sCD25 enhances Th17 cell responses <i>in vitro</i>.

<p>(<b>A&B</b>) Purified naive CD4+ T cells were activated under either Th17 or Th1 inducing conditions (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047748#s2" target="_blank">methods</a>) in the presence of a range of concentrations of sCD25 (20, 10, 5 or 1 µg/ml) or anti-IL-2 (10 µg/ml). Levels of IL-17A or IFNγ expression were determined after 96 hrs by (<b>A</b>) FACS and (<b>B</b>) ELISA. (<b>C</b>) Purified naive CD4+ T cells were activated under Treg inducing conditions, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047748#s2" target="_blank">methods</a>, in the presence or absence of sCD25 (20µg/ml) and FoxP3 expression determined by FACS. (<b>D</b>) Naive CD4+ T cells were stained with CFSE (2.5 µM) prior to activation under Th17 conditions in presence or absence of sCD25 (20 µg/ml). After 96 hours, levels of intracellular IL-17A expression and CFSE dilution or 7AAD incorporation were determined by FACS. (<b>E</b>) Purified naive CD4+ T cells were activated under Th0, Th17 and Th17& sCD25 (20 µg/ml) conditions for 72 hours and levels of P-Stat3 (pY705) determined by FACS. All data are representative of 3 independent experiments. Statistical Significance determined by unpaired student's t-test, ∗ p≤0.05, **p≤0.01, ***p≤0.001.</p

sCD25 acts early during Th17 development.

<p>Purified naive CD4+ T cells from IL-17AeGFP reporter mice activated under Th17 inducing conditions in the presence of sCD25 (20 µg/ml) or anti-IL-2 (10 µg/ml) for 48 hrs and examined for levels of (<b>A</b>) IL-17A and (<b>B</b>) RORγT expression by FACS. All data are representative of at least 3 independent experiments.</p

Exogenous sCD25 exacerbates autoimmunity.

<p>(<b>A</b>) MOG₃₃₋₅₅ immunized C57BL/6 mice developed clinical symptoms of EAE from day 12 after immunization with a peak of disease severity observed from day 19. Subcutaneous administration of recombinant sCD25 (25 µg/mouse) immediately prior to immunization and every 12 hours thereafter for 72 hrs resulted in a significant exacerbation in severity of symptoms during disease onset and induction. 6–7 mice used per group. (<b>B</b>) Mononuclear cells harvested from spinal cords of control (PBS) and sCD25 treated IL-17A-eGFP reporter mice (3 per group) on day 15 after immunization and analysed for expression of IL-17 and IFN-γ by CD4+ cells. (<b>C</b>) Cell numbers of CD4+IL-17+ and CD4+ IFNγ+ cells in spinal cords of IL-17A-eGFP reporter mice at day 15. Data representative of mean +/− std dev of 3 mice per group and 2 independent experiments.</p

Composition of the Schistosoma mansoni worm secretome: Identification of immune modulatory Cyclophilin A

<div><p>The helminth <i>Schistosoma mansoni</i> modulates the infected host’s immune system to facilitate its own survival, by producing excretory/secretory molecules that interact with a variety of the host’s cell types including those of the immune system. Herein, we characterise the <i>S</i>. <i>mansoni</i> adult male worm secretome and identify 111 proteins, including 7 vaccine candidates and several molecules with potential immunomodulatory activity. Amongst the molecules present in the secretome, a 17-19kDa protein analogous to human cyclophilin A was identified. Given the ability of cyclophilin A to modulate the immune system by regulating antigen presenting cell activity, we sought to determine whether recombinant <i>S</i>. <i>mansoni</i> Cyclophilin A (rSmCypA) is capable of modulating bone-marrow derived dendritic cell (BMDC) and T cell responses under <i>in vitro</i> conditions. rSmCypA was enzymatically active and able to alter the pro-inflammatory cytokine profile of LPS-activated dendritic cells. rSmCypA also modulated DC function in the induction of CD4⁺ T cell proliferation with a preferential expansion of Treg cells. This work demonstrates the unique protein composition of the <i>S</i>. <i>mansoni</i> male worm secretome and immunomodulatory activity of <i>S</i>. <i>mansoni</i> Cyclophilin A.</p></div

<i>S</i>. <i>mansoni</i> adult male WES proteins with immune-modulatory homologs.

