. After 10 days cells were restimulated with anti-CD3 mAb for 24 h. The levels of mRNA encoding c-maf and GATA-3 (A) and T-bet (B) were quantified by real-time RT–PCR. Transcript levels were normalized to the expression level of GAPDH. Syber green runs were performed with cDNAs from the same reverse transcription reaction from 400 ng of total RNA. The ΔΔC_T method was applied as a comparative method of quantification, using cells primed in neutral conditions (anti-CD3 mAb) as reference. The data are representative of 3 independent experiments, each in duplicate. ***<i>P</i>≤0.00001; **<i>P</i>≤0.0001; *<i>P</i>≤0.001. Error bars, SD.</p

Low CyaA or ET concentrations impair TCR–dependent Akt1 phosphorylation while enhancing Vav1 and p38 phosphorylation.

<p>(A) Immunoblot analysis, using a phosphospecific antibody, of Akt1 activation in postnuclear supernatants from PBL activated for 5 min by CD3 cross-linking in the presence or absence of 45 nM CyaA or 110 nM ET (CyaA hi, ET hi) or alternatively in the presence or absence of 0.28 nM CyaA or 0.11 nM ET (CyaA lo, ET lo). A representative experiment is shown (<i>n</i> = 3). (B) Immunoblot analysis, using phosphospecific antibodies, of Vav1 (<i>top</i>) or p38 (<i>bottom</i>) activation in postnuclear supernatants from PBL activated for 1 min (Vav) or 5 min (p38) by CD3 cross-linking in the presence or absence of 45 nM CyaA or 110 nM ET (CyaA hi, ET hi) or alternatively in the presence or absence of 0.28 nM CyaA or 0.11 nM ET (CyaA lo, ET lo). Filters were stripped and re-probed with control antibodies. Representative experiments are shown (<i>n</i>≥4). The migration of molecular mass markers is indicated. The graphs on the right of the immunoblots show the quantification by laser densitometry of the relative levels of Akt1, Vav1, and p38 phosphorylation (phosphorylation in anti-CD3 stimulated cells taken as 100%) in PBL activated by CD3 cross-linking in the presence or absence of 45 nM CyaA or 110 nM ET (CyaA hi, ET hi) or alternatively in the presence or absence of 0.28 nM CyaA or 0.11 nM ET (CyaA lo, ET lo). ***<i>P</i>≤0.001**<i>P</i>≤0.01; *<i>P</i>≤0.05. Error bars, SD.</p

Immunosuppressive concentrations of CyaA or ET prevent initiation of TCR signaling.

<p>(A) <i>Top left</i>, Immunoblot analysis, using a phosphospecific antibody, of Lck phosphorylation on the inhibitory C-terminal tyrosine residue (Y505) in postnuclear supernatants from PBL activated for 1 min by CD3 cross-linking in the presence or absence of either 45 nM CyaA or 110 nM ET (CyaA hi, ET hi). <i>Top right</i>, Immunoblot analysis, using an anti-phosphotyrosine antibody, of CD3ζ specific immunoprecipitates from PBL treated as above. <i>Bottom</i>, Immunoblot analysis, using phosphospecific antibodies, of ZAP-70 (left) and Erk1/2 (right) phosphorylation in postnuclear supernatants from PBL activated for 1 min (ZAP-70) or 5 min (Erk1/2) by CD3 cross-linking in the presence or absence of 45 nM CyaA or 110 nM ET (CyaA hi, ET hi). (B) Quantification by laser densitometry of the relative levels of Lck (phosphorylation in unstimulated cells taken as 100%), or CD3ζ, ZAP-70 and Erk1/2 phosphorylation (phosphorylation in anti-CD3 stimulated cells taken as 100%, indicated as a dotted line) in PBL activated by CD3 cross-linking in the presence of 45 nM CyaA or 110 nM ET (CyaA hi, ET hi) (<i>n</i>≥3). Where indicated, cells were activated in the presence of the adenylase cyclase deficient CyaA and ET mutants (45 nM CyaA-E5, 110 nM EL1) for 2 h or 6 h, respectively (<i>n</i> = 2). ***<i>P</i>≤0.001; **<i>P</i>≤0.01; *<i>P</i>≤0.05. Error bars, SD.</p

CyaA and ET promote Th2 cell differentiation through their cAMP elevating activity.

