Reduced IL-10 secretion by CD83mu B cells.

<p>Untouched B cells were isolated from pooled spleens of two 8–10 week old sex matched C57BL/6 (open bars), CD83Tg (black bars) and CD83mu (dark grey bars) mice. 2×10⁵ B cells were cultured in triplicates in the presence of titrated amounts of LPS as indicated on the x-axis. 6A: Total mouse Ig in the supernatant (dilution 1∶1024) was measured after seven days by ELISA. 6B: IL-10 in the supernatant was measured after 48 h. Presented are the combined results of 3 independent experiments, error bars show SEM. Asterisks indicate a significant difference of the mean (* p<0.05; ** p<0.005 *** p<0.0005) employing students t test.</p

CD83 is upregulated on activated B cells.

<p>C57BL/6 mice derived spleen cells (2×10⁶/ml) were stimulated with anti-BCR (1 µg/ml) and IL-4 (20 ng/ml) or with LPS (10 µg/ml) as indicated in the headline. Cells were triple stained for CD19, CD83 and CD69 at the indicated time points. 1A: Dot blots show all lymphocytes positive for surface expression of CD83 on the x-axis and CD19 expression on the y-axis. 1BC: Graphs show the percentage of CD83 positive B cells (1B) or the mean fluorescence intensity (MFI) of CD83 on B cells (1C) after stimulation with anti-BCR alone (open circle) anti-BCR and IL-4 (closed circle) or with LPS (closed square) in an independent experiment, error bars show SEM of duplicates. 1D: Dot blot shows 2×10⁴ CD19 positive cells derived from LPS activated spleen cells analyzed for CD83 (x-axis) and CD69 (y-axis) surface expression. 1E: 2×10⁶ purified C57BL/6 spleen derived B cells were stimulated with LPS (10 µg/ml). B cells were lysed at the indicated time points, deglycosylated and separated by SDS-PAGE. CD83 was detected by western blot with a polyclonal rabbit anti-CD83 serum. Results are representative for at least three independent experiments.</p

Purified CD83Tg B cells display reduced Ig and increased IL-10 response to LPS stimulation.

<p>Untouched B cells were isolated from pooled spleens of two 8–10 week old sex matched C57BL/6 (open squares) and CD83Tg (black squares) mice. 2×10⁵ B cells were cultured in 5 replicates in the presence of LPS (10 µg/ml) or no further stimulus, as indicated on the x-axis. Proliferation (4A) or IL-10 secretion (4B) were measured after 48h. 4C: Total mouse Ig in the supernatant (dilution 1∶1024) was measured after seven days by ELISA. Each dot represents an individual experiment, employing B cells purified from two pooled spleens (mean of five replicates the SEM not shown, being below 10%). The bars indicate the median and the asterisks indicate a significant difference of the median (***p<0.0005), employing students t test.</p

Reduced calcium signaling in CD83Tg B cells.

<p>Untouched B (7AB) or T cells (7CD) were isolated from pooled spleens of two 8–10 week old sex matched C57BL/6 (open bars) and CD83Tg (black bars) mice. Purified B and T cells were loaded with Fura-2-AM and stimulated with anti-ΒCR (Lo-MK) or anti-CD3 (145-2C11) respectively (10 µg/ml each). [Ca²⁺]_i was calculated from the fluorescence intensity ratio at 340 and 380 nm. 7AC: Bars represent the combined results from nine independent measurements, error bars show SEM. Asterisks indicate a significant difference of the mean. 7BD: Shown is a characteristic calcium tracing from a single experiment for B cells (7B) and T cells (7D) obtained from C57BL/6 (solid line) or CD83Tg (dotted line) mice.</p

CD83Tg spleen cells display reduced Ig and increased IL-10 response to LPS stimulation.

<p>3A-D: 2×10⁵ spleen cells derived from sex and age matched C57BL/6 (open squares) or CD83Tg founder 2 (closed squares) were cultured in the presence of LPS (10 µg/ml) or anti-CD3 (145-2C11, 1 µg/ml) or no further stimulus, as indicated on the x-axis. 3AB: Proliferation was estimated after 48h by ³H thymidine incorporation. 3C: Total mouse Ig in the supernatant was measured after seven days by ELISA. Results are presented as relative ELISA units (REU: OD₄₅₀ sample: OD₄₅₀medium control) of a 1∶1024 dilution. 3D: IL-10 in the supernatant was quantified by ELISA after 48 h. Each dot represents the analysis of an individual mouse (mean of five replicates the SEM not shown, being below 10%). The lines indicate the median of all mice analyzed and the asterisks indicate a significant difference of the median (*p<0.05; **p<0.005) employing students t test. 3E: HEL-specific Ig in the serum of six IgHEL Tg mice (open bar) or six IgHEL CD83double Tg mice (black bar) was quantified by ELISA, error bars indicate SEM. 3F: 2×10⁵ spleen cells derived from six IgHEL Tg mice (open bars) or from spleen cells derived from IgHEL CD83 double Tg mice (black bars) were cultivated <i>in vitro</i> for 48 h in the presence of indicated amounts of LPS. HEL-specific Ig in the supernatant was quantified by ELISA and is presented as REU of a 1: 40 dilution. Error bars show SEM of five replicates. This result is representative for five independent experiments.</p