<p><i>S</i>. <i>mansoni</i> adult male WES proteins with immune-modulatory homologs.</p

Proteomic analysis of WES molecules.

<p>(<b>A</b>) Comparative 2D proteomic analysis between adult male worm somatic molecules (AW) and WES molecules. AW and WES (200 μg) were electrofused in a 3–11 IPG strip and then electrophoresed in a 12% SDS-PAGE stained with colloidal-coomassie blue. Five spots (circled and numbered 1–5) were selected for identification by mass spectrometry. Protein hits: 1 –SmParamyosin (Smp_021920.1); 2–3 –SmFatty acid-binding protein (Smp_095360.1); 4–5 –SmCyclophilin (Smp_040130). (<b>B</b>) A representative gel of <i>S</i>. <i>mansoni</i> WES molecules, analysed as in (<b>A</b>), showing individual spots that were analyzed by mass spectrometry.</p

Vaccine candidates detected in the <i>S</i>. <i>mansoni</i> adult male worm secretome.

<p>Vaccine candidates detected in the <i>S</i>. <i>mansoni</i> adult male worm secretome.</p

Immune modulatory effects of rSmCypA on BMDC.

<p>(<b>A</b>) Representative histograms from flow cytometric analysis of LPS treated and LPS + rSmCypA co-treated BMDC for cell surface expression of CD86, CD80, MHCII and CD40. Data shown are representative of 5 independent experiments. (<b>B</b>) Representative histograms of BMDC for Alexa Fluor-647 labeled OVA_323-339 uptake following LPS induced stimulation with or without simultaneous treatment with rSmCypA. Data shown are representative of 2 independent experiments. (<b>C</b>) ELISA for the quantification of TNF-α and IL-12 in the supernatant of BMDC treated with LPS only or co-treated with LPS and rSmCypA, n = 3 per group. Data shown are representative of 2 independent experiments. Data are presented as mean and SEM and statistical difference between groups was determined using Student's <i>t</i> test.</p

Modulation of CD4⁺ T cells by SmCypA-treated BMDC.

<p>(<b>A</b>) Representative histograms from flow cytometric analysis of CFSE labeled TCR^OVA CD4⁺ T cell cultures with OVA and LPS treated or LPS and rSmCypA co-treated BMDC as APC. (<b>B</b>) Flow cytometric analysis for the identification of Treg (FOXP3⁺), Th17 (RORc⁺), Th2 (GATA3⁺) and Th1 (TBET⁺) CD4⁺ T cells following culture with OVA and BMDC as APC, activated under the outlined conditions, n = 4 per group. (<b>C</b>) Heatmap of ELISA for the detection of IL-10, IL-17A, IFN-γ and IL-4 in the supernatant of TCR^OVA CD4⁺ T cells and BMDC co-cultures. BMDC were activated, or not, under the indicated conditions prior to their co-culture with the TCR^OVA CD4⁺ T cells ± antigen (OVA), n = 6 per group. Data are presented as mean and SEM and statistical difference between groups was determined using Student's <i>t</i> test.</p

Production of rSmCypA.

<p><b>(A)</b> Coomassie stain of SDS-PAGE gel containing WES <b>(Lane 1)</b> and rSmCypA <b>(Lane 2),</b> with a subsequent Ponceau stain following western transfer and Anti-His tag expression to identify recombinant protein. Molecular Weight (MW) markers are shown. (<b>B</b>) Quantification of PPIase activity of rSmCypA in the presence or absence of PMSF. Figure is representative of three independent experiments.</p