ILT2-mediated signaling by B2M-HLA-G5 and B2M-HLA-G1s-Fc proteins.

<p>NFAT-GFP reporter cells expressing the ILT2-PILRβ chimera were stimulated for 16 h with 1.5 µg/ml of the indicated HLA-G recombinant proteins. Non-treated reporter cells were used as negative control, and tetrameric complexes of HLA-G1 (HLA-G1t, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021011#pone.0021011-Liang2" target="_blank">[31]</a>) were used as positive control. GFP expression on reporter cells was analyzed by flow cytometry. Numbers indicate the percentage of GFP-positive cells. Data shown are from one of four independent experiments.</p

Monomeric/multimeric status and conformation of recombinant proteins.

<p><b><i>A</i></b><i> Monomeric/multimeric status of recombinant proteins</i>. Western blot analysis of recombinant proteins immunoprecipitated from supernatants of HeLa B2M-HLA-G1s-Fc, HeLa Alpha1-Fc, HeLa B2M-HLA-G5, and of alpha1_peptide. <b><i>B</i></b><i> Quantification of B2M-HLA-G5 and B2M-HLA-G1s-Fc proteins</i>. Western blot analysis of recombinant B2M-HLA-G5 and B2M-HLA-G1s-Fc proteins immunoprecipitated from cell culture supernatants. Purified HLA-G5 recombinant protein was used as quantification standard. <b><i>C</i></b><i> Conformation of B2M-HLA-G5 and B2M-HLA-G1s-Fc proteins</i>. Recombinant HLA-G5 protein was used as a standard in HLA-G-specific ELISA. Curves represent the concentration of the proteins properly folded into the supernatant according to the dilution.</p

Primers used for fusion protein generation.

<p>Primers used for fusion protein generation.</p

Tolerogenic function of HLA-G fusion proteins and peptide.

<p><b>A.</b> C57BL/6 mice strongly recognize the MHC class II-disparate mutant bm12 mouse that carries the I-Abm12 alloantigen. The capability of HLA-G-coated beads to delay rejection was evaluated. Control treatment (dotted lines): beads coated with mAb but without HLA-G proteins. Results are expressed as Median of graft survival time. Kaplan-Meier curves representing graft survival are shown for each HLA-G protein/peptide for controls (plain lines). Associated values are indicated as a table underneath the curves. <b>B.</b> The same experiments were performed using ILT4-transgernic mice as skin graft recipients, and for Alpha1-Fc and B2M-HLA-G5. <b>Tables</b>: Median survival of transplant, number of animals, and significance are indicated below the corresponding survival graphs.</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

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

Gating strategy and overview of T_reg (CD4⁺/CD25⁺/FoxP3⁺) isolated from gMLN with and without HLA-G treatment after allogenic intestinal transplantation.

<p>(A) Gating strategy and representative result for Treg analysis. (B) Overview of classical Treg (CD4⁺/CD25⁺/FoxP3⁺ Treg), isolated from gMLN. A tendency to higher abundance in HLA-G treated animals, without reaching statistical significance (Mann-Whitney U test p>0.05), was observed.</p