Signals from inhibitory peptides dominate to suppress NK cell activation.

<p>721.221-ICP47-A1*002 cells were pulsed with mixtures of Gag GY9 and GY9 L8W (A, B & E) or Env RY8 and RY8 V7W (C, D & F) and tested for susceptibility to killing by Mamu-KIR3DL05⁺ NK cells in CAM cytotoxicity assays. Representative data (A & C) and mean percent specific lysis (B & D) are shown for three independent experiments using NK cells from different animals. In panels A-D, the percentages of inhibitory versus disinhibitory peptides varied, keeping the total peptide concentration constant at 0.5 μM for GY9/GY9 L8W and 5 μM for RY8/RY8 V7W. Error bars indicate +1 SD and asterisks indicate significant differences in the lysis of target cells pulsed with GY9 or RY8 compared to target cells pulsed with the indicated peptide mixtures (*p<0.05, ****p<0.001 by two-way ANOVA with Dunnett’s test). In panels E-F, target cells were pulsed with increasing concentrations of Gag GY9 (E) or Env RY8 (F) in combination with a fixed concentration of their respective disinhibitory variants (0.375 μM GY9 L8W or 3.75 μM RY8 V7W). The dashed line indicates 50% inhibition where GY9 or RY8 alone defines 100% inhibition and GY9 L8W or RY8 V7W alone defines 0% inhibition. Mamu-A1*002 stabilization on the surface of 721.221-ICP47-A1*002 cells was verified by flow cytometry using the MHC class I-specific monoclonal antibody W6/32 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005145#ppat.1005145.s005" target="_blank">S5 Fig</a>).</p

Peptide-dependent inhibition of Mamu-KIR3DL05⁺ NK cells.

<p>(A) Mamu-KIR3DL05⁺ and -KIR3DL05^- NK cells from the same animal were incubated with CAM-labeled 721.221-ICP47-A1*002 target cells pulsed with the indicated SIVmac239 peptides. Percent specific lysis was calculated from the amount of CAM released into the culture supernatant after 4 hours at the indicated effector to target (E:T) ratios. The results shown are representative of data obtained with NK cells from three different animals. Controls include target cells incubated without peptide (No peptide) or with a GY9 variant with substitutions at anchor positions (S2A & Y9G) that abrogate binding to Mamu-A1*002 (Control). (B) Bar graphs summarize the mean percent specific lysis for independent experiments with Mamu-KIR3DL05⁺ NK cells from three different animals. Error bars indicate +1 standard deviation (SD) and asterisks indicate significant differences in the lysis of target cells pulsed with each SIV peptide compared to control cells without peptide (****p<0.001 by two-way ANOVA with Dunnett’s test). (C) Stabilization of Mamu-A1*002 on the surface of 721.221-ICP47-A1*002 cells was determined by staining with the pan-MHC class I monoclonal antibody W6/32 and the relative geometric mean fluorescence intensity (gMFI) normalized to cells incubated without peptide is shown. Data is representative of three independent experiments.</p

Substitutions at position 8 of Gag GY9 or Nef YY9 alter binding of Mamu-A1*002 to Mamu-KIR3DL05.

<p>(A) Jurkat cells expressing HA-tagged Mamu-KIR3DL05 were stained with an anti-HA antibody and Mamu-A1*002 tetramers folded with Gag GY9, Nef YY9, or variant peptides with alanine or tryptophan substitutions at position 8 as indicated. (B) Tetramer integrity was confirmed by staining LILRB1-expressing Ba/F3 cells with each tetramer.</p

SIVmac239 peptides bound by Mamu-A1*002.

<p>¹Position indicates the location of the peptide N-terminal residue within the respective SIVmac239 protein.</p><p>²Relative binding affinities of SIV peptides for Mamu-A1*002 reported by Loffredo et al. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005145#ppat.1005145.ref031" target="_blank">31</a>].</p><p>SIVmac239 peptides bound by Mamu-A1*002.</p

Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 inhibit Mamu-KIR3DL05⁺ NK cells.

