Rice University Class of 2014 Undergraduate Convocation program

4 page program for the One-hundred first Commencement Undergraduate Convocation Ceremony, May 16, 2014, including the location and dates of events, speakers' names and subjects and music

Schistosome antigens bind to B cells and are internalized into acidic compartments.

<p><b>(A)</b> C57BL/6 mice were i.v. injected with 200 μg of fluorescently labeled SEA and spleens snap-frozen 30 minutes later. Fluorescence microscopic image shows localization of SEA to B220⁺ B cells and is representative for 2 experiments with N = 5 mice and 3 viewing fields per section imaged. <b>(B, C)</b> Splenic B cells were analyzed by flow cytometry at 30 minutes to 24 hours after i.v. injection of labeled SEA for <b>(B)</b> frequency of SEA⁺ cells within total B cells or B cell subsets as gated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006539#ppat.1006539.g002" target="_blank">Fig 2A</a>, and for <b>(C)</b> fold increase of CD86 expression on total MZ B cells compared to MZ B cells gated for SEA⁺ and SEA^- populations. Summary of 2 experiments with N = 2. <b>(D, E)</b> Splenic B cells from naïve mice were cultured <i>in vitro</i> with SEA (20 μg/ml) labeled with a green dye (PF-488) alone, or co-labeled with a pH-sensitive dye (pHrodo Red). After 60 minutes, the frequency of SEA⁺ B cells was determined by flow cytometry. <b>(D)</b> Gated for total CD19⁺ B cells (1 representative out of 2 experiments shown). <b>(E)</b> Gated for follicular (FO) and marginal zone (MZ) B cell subsets (summary of 3 experiments). Significant differences indicated with ^# p < 0.05 and ^## p < 0.01 are determined by one-sample t-test of log-transformed data (C) or between B cell subsets by Wilcoxon paired test.</p

Schistosome eggs and SEA (soluble egg antigen) drive development of Breg cells <i>in vivo</i>.

<p>C57BL/6 mice were i.p. injected with two doses of 5000 <i>S</i>. <i>mansoni</i> eggs or 100 μg SEA in PBS, or PBS as control. At day 14, CD19⁺ MACS-isolated splenic B cells were restimulated with SEA (20 μg/ml) for 2 days. <b>(A)</b> Cytokine concentration in culture supernatants as determined by ELISA. <b>(B, C)</b> Representative FACS plots (B) and summary (C) for intracellular IL-10 expression of B cells after addition of Brefeldin A to the last 4 hours of the culture. <b>(D)</b> Mean fluorescence intensity of CD40 and CD86 expression. <b>(E-G)</b> SEA-restimulated B cells were co-cultured for 4 days with CD25-depleted CD4 T cells. Frequency of CD25⁺Foxp3⁺ Treg cells after co-culture in representative FACS plots <b>(E)</b> and summary <b>(F)</b> is shown. <b>(G)</b> IL-10 concentration in culture supernatants after co-culture. Summary of 4 experiments with N = 12–16 (A-D) or 2 experiments with N = 8–9 (E-G). Significant differences by Mann-Whitney test are indicated with * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Human CD1d⁺ Breg cells increase IL-10 expression after SEA and IPSE/alpha-1 stimulation.

<p>B cells were isolated from PMBCs of healthy volunteers and stimulated for 3 days with SEA (20 μg/ml), natural IPSE (nIPSE, 10 μg/ml), plant-derived IPSE (pIPSE, 10 μg/ml), or left untreated. Different Breg cell subsets and intracellular IL-10 expression were assessed by FACS. Summary of 3 donors. Significant differences to the respective medium control are indicated with ^# p < 0.05, or between conditions with and without anti-CD40, with * p < 0.05, ** p < 0.01 as tested by paired t-test.</p

The secretory SEA component IPSE/alpha-1 induces Breg cell development.

