Intraspecies polymorphisms in <i>M. sylvanus</i> TRIM5α.

<p>Nonsynonymous SNPs are marked in <b>bold</b>.</p><p>nt  =  nucleotide residue number.</p><p>aa  =  amino acid residue number.</p

TRIMCyp-linked alleles form a monophyletic group.

<p>Phylogenetic tree of <i>TRIM5</i> genomic sequences from the exon 6–8 region. Tree was built using a neighbor-joining algorithm using the homologous human sequence as an outgroup (not shown). Bootstrap values from 1000 replicates are shown. Scale bar, substitutions per site.</p

Model for TRIMCyp evolution in Old World primates.

<p>Schematic dendrogram showing the history of TRIMCyp evolution inferred here, in the context of established phylogenetic relationships among Old World primate species studied. Filled star, evolution of TRIMCyp. Open star, fixation of TRIMCyp. Asian macaque phylogenetic groups are indicated at right <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014019#pone.0014019-Li1" target="_blank">[23]</a>. This graphical representation is not to scale and is not intended to reflect relative divergence. Relationships are adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014019#pone.0014019-Li1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014019#pone.0014019-Tosi1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014019#pone.0014019-Page1" target="_blank">[37]</a>.</p

Baboons lack TRIMCyp.

<p>(A) Sequence of the intron 6/exon 7 junction, showing the splice site SNP and the <i>NsiI</i> polymorphism, in a macaque that expresses TRIMCyp (<i>M. nemestrina</i>, GenBank EU371641.1); a macaque lacking TRIMCyp (<i>M. mulatta</i> sequenced genome, GenBank NC_007871.1); and baboons (<i>Papio</i>, GenBank HM468444-HM468446). Capital letters, exon; lowercase letters; intron. Box, splice acceptor site. DNA from both <i>P. cynocephalus anubis</i> and <i>P. hamadryas</i> was sequenced, and all sequences were identical in the region shown here. (B) PCR across the <i>TRIM5</i> 3′ UTR (Primers 3 and 6) in <i>P. cynocephalus anubis</i> (4 individuals, lanes 1–4) and <i>P. hamadryas</i> (lane 5). Three <i>M. fascicularis</i> of known genotypes were used as controls. Lane 6, <i>CypA</i> insertion heterozygote. Lane 7, homozygote with <i>CypA</i> insertion. Lane 8, homozygote lacking <i>CypA</i> insertion. (C) RT-PCR for TRIM5α (lanes 1, 3, and 5) and TRIMCyp (lanes 2, 4, and 6) in cDNA from 3 <i>P. cynocephalus anubis</i>.</p

Interspecies comparison of predicted TRIM5α amino acid sequence from <i>M. sylvanus</i>, <i>M. mulatta</i>, and <i>Papio cynocephalus anubis</i>.

<p><b>Bold</b>  =  not found in P. cynocephalus or M. mulatta.</p

<i>M. sylvanus</i> lack TRIMCyp.

<p>(A) <i>NsiI</i> restriction site assay on 36 <i>M. sylvanus</i>. (B) PCR across the <i>TRIM5</i> 3′ UTR in 36 <i>M. sylvanus</i>. Three <i>M. fascicularis</i> of known genotype were used as controls (last 3 lanes of both parts A and B). (C) RT-PCR for TRIM5 (left lanes) and TRIMCyp (right lanes) in cDNA from 36 <i>M. sylvanus</i>. Mfa, <i>M. fascicularis</i> heterozygote expressing both TRIM5 and TRIMCyp. –RT, <i>M. sylvanus</i> sample run without reverse transcriptase.</p

Vβ11 and 6B11 phenotypes of Vα24+ cells among PBMCs from pig-tailed macaques.

<p>One million PBMCs were incubated with anti-Vα24-PE together with either anti-Vβ11-FITC or 6B11-FITC followed by flow cytometric analysis, as described in Fig. 1.</p

α-GalCer-CD1d-Tet+CD3+ cells among PBMCs from pig-tailed macaques.

<p>Peripheral blood mononuclear cells (PBMCs) obtained from each pig-tailed macaque were incubated with anti-CD3-PerCP together with α-GalCer-loaded human CD1d-tetramer-PE (α-GalCer-CD1d-Tet-PE) in upper panel or unloaded human CD1d-tetramer-PE as a negative control in lower panelData were analyzed using Flowjo software (Tree Star). For all figures, the data represent one of three similar experiments.</p

CD4/CD8 phenotype of α-GalCer-CD1d-Tet+CD3+ <i>i</i>NKT cells derived from pig-tailed macaques.

<p>(A) One million PBMCs were first incubated with α-GalCer-CD1d-Tet-PE and anti-CD3-PerCP. Cells were also stained with anti-CD4-APC and anti-CD8-FITC then subjected to flow cytometric analysis, as described in Fig. 1. Data represent one of two similar experiments. (B) The percentage of α-GalCer-CD1d-Tet⁺CD3⁺ cells among PBMCs and the percentage of CD4⁺ or CD8⁺ cells among α-GalCer-CD1d-Tet⁺CD3⁺<i>i</i>NKT cells of each pig-tailed macaque are listed. (C) One million PBMCs were first incubated with anti-Vα24-PE then stained with CD8α-perCp anti-CD8β-APC and subjected to flow cytometric analysis, as described in Fig. 1.</p

Secretion of IFN-γ and TNF-α by pig-tailed macaque <i>i</i>NKT cells upon stimulation with α-GalCer.

<p> One million PBMC cells were stimulated with 0.1 µg/ml or 5 µg/ml of α-GalCer followed by the addition of 5 µg/ml Brefeldin A for the last 4 hours of incubation. Invariant natural killer T (<i>i</i>NKT) cells were then gated with α-GalCer-CD1d-Tet⁺ and CD3⁺ followed by flow cytometric analysis. (A) Flow cytometric figure shows the pattern of IFN-γ and TNF-α expression by α-GalCer-activated, IFN-γ-secreting <i>i</i>NKT cells gated from PBMCs from one representative pig-tailed macaque. (B) The graph shows the percentages of <i>i</i>NKT cells secreting the respective cytokines among total <i>i</i>NKT cells derived from the PBMCs of five pig-tailed macaques.</p