B7-H1^−/− DC induce more protective cytokines than WT DC.

<p>MOG_35–55 loaded and TNF-matured WT or B7-H1^−/− DC (2,5×10⁶) were injected i.v. into WT mice 7, 5 and 3 days before EAE induction. Control mice were injected with PBS. 10 days after EAE induction spleen cells were isolated and restimulated with different concentrations of MOG_35–55. After 4 days of culture supernatants were tested by ELISA for IL-10, IL-13, IL-17 and IFN-γ. Results show the mean of 4 mice per group and are representative for 2 independent experiments with 4 mice per group.</p

Strong increase of protective serum cytokines after injection of B7-H1^−/− DC injection is mainly produced by type II NKT cells.

<p>A) Splenocytes from WT and Jα281^−/− mice were stained for CD3 and NK1.1 and investigated by flow cytometry. Double positive cells (NKT cells) were analyzed for PD-1 expression. B) MOG_35–55 loaded and TNF-matured WT or B7-H1^−/− DC (2,5×10⁶) were injected i.v. into WT, CD1d−/− or Jα281^−/− mice three times (on day −4, −2 and 0). Similarly, control mice were injected with PBS. Blood was collected 2 hours after the last DC injection and serum cytokines were measured by ELISA for IFN-γ, IL-17, IL-4 and IL-13. The results represent the average of 3 mice per group and are representative for 4 independent experiments for the WT mice and for 2 independent experiments for CD1d−/− and Jα281^−/− mice. * p<0.05, ** p<0.01, *** p<0.001, n.s. not significant.</p

Injection of B7-H1^−/− DC results in reduced frequency of neurantigen-specific IFN-γ and IL-17 secreting cells in the CNS.

<p>MOG_35–55 loaded and TNF-matured WT or B7-H1^−/− DC (2,5×10⁶) were injected i.v. into WT mice 7, 5 and 3 days before EAE induction. Control mice were injected with PBS. After 15 days CNS mononuclear cells were harvested and CNS T cell infitration and cytokine production were investigated using flow cytometry and ELISPOT assay. A) Dot blots are gated for live lymphocytes and inserted numbers represent the percentage of CD4⁺ and CD8⁺ CNS infiltrating T cells of 3–5 pooled mice per group of one experiment. Results are representative for 2 independent experiments. B) ELISPOT analysis of IFN-γ (left) or IL-17 (right) production by infiltrating CNS cells after MOG_35–55 peptide restimulation. Results are representative for 2 independent experiments with 3–5 pooled mice per group. * p<0.05, ** p<0.01, n.s. not significant.</p

WT and B7-H1^−/− DC show equal expression of surface markers and have the same potential to stimulate CD4⁺ T cells.

<p>A) BM-derived DC from WT and B7-H1^−/− mice were matured with TNF over night, stained for different surface markers and analyzed by flow cytometry. The shaded histograms show the unstimulated control and the black lines show TNF-matured DC. Results are representative for 3 independent experiments. B) TNF-stimulated and MOG_35–55-loaded DC from WT and B7-H1^−/− mice were co-cultured with CFSE-stained CD4⁺ T cells with MOG_35–55 transgenic TCR. Proliferation of the T cells was analysed by the dilution of the CFSE-staining. The dottet line shows the control without DC, the shaded histogram shows the co-culture with 1×10⁴ DC and the filled line shows the co-culture with 3×10⁴ DC. Results are representative for 3 indepentent experiments.</p

NKT cell hybridoma cells express PD-1 but only the type II NKT cells are stimulated by DC and are negatively regulated by B7-H1.

<p>A) NKT cell hybridoma cells s were stained for PD-1 and analyzed by FACS. The shaded histogram shows the isotype control staining and the black line shows PD-1 staining. B) DC from WT and B7-H1^−/− mice were co-cultured with the indicated NKT hybridoma cells in the presence or absence of αGC (10 ng/ml). After 24 hours, the supernatants were tested for IL-2 content by ELISA. Results show 1 out of 4 representative and independent experiments. ** p<0.01.</p

Statistics of EAE.