<p><b>(A)</b> Splenic B cells were incubated with PF-647-labeled SEA (20 μg/ml), natural IPSE/alpha-1 (nIPSE, 1–5–10 μg/ml) or left untreated and the frequency of SEA- and IPSE-positive cells measured after 60 minutes by flow cytometry. Representative FACS plots and summary of 3 experiments is shown (N = 3). <b>(B)</b> Splenic B cells from naïve mice were cultured for 3 days with 10 μg/ml nIPSE, 20 μg/ml SEA, 20 μg/ml SEAΔIPSE or medium as control, with or without addition of anti-CD40 mAb (2 μg/ml). Cytokine concentration in supernatants after 3 days as determined by ELISA. Summary of 4 experiments. <b>(C)</b> Splenic B cells from naïve mice were cultured for 3 days with 10 μg/ml recombinant, plant-derived IPSE (pIPSE), 20 μg/ml SEA or medium as control. Cytokine concentration in supernatants after 3 days as determined by ELISA. Summary of 4 experiments. <b>(D)</b> Splenic B cells were cultured for 3 days with nIPSE (10 μg/ml), SEA (20 μg/ml) or medium alone as control, and subsequently co-cultured with CD4⁺CD25^- sorted splenic T cells from C57BL/6 or DEREG mice. After 4 days, the frequency of CD25⁺Foxp3⁺ Treg cells within the CD4 T cell population was determined by flow cytometry. Summary of 3 experiments. Significant differences to medium control are indicated with * p < 0.05, as tested by Mann-Whitney test. ^# p < 0.05 indicates significant difference by one-sample t-test of log-transformed data.</p

Schistosome antigens mainly activate the marginal zone B cell subset of the spleen.

<p>C57BL/6 were treated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006539#ppat.1006539.g001" target="_blank">Fig 1</a>, splenic follicular (FO) B cells (CD19⁺CD23⁺CD21^low) and marginal zone (MZ) B cells (CD19⁺CD23^-CD21^hi) FACS sorted, and restimulated for 2 days with SEA (20 μg/ml). <b>(A)</b> Gating scheme for both B cell subsets within the CD19⁺ gated B cell population. <b>(B, C)</b> Cytokine concentration in culture supernatants as determined by ELISA. <b>(D)</b> Intracellular IL-10 expression of B cell subsets after addition of Brefeldin A to the last 4 hours of the culture. <b>(E)</b> Mean fluorescence intensity of CD86 expression of B cell subsets. Summary of 2 experiments with N = 6 (B, C) or 3–5 experiments with N = 13–21 (D, E). Significant differences by Mann-Whitney test are indicated with * p < 0.05, *** p < 0.001. Significant differences between B cell subsets by Wilcoxon paired test are indicated with ^# p < 0.05, ^## p < 0.01, ^### p < 0.001.</p

Breg cells can be generated <i>in vitro</i> by stimulation with schistosome egg antigens which involves lysosomal processing.

<p><b>(A-E)</b> Splenic B cells from naïve mice were cultured for 3 days with 20 μg/ml SEA or medium as control. Cytokine concentration in culture supernatants of <b>(A)</b> total B cells (6 experiments) or <b>(B)</b> sorted B cell subsets (2 experiments with N = 6). <b>(C)</b> Intracellular IL-10 expression of total B cells after addition of PMA, ionomycin and Brefeldin A to the last 4 hours of the culture (4 experiments). <b>(D)</b> Mean fluorescence intensity of the activation markers CD40 and CD86 (4 experiments). <b>(E)</b> Splenic B cells were stimulated <i>in vitro</i> as above, and subsequently co-cultured with CD4⁺CD25^- sorted splenic T cells. After 4 days, the frequency of CD25⁺Foxp3⁺ Treg cells within the CD4 T cell population was determined by flow cytometry. Summary of 4 experiments. <b>(F, G)</b> Splenic B cells from naïve mice were cultured <i>in vitro</i> with SEA (20 μg/ml), CpG (5 μg/ml) or Pam3Cys (10 μg/ml) for 2 days. Every day, chloroquine (5 μM) was added to the culture. Intracellular IL-10 expression after addition of PMA, ionomycin and Brefeldin A during the last 4 hours of the culture <b>(F)</b>, and cytokine concentrations in culture supernatants <b>(G)</b>. Summary of 2 experiments with N = 3–4. Significant differences are indicated with * p < 0.05, ** p < 0.01 and tested by Mann-Whitney. ^# p < 0.05 indicates significant difference between B cell subsets by Wilcoxon paired test.</p

CD40 ligation enhances SEA-induced Breg cell development.

