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

Primers used for fusion protein generation.

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

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