<p>Mamu-KIR3DL05⁺ NK cells were incubated at a 5:1 E:T ratio with CAM-labeled 721.221-ICP47-A1*002 target cells pulsed with the indicated SIVmac239 peptides. Percent specific lysis was calculated from the amount of CAM released into the culture supernatant after a 4-hour incubation. Bars represent the mean percent specific lysis for experiments using NK cells from three different animals. Peptides are ordered from highest to lowest affinity for Mamu-A1*002 according to Loffredo et al. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005145#ppat.1005145.ref031" target="_blank">31</a>]. Previously defined CD8⁺ T cell epitopes are indicated by purple bars and controls include target cells incubated without peptide (blue) or with a GY9 variant with substitutions at anchor positions that abrogate binding to Mamu-A1*002 (red). Error bars indicate +1 SD and asterisks indicate significant differences in the lysis of target cells pulsed with each SIV peptide compared to control cells without peptide (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001 by one-way ANOVA with Dunnett’s test). Stabilization of Mamu-A1*002 on the surface of 721.221-ICP47-A1*002 cells was verified by flow cytometry using the MHC class I-specific monoclonal antibody W6/32 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005145#ppat.1005145.s001" target="_blank">S1 Fig</a>).</p

Modeling of amino acid changes in SIV peptides bound by Mamu-A1*002.

<p>Three-dimensional crystal structures of Mamu-A1*002 in complex with Gag GY9 (A) and Nef YY9 (B) are shown with alanine or tryptophan substitutions modeled at the positions indicated in red [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005145#ppat.1005145.ref035" target="_blank">35</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005145#ppat.1005145.ref036" target="_blank">36</a>]. The α1- and α2-domains of the MHC class I heavy chain (blue), β2-microglobulin (grey), and SIV peptide (yellow) are depicted.</p

Acute phase CTL responses against ARF-10 in two SIV-infected Rhesus macaques.

<p>A) and B) Magnitude of CTL responses against 15-mers in ARF-10 in Rhesus macaques rh2261, A, and r04028, B, as measured by IFN–γ ELISPOT using cryopreserved PBMC harvested 3 weeks after initial SIV infection. The SIV infection history of the animals is described in the text. Bars are color coded to match with remaining panels in the figure. C) and D) Peptide dilutions to fine map the minimal optimal epitopes targeted in ARF-10 by rh2261, C, and r04028, D. For rh2261, we used PBMC harvested in the acute phase of infection. For animal r04028, due to PBMC sample limitations, we expanded antigen-specific CTL by exposing PBMC to autologous irradiated BLCL pulsed with the responsive 15-mer for several weeks and used these cells in an ELISPOT. For panels A–C, we used 100,000 PBMC per well, in duplicate, in ELISPOT plates. For the epitope mapping with r04028, we used 20,000 antigen-specific cells per well combined with 10,000 autologous BLCL per well as antigen presenting cells. Peptides tested are shown at right of each mapping panel and the peptides we determined represented the minimal epitopes are shown in color to match panels A and B. E) The mapped epitopes within ARF-10 are boxed using the color scheme described above.</p

The genomic locations of ARFs -1 and -10 as well as the primers used to amplify this region for pyrosequencing, within the SIVmac239 genome.

<p>The SIVmac239 genome is 10,535 nt in length. The ORFs encoding the classical viral proteins are shown in blue and the ORFs encoding the ARFs studied in this report are shown in orange. Note that ARF-10 is depicted as separate from Rev exon 1 to emphasize its independent translation. However, ARF-10 is a composite of the first exon of Rev and the first 50 amino acids translated from the Rev intron. The relative locations of the forward and reverse primers used to amplify viral RNA for pyrosequencing are shown as labeled black arrows. This image was created using the software Geneious version 5.6.4 created by Biomatters, available from <a href="http://www.geneious.com" target="_blank">www.geneious.com</a>.</p

Acute phase CTL responses against ARF-1 in two SIV-infected Rhesus macaques.

<p>A) and B) Similar to panels A and B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061383#pone-0061383-g002" target="_blank">figure 2</a>. Here, we depict the week 3 responses to 15-mers within ARF-1 in two animals, r97111, A, and r97035. The infection history of these animals is described in the text. C) and D) The minimal epitope was mapped to the RP9 peptide in both animals using serial 10-fold dilutions of peptide in IFN–γ ELISPOT assays. The peptides used are shown at right and the RP9 peptide, which was determined to be the minimal epitope is shown in orange. E) The location of the RP9 epitope within ARF-1.</p

MID-tagged oligonucleotides used to generate ARF1/ARF10/Env specific amplicons.

<p>MID tags are in bold italics. Sequence-specific primer is 3′ to the MID tag while adaptor sequence is 5′ to the tag in each oligonucleotide. The number of MID-tagged primers is less than the number of samples as some primers were re-used in separate runs.</p