<p><b>(A, B)</b> Splenic B cells from naïve mice were cultured with 20 μg/ml SEA or medium as control, with or without addition of anti-CD40 co-stimulatory antibody. After 3 days of culture, supernatants were analyzed for IL-10 and IL-6 by ELISA <b>(A)</b> and B cell CD86 expression by flow cytometry <b>(B)</b>. Summary of 6 experiments. <b>(C)</b> Splenic B cells were stimulated for 3 days with anti-CD40 mAb (2 μg/ml) and SEA (20 μg/ml) or anti-CD40 alone, and subsequently co-cultured with CD4⁺CD25^- sorted splenic T cells. After 4 days, the frequency of CD25⁺Foxp3⁺ Treg cells within the CD4 T cell population was determined by flow cytometry. Summary of 4 experiments. <b>(D-F)</b> Mice were i.p. injected with two doses of 100 μg SEA or PBS as control. CD40 ligand was blocked <i>in vivo</i> by i.p. injection of 200 μg hamster anti-mouse CD40 ligand or 200 μg hamster IgG as control (Ctrl) for 4 times, every 4 days starting at day -1 before SEA/PBS treatment. At day 14 after the first SEA/PBS injection, splenic B cells were <i>in vitro</i> restimulated with SEA for 2 days. Summary of 2 experiments (N = 8). <b>(D)</b> Fold increase of IL-10 in supernatants from B cells of SEA-injected versus PBS-injected mice. <b>(E)</b> Intracellular IL-10 expression of B cells after addition of Brefeldin A to the last 4 hours of the culture. <b>(F)</b> Mean fluorescence intensity of CD86 on B cells. Significant differences are indicated with * p < 0.05, ** p < 0.01, *** p < 0.001 as tested by Mann-Whitney test. ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 indicates significant difference by one-sample t-test of log-transformed data (D) or to respective medium control by Mann-Whitney test.</p

Macrophage subsets of the MZ bind SEA but are dispensable for schistosome antigen-mediated Breg cell induction.

<p><b>(A, B)</b> Spleens were snap-frozen 30 minutes after i.v. injection of 200 μg fluorescently labeled SEA, and binding of SEA analyzed by fluorescence microscopy. Images are representative for 2 experiments with N = 5 mice and 3 follicles per section imaged. <b>(A)</b> SEA clustered around the marginal zone of the spleen (indicated by white dashed line). <b>(B)</b> SEA localized in the marginal zone to macrophages expressing Siglec-1 (marginal metallophilic macrophages) and SIGN-R1 (MZ). <b>(C, D)</b> Mice were i.v. injected with fluorescently labeled SEA and splenocytes harvested 30 minutes-24 hours later. <b>(C)</b> Frequency of SEA-positive cells in various splenocyte subsets as determined by flow cytometry (gating schemes see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006539#ppat.1006539.s004" target="_blank">S4 Fig</a>). MΦ, macrophages; RP, red pulp; MM, marginal metallophilic; DC, dendritic cells. <b>(D)</b> Mean fluorescence intensity of surface markers on MM macrophages calculated as fold increase versus the expression at time-point 0 hours. Summary of 2 experiments with N = 2. <b>(E-G)</b> Mice were i.p. injected with 200 μl clodronate-containing liposomes or PBS control liposomes. Three weeks later, mice were i.p. injected with two doses of 5000 eggs. Seven days after the last egg injection, splenic B cells were restimulated for 2 days with SEA. <b>(E)</b> Absence of splenic Siglec-1 and SIGN-R1-expressing macrophage subsets at the time-point of spleen collection was confirmed by fluorescence microscopy. B cells were stained with B220. <b>(F)</b> Cytokine concentration in culture supernatants as determined by ELISA. <b>(G)</b> Intracellular IL-10 expression of B cells after addition of Brefeldin A to the last 4 hours of the culture. One representative (N = 5) out of 2 similar experiments is shown. Significant difference by Mann-Whitney test is indicated with * p < 0.05, ** p < 0.01. Significance as determined by one-sample t-test of log-transformed data is indicated with ^# p < 0.05, ^## p < 0.01.</p