<i>In vitro</i> stimulation of CD83mu spleen cells.

<p>2×10⁵ spleen cells derived from C57BL/6 (open squares) or CD83mu (closed circles) mice were cultured in the presence or absence of LPS (1 µg/ml) as indicated on the x-axis. 5A: Proliferation was estimated by ³H thymidine incorporation after 48h culture. 5B: Total mouse Ig in the supernatant was measured after seven days by ELISA and results are presented as REU of a 1:200 dilution. 5C: IL-10 in the supernatant was quantified by ELISA after 48 h. 5DE: spleen cells were cultured in the presence or absence of anti-CD3 (1 µg/ml). Proliferation (5D) and IL-2 secretion (5E) were measured after 48h culture. Each dot represents an individual experiment performed in five replicates SD being below 10%, the bars indicate the median of all five experiments performed.</p

Positive correlation of CD83 expression to CD86 and MHC-II expression on CD83Tg and CD83mu B cells.

<p>C57BL/6 and CD83 negative littermates to CD83Tg founder 1 mice (open bars), CD83Tg (black bars) or CD83mu (dark grey bars) derived spleen cells (2×10⁶/ml) were double stained for CD19 and either CD83 (2A), CD86 (2B), MHC-II (2C), CD80 (2D), CD40 (2E), CD69 (2F) or IgM (2G) <i>ex vivo</i> or after 48 h incubation with 10 µg/ml LPS as indicated on the x axis. 2×10⁴ CD19 positive cells were analyzed by FACScan. Please note that each bar represents the combined results of five independent experiments employing two female age matched mice of each group per experiment, error bars show SEM. Asterisks indicate a significant difference of the mean (* p<0.05; ** p<0.005; *** p<0.0005) employing students t test.</p

P2X7(k) is the dominant transcript in T cells, P2X7(a) is the dominant transcript in macrophages and muscle cells.

<p>Total RNA was prepared from primary splenic and lymph node T cells, peritoneal macrophages, and skeletal muscle tissue from C57BL/6 mice (carrying the 451L allelic variant of P2X7). RNA samples were subjected to quantitative RT-PCR analyses with primers specific to exon one of P2X7(a) or P2X7(k) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041269#pone.0041269-Nicke1" target="_blank">[23]</a>. Results are representative of four independent experiments for lymphocytes and three independent experiments for macrophages and skeletal muscle. Error bars represent SEM. *, p<0.05; ***, p<0.001.</p

ADP-ribosylation of P2X7(k) induces stable Ca²⁺ signals.

<p>HEK cells were co-transfected with expression constructs for mRFP, ART2.2, and the indicated splice variants of P2X7. 20 h post transfection, cells were loaded with the Ca²⁺-sensitive fluorophore Fura-2 before live-cell imaging by fluorescence microscopy at 37°C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041269#pone.0041269-Adriouch1" target="_blank">[8]</a>. Images were captured every 5 sec. At the indicated times, the perfusion buffer was changed to perfuse cells with increasing doses of NAD⁺ (panels 1–3) or ATP (panels 4–6). Ratio images (340/380 nm) were constructed pixel-by-pixel and single cell tracings were obtained using the Openlab software. Grey lines show single cell tracings, red lines the calculated mean. Results are representative of two independent experiments.</p

ADP-ribosylation of P2X7(k) induces permeabilization of the cell membrane to the amine-reactive dye PacO.

<p>HEK cells were co-transfected with expression constructs for ART2.1 and the indicated splice variants of P2X7, harvested 48 post transfection, and incubated for 30 min in the presence of the indicated concentrations of NAD⁺ (A) or ATP (B) as in Fig. 3. Cells were stained with Alexa647-conjugated anti-P2X7 (Hano44) and amine-reactive Pacific Orange (PacO) and subjected to FACS-analyses. Similar results were obtained with constructs on the BALBc background (451P). Results are representative of two independent experiments.</p