Suppressive capacity of monocytic and granulocytic MDSC subsets.

<p>C57BL/6 mice were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119662#pone.0119662.g001" target="_blank">Fig. 1</a>. Gating strategy for sorting (A). At the indicated times myeloid subsets from liver (B), kidney (C), spleen (D) or in vitro bone marrow culture (E) were isolated and CD11b⁺ cells were sorted on the basis of their Ly6C or Ly6G expression and additional expression of IFNγRβ (A), yielding 4 separate subsets of CD11b⁺ myeloid cells. Naïve CFSE-labelled CD8 T cells were stimulated using αCD3/αCD28 coated beads and the different subsets of sorted myeloid cells were added at a 3:1 ratio (B-E). After 72h T cell proliferation was analysed by flow cytometry and the percentage of proliferated T cells is depicted (B-E). Cumulative data from 2 independent experiments are shown. Data are depicted as mean +/- SEM. Significance was calculated by ANOVA. *p≤0.05, **p≤0.01, ***p≤0.001. ND = not detectable.</p

Differential distribution of monocytic and granulocytic myeloid derived suppressor cells within Gr-1 positive cells in liver and kidney inflammation and fibrosis.

<p>C57BL/6 mice underwent bile-duct ligation, were fed an adenine-enriched diet, or were injected i.v. with LPS/IFNγ. Furthermore, BM-MDSC were generated <i>in vitro</i> by culture of bone marrow cells with CSF2 (GM-CSF) (A, B). After the indicated times liver, kidney, spleen or bone marrow cells were isolated and analysed by flow cytometry. Histograms depict viable (Hoechst negative), non-parenchymal cells stained with CD11b and Gr-1 (A) or viable, CD11b^pos cells stained for Ly6G and Ly6C (B). Representative (A, B) and cumulative (C) data of 4 (liver, spleen) or 3 (kidney, bone marrow) independent experiments are shown (n>9). Absolute numbers of CD11b⁺Ly6C⁺ and CD11b⁺Ly6G⁺ meyloid cells in the liver (D) and kidney (E) at the indicated time-points after BDL or adenine-feeding, respectively. Data are depicted as mean +/- SEM. Significance was calculated by ANOVA. *p≤0.05, **p≤0.01, ***p≤0.001.</p

Fibrosis markers in the liver and kidney after all-trans-retinoic acid treatment.

<p>C57BL/6 mice underwent bile-duct ligation, were fed an adenine-enriched diet or as a control were left untreated. After 7 days mice were treated with 1g/L all-trans-retinoic acid (ATRA) in their drinking water (BDL: n = 7, Adenine: n = 4) or not (BDL: n = 8, adenine: n = 4)) for the remaining time until analysis. At day 14 (BDL and adenine feeding), total liver and kidney RNA was isolated for real-time PCR of fibrosis markers. Shown are mRNA expression levels for α-SMA, collagen IV, TGF-β and vimentin relative to the levels in non-treated mice (n = 3), which was set to 1. Data are depicted as mean +/- SEM. Significance was calculated by ANOVA. *p≤0.05, **p≤0.01, ***p≤0.001.</p

Surface marker expression on suppressive and non-suppressive myeloid subsets.

<p>C57BL/6 mice were treated and cells were isolated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119662#pone.0119662.g001" target="_blank">Fig. 1</a>. Myeloid cells from non-treated mice served as controls (steady state). Flow cytometric analysis of surface markers associated with MDSC induction/function on CD11b⁺Ly6C⁺ and CD11b⁺Ly6G⁺ myeloid cells are depicted in the histograms. Specific staining: black lines. Isotype controls: filled grey. Representative data of 3 independent experiments is shown.</p

MPO⁺ cells in graft muscularis whole-mounts stained by Hanker-Yates reaction.

<p>At seven days after ITX, HLA-G treatment caused a significant reduction in MPO⁺ cell infiltrate in the graft muscularis.</p

Morphologic characteristics and histologic grading of ACR using the Wu-score.

<p>(A) Representative H/E stained intestinal specimens with and without HLA-G treatment. Arrows indicate stronger leucocytic infiltration in graft mesentery, sites of crypt epithelial injury, propria fibrosis and increased crypt apoptotic body counts, respectively. (B) Overview of severity of ACR as assessed by Wu-score (one reviewer shown). n = 5 in each group.</p

Gating strategy and overview of CD4⁺ and CD8⁺ T-cells isolated from gMLN with and without HLA-G treatment after allogenic intestinal transplantation.

<p>A) Representative gating strategy for recipient(LEW)-derived (MHC I/RT1.Ac-negative), CD45⁺ and CD8⁺ and likewise, CD4⁺ T-cells. Percentages of CD4⁺ and CD8⁺ are given as percentages of CD45⁺ parent population, shown is a representative result for CD8⁺ with and without HLA-G treatment at POD 7. (B) Overview of CD4⁺ and CD8⁺ T-cells with and without HLA-G treatment at both timepoints. A significant increase in CD8⁺ T-cells in the CTL groups is shown without treatment, as well as a significant reduction of this CD8⁺ T-cell population at day seven with HLA-G treatment.</p

Experimental groups and study design.

<p>Experimental groups and study design.</p

In-vitro graft contractility measured as a dose-response curve under betanechol stimulation.

<p>Dose response of graft contractility to rising bethanechol concentrations, curves fitted to a logistic four-parameter sigmoidal model. Both midpoint (logEC₅₀₎ and slope were significantly ameliorated by HLA-G treatment at seven days after ITX.</p

ACR-related gene expression in graft muscularis.

<p>The first time point at four days after allogenic ITX showed a significant downregulation of both TNFα and IL-10 by HLA-G treatment (Mann-Whitney U test p<0.05). At seven days after allogenic ITX, TNFα expression remained significantly reduced by HLA-G treatment.</p