Effects of Ag85B stimulation and 4-1BB ligation on in vivo frequency of CD4⁺ and CD8⁺ T cells and 4-1BB expression of cells from NP-mice adoptively transferred into C57BL/6Ly5.1 recipient mice.

<p>C57BL/6Ly5.1 mice adoptively transferred with 10⁷ spleen cells from NP-mice (cells CD45.2⁺) were treated or not with antigen Ag85B and mAbs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011019#s4" target="_blank">Materials and Methods</a>. Slpeen cells were harvested on post-infection day 10, stained with CD45.2-FITC, CD4-PE, CD8-PerCP, CD4PerCPCy5.5 and 4-1BB-PE mAbs and then analyzed by flow cytometry. The data, obtained by gating CD45.2⁺ cells on lymphocyte population, show the frequency of CD4⁺ and CD8⁺ T cells originating from NP-mice and the percentage of T cells expressing 4-1BB. The results are presented as mean ± SEM of 3 mice per group.</p><p>#, § statistical significance for differences between groups determined by ANOVA and Bonferroni-type multiple t-test (# Ag85B/control IgG vs none; § Ag85B/control IgG vs Ag85B/anti-4-1BB mAb).</p

Ag85B protein stimulation in spleen cells of NP-mice induces expression of 4-1BB on both CD4⁺ and CD8⁺ T cells.

<p>Pooled spleen cells of NP-mice (panel A) or P-mice (panel B) were re-stimulated with Ag85B protein (5 µg/ml). After 3 or 6 days of cultures, cells were labelled with the following mAb: CD4 PE-Cy7, CD8 PE-Cy7, CD3 FITC and 4-1BB PE. Cell surface molecule expression was analyzed by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011019#s4" target="_blank">Materials and Methods</a>. Within each panel, the percentage indicates the number of cells expressing the 4-1BB receptor. Plots, gated on CD4⁺ or CD8⁺ T cells in the lymphocyte-gate, are representative of one experiments out of 3.</p

Aspartyl Proteinases of Eukaryotic Microbial Pathogens: From Eating to Heating - Fig 1

<p><b>Left</b>. The molecular ribbon-like structure of Sap2, a major AP of <i>Candida albicans</i>. Note the flexible flaps that control the access to the central region faced and delimited by the two active sites DTGS and DSGT and accommodating an enzyme inhibitor. N-ter is the N-terminus and C-ter the C-terminus of the amino-acid sequence. <b>Right</b>. Sequences of Sap2 and plasmepsin II of <i>Plasmodium falciparum</i>, which is most similar to Sap2 among the APs of eukaryotic microbial pathogens, showing two regions of high similarity (highlighted in red). The identity of the two whole sequences is 28.2% and their similarity 57.4% (FASTA; MBL Swiss-Prot).</p

Effects of 4-1BB ligation in spleen cells of NP- and P-mice stimulated with Ag85B protein on expression of activation markers on CD8⁺ T cells.

<p>Unfractionated spleen cells of P- or NP-mice were cultured with Ag85B protein (5 µg/ml) in the presence of IgG2a control Ab or agonist anti-4-1BB mAb (5 µg/ml). After 6 days of culture cells were labeled with the indicated mAbs. Cell surface molecule expression was analyzed by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011019#s4" target="_blank">Materials and Methods</a>. The results are presented as mean ± SEM of the percentage of CD8⁺ T cells staining for the indicated markers. Pooled data of 3 independent experiments are shown.</p><p>#, § statistical significance (p<0.05 or <0.01) for differences between groups determined by ANOVA and Bonferroni-type multiple t-test (# Ag85B/control IgG vs unstimulated; § Ag85B/control IgG vs Ag85B/anti-4-1BB mAb).</p

Role of CD8⁺ T cells in modulating Ag85B-mediated IFN-γ secretion associated with protection and non-protection against MTB infection.

<p>Untouched purified CD4⁺ T cells (5×10⁴ cells/well) and CD8⁺ T cells (3×10⁴ cells/well) obtained by negative magnetic bead separation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011019#s4" target="_blank">Materials and Methods</a> from spleen cells of NP-mice (panel A) or P-mice (panel B) were cultured, alone or together (using the CD4/CD8 ratio of 1∶0.6 as found in fresh spleen cells) on a feeder of CD3⁺ T cell-depleted spleen cells of naive mice (1.5×10⁵ cells/well). Untouched purified CD8⁺ T cells recovered from naïve unvaccinated (3×10⁴ cells/well) were also added to CD4⁺ T cell co-culture. Cells were re-stimulated with Ag85B protein (5 microg/ml). Culture supernatants were harvested after 3 days for IFN-g detection by a specific quantitative sandwich ELISA Kit. Data are presented as mean of 4 independent experiments. Error bars indicate SEM. The level of statistical significance for differences were determined by a two-tailed Student <i>t</i> test (**, p<0.01) between Ag85B-induced responses by CD4⁺ T cells alone and co-cultured with CD8⁺ T cells. Purified CD8⁺ T cells of both naïve and Ag85B-immunized mice or cells used as feeder did not release detectable amounts of IFN-g (data not shown).</p

The proposed view of Sap-induced inflammasome activation and inflammasome-dependent cytokine production.