<p>Mice were injected i.v. (days −7, −5, −3) with PBS, WT DC or B7-H1^−/− DC and on day 0 EAE was induced. The incidence, mean maximal score and mean day of onset is shown.</p>a<p>Statistically significant when compared with PBS group: p<0.001 by Student's t-test.</p>b<p>Statistically significant when compared with WT DC group: p<0.05 by Student's t-test.</p

Maximum binding of differently loaded CD1d dimers.

<p>(A+B) Highest relative staining reached with each CD1d dimer loaded with indicated glycolipid as presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143449#pone.0143449.g002" target="_blank">Fig 2</a>. Presented (A) as absolute or (B) as relative to the highest value reached for each CD1d. Human CD1d: Binding maxima are for all glycolipids are reached with Tyloxapol as surfactant. Mouse CD1d: Binding maxima with αGC and PBS57 are reached with Tween 20, forDB01-1 with Triton X-100 and for PBS44 with Tyloxapol. Rat CD1d: Binding maxima for αGC, DB01-1 and PBS44 are reached with Triton X-100, and for PBS57 with Tyloxapol. Cotton Rat: All maxima reached using Tyloxapol. Statistical analysis can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143449#pone.0143449.s003" target="_blank">S2 Table</a>. Hierarchy of surfactant efficacy between experiments are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143449#pone.0143449.s001" target="_blank">S1 Fig</a>. Data were computed from results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143449#pone.0143449.g002" target="_blank">Fig 2</a>.</p

<i>In vitro</i> loading analysis of CD1d dimers.

<p>CD1d dimers were incubated with 40x molar excess of indicated glycolipid in presence of 0.05% of indicated surfactant or without surfactant o.n. at 37°C. After <i>in vitro</i> loading, the indicated amount of CD1d dimers was used for staining of 10⁵ iNKT TCR transduced cells, dimer binding was revealed by staining with PE labeled donkey F(ab′)₂ fragment anti-mouse IgG (H+L). Binding presented as relative staining, i.e. ratio between geometric means of CD1d dimer and CD3 antibody staining was performed simultaneously. In total, staining was performed three times independently using oligomers derived from two independent loadings. Hierarchy of surfactant efficacy between experiments is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143449#pone.0143449.s001" target="_blank">S1 Fig</a>. Note that scales differ in order to facilitate visualization different hierarchies of binding caused by the different surfactants.</p

Experimental setup.

<p>(A) α1 and α2 domain amino acids of CD1d molecules used in this study. Alignment was performed using BioEdit. Human: GenBank BC027926.1, Mouse: GenBank AK002582.1, Rat: GenBank AB029486.1, Cotton Rat: GenBank KM_267558. (B) <i>In vitro</i> loading analysis of CD1d dimers presented as schematic overview. (C) Scheme explaining the emergence of different saturating curves depending on loading efficacy of dimers. Upper panel: Inefficient loading leads to low proportion of functionally bivalent CD1d dimers, Lower panel: Efficient loading leads to high proportion of functional bivalent CD1d dimers. Drawing represents several step flow cytometry analysis of incubation/wash/detection. (D) Structural features of all glycolipids used in this study.</p

<i>In vitro</i> loading analysis of rat CD1d tetramers.

<p>PE conjugated CD1d tetramers were incubated with 40x molar excess of indicated glycolipid in presence of 0.05% indicated surfactant or without surfactant o.n. at 37°C. After <i>in vitro</i> loading, the indicated amount of CD1d tetramers was used for staining of 10⁵ iNKT TCR transduced cells. Binding presented as relative staining, i.e. ratio between geometric means of CD1d tetramer and CD3 antibody staining performed simultaneously. In total, staining was performed three times independently using oligomers derived from two independent loadings. Hierarchy of surfactant efficacy between experiments is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143449#pone.0143449.s001" target="_blank">S1 Fig</a>.</p