Summary of the changes in cytokine expression.

<p>Cytokine release was measured 24 hrs or 3 days after incubation of normal PBMCs with isu-peptide homopolymers alone or in the presence of a mitogen, respectively. The full name of the abbreviated molecules is given in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s008" target="_blank">Table S4</a>.</p

Localisation and activity of the immunosuppressive (isu) domain of gp41 of HIV-1.

<p>(a) Functional domains in gp41 of HIV-1 (accession-nr. NCBI K03455): FP, fusion peptide; FPPR, fusion peptide proximal region; NHR, N-terminal helical region; ISU, isu domain; S-S, cystein-cystein loop; CHR, C-terminal helical region; MPER, membrane proximal external region; MSD, membrane spanning domain, 3S, domain binding to the gC1qR and inducing NKp44L expression. In the amino acid sequence of the isu domain stars (*) indicate NH₂-groups, points (.) mark COOH groups relevant for polymerisation. (b) Localisation of the isu domain after interaction of the NHR with the CHR generating a six helix bundle (only one molecule of the trimer is shown). (c) Influence of the isu-peptide on the proliferation of PHA stimulated PBMCs from a healthy donor. Cell proliferation was measured by ³H-thymidine incorporation. ³H-thymidine was added on day zero, one, two or three and cells were then harvested one the next day and the counts per minute were determined, gray - medium control, dark gray – isu peptide-BSA conjugates, added at day 0. (d) Dose dependent induction of IL-6 and Il-10 release by the isu peptide homopolymer (triangle) as measured in ELISAs. In contrast, the amidated control peptide (square) is inactive. (e) Comparative ELISA analysis of IL-10 release from PBMCs of seven donors all treated with the same amount and batch of the isu-peptide homopolymer, the IL-10 release of their PBMCs incubated with medium alone was zero. The p values were calculated using the Student's t-test, n = 3. The p value for the high responder donor 1 was p = 0.001, that of the low responder donor 7 p = 0.03.</p

Influence of the isu peptide on cytokine release and gene expression.

<p>(a) PBMCs were incubated for 24 hrs with or without the isu peptide homopolymer. The cytokine array VI (see Material and Methods) was used. The up-regulated cytokines are circled. (b) PBMCs were treated with Con A and incubated with and without the isu peptide homopolymer for three days and the cytokine array I was used. The down-regulated cytokines are circled. Similar results were obtained with PBMCs from ten other donors. (c) Cytokine array measuring simultaneous release of ten different cytokines after incubation of PBMCs from one donor with and without the isu peptide homopolymer at different time points (6 to 24 hrs), confirming and extenting the results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone-0055199-g002" target="_blank">Figure 2a</a>. Control PBMCs were incubated with medium. Compare the increase in IL-10 expression with that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone-0055199-g003" target="_blank">Figure 3a</a>. (d) Genes with the highest up-regulation (upper part) and down-regulation (lower part) of expression in PBMC of one donor in response to the isu peptide treatment. Using specific real-time PCRs for the up- and down-regulated genes, the changes were confirmed in PBMCs of other donors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone-0055199-g003" target="_blank">Figure 3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone-0055199-g004" target="_blank">4</a>, Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s002" target="_blank">Figure S2</a>). Fold changes (Fc) indicates gene expression compared to control cells incubated in medium. The full names of the genes are given in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s008" target="_blank">Table S4</a>.</p

Kinetics of the modulation of cytokine release and gene expression induced by the isu peptide.

<p>a, Kinetics of the IL-10 and IL-6 release and expression of IL-10 and IL-6 mRNA. PBMCs were incubated with (gray) or without (light gray) isu peptide homopolymers and supernatants as well as mRNA were collected at different time points between 0–24 hours. The p values were calculated using the Student’s t-test, n = 3. When comparing the IL-10 release induced by the isu peptide homopolymer and that by medium alone, the p value at the peak release (15 hrs) is p = 2.16E-05, the p value for IL-10 RNA at 24 hrs is p = 0.09. All other values were accordingly. b, c, Kinetics of the expression of MMP-1, TREM-1, FCN1, CXCL9 and SEPP-1 in PBMCs incubated with (dotted line) or without (straight line) isu-peptide homopolymers. The figures show a representative result obtained with PBMCs from more than five donors. The p values were calculated using the Student’s t-test, n = 3, the p-value of sTREM-1 release at 24 hrs is p = 0.02.</p

Donor-dependence of IL-10 and MMP-1 release.