<p>Sap2 and Sap6 activate the NLRP3 inflammasome pathway through an early cascade of events, causing upstream NLRP3 inflammasome activation and downstream caspase-1-mediated cytokine production. Late events depend on Sap endocytosis inducing the translocation of NF-κB (p50/p65) into the nucleus, pro-IL-1β and pro-IL-18 synthesis, then (through type I IFN production) caspase-11 activation that cooperates with the NLRP3 inflammasome in triggering downstream caspase-1-mediated cytokine production. For details about this proposed scheme of Sap/inflammasome/caspases activation, see [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005992#ppat.1005992.ref038" target="_blank">38</a>], [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005992#ppat.1005992.ref039" target="_blank">39</a>], and [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005992#ppat.1005992.ref042" target="_blank">42</a>].</p

The <i>SAP</i> family of <i>C</i>. <i>albicans</i> contains at least ten proteins with a signal peptide and are secreted, except Sap9 and Sap10, which remain bound to the cell wall.

<p>They are characterized by broad spectrum proteolytic ability and virulence properties that are reported to be differentially expressed at different stages and forms of fungus growth and disease. Sap2 (alike Sap1 and Sap3) is active at acidic pH and is dominantly associated with yeast form of growth while Sap6 (alike Sap4 and Sap5) is more active at neutral to slightly alkaline pH Together with the dominant Sap5, Sap6 has been associated with hyphal growth. For details, see [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005992#ppat.1005992.ref007" target="_blank">7</a>] and [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005992#ppat.1005992.ref028" target="_blank">28</a>].</p

Effects of 4-1BB ligation in spleen cells of NP- and P-mice stimulated with Ag85B protein on expression of activation markers on CD4⁺ T cells.

<p>Unfractionated spleen cells of P- or NP-mice were cultured with Ag85B protein (5 µg/ml) in the presence of IgG2a control Ab or agonist anti-4-1BB mAb (5 µg/ml). After 6 days of culture cells were labeled with the indicated mAbs. Cell surface molecule expression was analyzed by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011019#s4" target="_blank">Materials and Methods</a>. The results are presented as mean ± SEM of the percentage of CD4⁺ T cells staining for the indicated markers. Pooled data of 3 independent experiments are shown.</p><p>#, § statistical significance (p<0.05 or <0.01) for differences between groups determined by ANOVA and Bonferroni-type multiple t-test (# Ag85B/control IgG vs unstimulated; § Ag85B/control IgG vs Ag85B/anti-4-1BB mAb).</p

Effects of 4-1BB ligation in Ag85B-activated spleen cells of P-mice on cytokine/chemokine production.

<p>Unfractionated spleen cells of P-mice were cultured with Ag85B protein (5 µg/ml) in the presence of IgG2a control Ab or agonist anti-4-1BB mAb (5 µg/ml). Culture supernatants were harvested at 48 h for IL-2, IL-4; at 72 h for IL-6, IL-10, XCL-1, MIP-1β, TGF-β1, TNF-α and at 96 h for IL-17 detection by specific quantitative sandwich ELISA Kits. The results are presented as mean ± SEM in pg/ml for cytokine detection. Pooled data of 3 independent experiments are shown.</p

Effects of 4-1BB ligation in spleen cells of NP- and P-mice stimulated with Ag85B protein on IFN-γ-producing CD4⁺ T cells and on proliferation of CD4⁺ and CD8⁺ T cells.

<p>To analyze IFN-γ- or IL-10-producing CD4⁺ T cells, unfractionated spleen cells of P- or NP-mice were cultured with Ag85B protein (5 µg/ml) in the presence of IgG2a control Ab or agonist anti-4-1BB mAb (5 µg/ml). After 2 days (for IL-10 analysis) or 4 days (for IFN-γ analysis) of culture and an overnight incubation with brefeldin A cells were stained with CD4-PE-Cy5 and then intracellular stained with PE anti-IL-10 or FITC anti-IFN-γ Ab as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011019#s4" target="_blank">Material and Methods</a>. Cells were then analyzed by flow cytometry and the data show frequency of IFN-γ- or IL-10-producing CD4⁺ T cells.</p><p>To study cell proliferation, unfractionated spleen cells of NP- or P-mice, were labelled with CFSE as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011019#s4" target="_blank">Materials and Methods</a> and then stimulated with Ag85B protein (5 µg/ml) in the presence of agonistic anti-4-1BB mAb (5 µg/ml) or rat IgG2a control Ab (5 µg/ml) for 4 days. At this time point cells were labelled with CD4-PE and CD8-PerCP and analyzed by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011019#s4" target="_blank">Materials and Methods</a>. The percentage of CD4⁺CFSE^low and CD8⁺CFSE^low indicates the number of replicating CD4⁺ or CD8⁺ T cells, respectively.</p><p>The results are presented as mean ± SEM of the percentage of CD4⁺ T cells or CD8⁺ T cells staining for the indicated markers. Pooled data of 3 independent experiments are shown.</p><p>#, § statistical significance for differences between groups determined by ANOVA and Bonferroni-type multiple t-test (# Ag85B/control IgG vs unstimulated; § Ag85B/control IgG vs Ag85B/anti-4-1BB mAb).</p><p>nd = not done.</p