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

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

SIV infection induces ISG expression during both phases of infection in PLNs.

<p>(<b>A</b>) Venn diagram showing the number of ISGs expressed at D9, M3, or both time points. (<b>B</b>) Comparison of mean ISG expression FC at D9 and M3 p.i. for the 51 ISGs expressed at both time points. (<b>C</b>) Venn diagram of ISGs expressed at D9 p.i., splitting them into those only inducible by type I IFN (Type I), only inducible by type II IFN (Type II), and those inducible by both IFN types (left) and comparison of their mean induction levels (right). (<b>D</b>) Venn diagram of ISGs expressed at M3 p.i., splitting them into those only inducible by type I IFN, those only inducible by type II IFN, and those inducible by both IFN types (left) and comparison of their induction levels (right). (<b>E</b>) Venn diagram of ISGs expressed at D9 p.i., splitting them into those displaying at least one ISRE, one GAS, or both sequence motifs in their promoters (left) and comparison of their induction levels (right). (<b>F</b>) Venn diagram of ISGs expressed at M3 p.i., splitting them into those displaying at least one ISRE, one GAS, or both in their promoters (left) and comparison of their induction levels (right). (<b>G</b>) Functional enrichment of type I-, type II-, and type I&II ISG subsets. The p value was calculated by the Fisher test with the Benjamini-Hochberg correction for multiple comparisons. The -log(p-value) is given for the most significant processes. In C, D, E, and F, the p value is given for Welch's unequal variances <i>t</i>-test, considering p < 0.05 to be significant.</p

Infection of six cynomolgus macaques with SIVmac251 and establishment of chronic infection.

<p>(<b>A</b>) vRNA load in plasma. (<b>B</b>) Evolution of total CD4 T cell numbers in the blood over time (percentage of baseline). (<b>C</b>) vRNA in RB. (<b>D</b>) vRNA in PLNs. ULD = under the limit of detection. P values are given for non-parametric Wilcoxon rank sum test and was considered significant if p < 0.05.</p

Confirmation of differential expression of IFNs in tissues by RT-qPCR and at the protein level in plasma.

<p>(<b>A</b>) IFN-α mRNA expression in PLNs by RT-qPCR. (<b>B</b>) IFN-β mRNA expression in PLNs by RTqPCR. (<b>C</b>) IFN-α mRNA expression in RB by RT-qPCR. (<b>D</b>) IFN-γmRNA expression in PLNs by RT-qPCR. (<b>E</b>) MXA mRNA expression in PLNs by RT-qPCR. (<b>F</b>) IRF9 mRNA expression in PLNs by RT-qPCR. (<b>G</b>) Type-I IFN antiviral activity measured in plasma (MDBK/VSV biological assay) and (<b>H</b>) plasma IFN-γ concentration (measured by Luminex). ULD = Under the Limit of Detection. The Lower Limit of Quantification (LLOQ) is represented as a red dotted line in figure 3H. P values are given for non-parametric Wilcoxon rank sum test and was considered significant if p < 0.05.</p

Evolution of the ISG-associated functional signature during infection.

<p>Functional enrichment of ISGs differentially expressed at either (<b>A</b>) D9 p.i., (<b>B</b>) M3 p.i., or (<b>C</b>) both time points (D9&M3), was performed using IPA. The right-tailed Fisher Exact Test with the Benjamini-Hochberg correction for multiple tests was used to identify processes showing statistically significant over-representation of focus ISGs. Over-represented functional or pathway processes have more focus genes than expected by chance and the–log(p-values) of the most significant functions are plotted in the radar plot.</p

Heat-map signatures of differentially expressed ISGs.

<p>The differentially expressed genes of the ISG cluster with the most significant functions are represented as heat-maps. The color intensity shown at each time point is related to the FC in RNA expression: grey for not differentially expressed (not DEG), yellow for FC values between 1 and 2, orange for FC values between 2 and 5, and red for FC values above 5.</p

Transcriptome profiling reveals inter-tissue differences and the evolution of differential gene expression post-infection.

<p>Two critical time points, day 9 (D9) and month 3 (M3) p.i., were compared to baseline (before viral infection) (<b>A</b>) Number of genes with significant differential expression relative to baseline in RBs, PLNs, and PBMCs. A two-sample t-tests (p < 0.05) was used to identify differentially expressed genes (<b>B</b>) Multidimensional scaling (MDS) representation showing the segregation of biological samples based on the altered expression of the 1,638 genes with FC > 2. (<b>C</b>) Venn diagram showing the distribution of differentially expressed genes over time in RBs, PLNs, and PBMCs. (<b>D</b>) Evolution of RNA expression of interferons and interferon receptors in RBs, PLNs, and PBMCs at D9 and M3 p.i. Heatmap representing the FC from baseline for the six SIVmac251 infected macaques.</p