<p>a, PBMCs from three donors were incubated with the same batch of the isu peptide polymer or medium and IL-10 release and MMP-1 mRNA expression were measured simultaneously. The donor-dependence of IL-10 release was shown using PBMCs from more than 50 donors some are shown in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s002" target="_blank">Figure S2</a>. b, Kinetic date of IL-10 release from PBMCs from a high responder donor A (column 1, dark grey) and low responder donor B (column 3, dark grey), both treated with the isu peptide homopopymer. Untreated medium control cells from both donors (Colum 2 and 4, light grey) did not release IL-10. The p values were calculated using the Student’s t-test, n = 3, p = 0.00015 in the case of a high responder and p = 0.03 in the case of a low responder.</p

Variation in CD8-TL epitopes in virus populations from non-controller M3/M3 MCM and an SIVmac239 stock.

<p>Amino acid variation detectable by pyrosequencing in 12 CD8-TL epitopes in viruses isolated from three M3/M3 MCM (CY0163, CY0164, and CY0166) is compared to variation previously reported by Sanger sequencing bulk PCR amplicons <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047818#pone.0047818-OConnor2" target="_blank">[18]</a>. Amino acid variants detectable within an SIVmac239 stock were characterized here and elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047818#pone.0047818-OConnor3" target="_blank">[34]</a>. Sequences that match the inoculum are represented with a “.”. Mixed populations are represented with an “X”. The number of high quality reads with each specific sequence is shown. The total number of reads for each epitope can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047818#pone.0047818.s004" target="_blank">Table S2</a>. The frequency of reads with a specific amino acid sequence is shown as “% Var.” The sequences labeled as “other” include variants that were individually present at less than 1% and variants that were also detected in the inoculum, which included the A2V mutation in Gag_221–229PR9. The relative shading of each box reflects the frequency of reads, as indicated in the legend.</p

Correlation coefficients between proportion variant at SNP sites in SIV from different hosts.

1<p>Only sites synonymous in all reading frames and showing one or more differences from the inoculum in at least one host (N = 451 sites) were included.</p>2<p>Only sites nonsynonymous in all reading frames and showing one or more differences from the inoculum in at least one host (N = 420 sites) were included.</p>*<p>Significance of correlation coefficient: P<0.05 (Bonferroni-correction for multiple testing).</p>**<p>Significance of correlation coefficient: P<0.01 (Bonferroni-correction for multiple testing).</p>***<p>Significance of correlation coefficient: P<0.001 (Bonferroni-correction for multiple testing).</p>†<p>The correlation for nonsynonymous sites was significantly different (P<0.001 in each case; Bonferroni-correction for multiple testing) from the corresponding correlation for synonymous sites.</p

Synonymous (π_S) and nonsynonymous (π_N) nucleotide diversity in epitope and non-epitope regions from all animals.

<p>Paired t-tests of the hypothesis that π_S = π_N; ^a P<0.05; ^b P<0.01.</p><p>Nucleotide diversity is analyzed in all 12 epitope and non-epitope regions of the SIV genomes isolated from all four animals (mean values by gene). T-tests of the hypothesis that π_S or π_N for CY0165 equals the corresponding value for the other animals: ^c P<0.01; ^d P<0.001.</p

Synonymous (π_S) and nonsynonymous (π_N) nucleotide diversity in variant epitopes from CY0163, CY0164, and CY0166.

<p>Paired t-tests of the hypothesis that π_S = π_N: ^aP<0.05; ^bP<0.01.</p><p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047818#pone.0047818.s005" target="_blank">Table S3</a> for the value of nucleotide diversity for individual epitopes and remainders.</p

Synonymous (<i>π_S</i>) and nonsynonymous (<i>π_N</i>) nucleotide diversity in non-overlapping coding regions of SIV populations.

*<p><i>π_S</i> or <i>π_N</i> significantly different from corresponding value for CY0165 (P<0.05; Z-test).</p>**<p><i>π_S</i> or <i>π_N</i> significantly different from corresponding value for CY0165 (P<0.01; Z-test).</p>***<p><i>π_S</i> or <i>π_N</i> significantly different from corresponding value for CY0165 (P<0.001; Z-test).</p