<p>Enriched CD4⁺ T cells from 6 healthy donors were primed with anti-CD3 mAb, as such or following pretreatment for 2 h with 0.28 nM CyaA/CyaA-E5, or with 0.11 nM ET/EL1. Cells primed in Th2- (IL-4) or Th1- (IL-12) inducing conditions were included as controls. After 10 days cells were washed and restimulated with anti-CD3 mAb for 48 and 24 h respectively, and the levels of IL-4 and IL-13 (A) and IFN-γ and TNF-α (B) were quantified by ELISPOT. The results, obtained on duplicate samples, are expressed as % spot-forming units by CyaA or ET treated cells compared to control cells primed in the absence of either toxin (taken as 100%). ***<i>P</i>≤0.0001; **<i>P</i>≤0.001; *<i>P</i>≤0.01. Error bars, SD.</p

Low CyaA or ET concentrations do not affect initiation of TCR signaling.

<p>(A) Immunoblot analysis, using phosphospecific antibodies, of CD3ζ, ZAP-70, or Erk1/2 phosphorylation in postnuclear supernatants of cells activated as above in the presence or absence of either 0.28 nM CyaA or 0.11 nM ET (CyaA lo, ET lo). Filters were stripped and re-probed with control antibodies. Representative experiments are shown (<i>n</i>≥3). The migration of molecular mass markers is indicated. (B) Quantification by laser densitometry of the relative levels of CD3ζ, ZAP-70, and Erk1/2 phosphorylation (phosphorylation in anti-CD3 stimulated cells taken as 100%, indicated as a dotted line) in PBL activated by CD3 cross-linking in the presence or absence of either 0.28 nM CyaA or 0.11 nM ET (CyaA lo, ET lo) (<i>n</i>≥3). Error bars, SD.</p

Concentration dependent suppression of T cell proliferation by CyaA and ET.

<p>(A) [³H]-thymidine uptake by PBL stimulated for 48 h by CD3 cross-linking in the presence or absence of the indicated concentrations of CyaA (<i>top</i>) or ET (<i>bottom</i>). The results, obtained on triplicate samples of PBL from 5 independent donors, are expressed as % [³H]-thymidine uptake (cpm) by CyaA or ET treated cells compared to control cells stimulated in the absence of either toxin (taken as 100%). The arrow shows the toxin concentration selected for the polarization experiments (CyaA, 0.28 nM; ET, 0.11 nM). (B) [³H]-thymidine uptake by PBL stimulated for 48 h by CD3 cross-linking in the presence or absence of either CyaA and ET or the respective adenylase deficient mutants (45 nM CyaA/CyaA-E5, 110 nM ET/EL1). The results are expressed as in A. ***<i>P</i>≤0.001; **<i>P</i>≤0.01; *<i>P</i>≤0.05. Error bars, SD.</p

Characteristics of the used antigens: all the used antigens were recombinant proteins (from Hoghart et al. [14], modified).

<p>Characteristics of the used antigens: all the used antigens were recombinant proteins (from Hoghart et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046041#pone.0046041-Hogarth1" target="_blank">[14]</a>, modified).</p

A receiver operator characteristic (ROC) plot is shown, illustrating sensitivity and specificity of AlaDH IFN-γ and IL-2 ELISpot results in discriminating children with latent (n = 21) and overt (n = 25) tuberculosis.

<p>Area under the ROC curve was 0.700 (95%IC: 0.547-0.853; p = 0.021 <i>vs.</i> the identity - diagonal - line) considering IFN-γ ELISpot and 0,896 (95%IC: 0.785–1.008; p<0.0001 <i>vs.</i> the identity - diagonal - line) for IL-2 ELISPOT.</p

ELISPOT assay results from 75 study children according to final diagnosis.

<p>Note.</p>*<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046041#s4" target="_blank">Results</a> are expressed as median and interquartile range of spot forming colonies per million PBMCs; TB:tuberculosis.</p