Modulation of CADM1 expression by HTLV-1 proteins.

<p>CEM cells were transfected with plasmids encoding Tax, HBZ-TTG, HBZ or GFP alone. Cells were cultured in the presence of medium only or in the presence of PMA/CAI. After 16h, cells were harvested and stained with a viability stain and anti-CADM1. The frequency of CADM1 expressing transfected cells was determined by gating on live GFP⁺ cells. (A) Data from three independent experiments. (B) Representative flow plots. Numbers indicate the percentage of live cells in each quadrant. Statistical analysis: Repeated measures Anova with Tukey post-test. *,p<0.05 versus GFP+PMA/CAI; ##, p<0.01 versus HBZ+PMA/CAI.</p

Concordance of Tax expression with putative markers of infected cells.

<p>PBMCs from the same cohort as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005560#ppat.1005560.g001" target="_blank">Fig 1</a> were cultured for 18 hours at 1 x 10⁶ cells/ml in the presence of 20 ng/ml CMA. Cells were stained with antibodies specific for CADM1, CD25, ICAM-1, CCR4 and CD4 followed by intracellular staining with an antibody specific for Tax, then analysed by flow cytometry. <b>(A)</b> Representative dot plots showing Tax and CADM1 expression in total CD4⁺ T cells in uncultured (0 hr) and cultured (18 hr) PBMCs (donor PVL 20.18%). Numbers indicate percentages of total live CD4⁺ cells. <b>(B)</b> Representative dot plots to compare the expression of CD25, CCR4 and ICAM-1 on Tax⁺ or Tax^<b>−</b>CD4⁺ T cells. Numbers indicate percentages of the total Tax⁺CD4⁺ or Tax^<b>–</b>CD4⁺ population respectively.</p

Proportion of proviral copies carried by CADM1⁺ lymphocytes.

<p><b><i>(A)</i></b><i>CADM1</i>^<i>+</i><i>CD4</i>^<i>+</i><i>T cells carry the bulk of the proviral load</i>. Purified CD4⁺ T cells from 12 individuals (AC, n = 6; HAM, n = 6,) were stained for surface markers and flow sorted as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005560#ppat.1005560.s003" target="_blank">S2 Fig</a>. Genomic DNA was extracted from sorted populations and the PVL of each population was quantified. The frequency of each population in PBMC was used to calculate the relative contribution of each population to the proviral load. Bar denotes mean ± SEM. Numbers denote the median number of HTLV-1 copies detected per cell. <b><i>(B)</i></b> <i>CADM1 identifies ~65% of infected T cells</i>. Purified CD4⁺ T cells from 6 individuals were cultured for 16 hours, stained with antibodies specific for cell surface markers and Tax, then analysed by flow cytometry. Combining PVL data and the observed frequency of Tax⁺ cells, the median distribution of load among Tax^+/–CADM1^+/–CD4⁺ T cells was calculated. Pie chart represents the total proviral burden in CD4⁺ T cells. The white segment indicates infected cells that did not express Tax or CADM1.</p

The frequency of CADM1⁺CD4⁺ T cells is positively correlated with proviral load.

<p>Uncultured PBMC from 32 individuals (uninfected (UI), n = 8; AC, n = 13; HAM, n = 11), were stained with antibodies specific for CADM1, CD25, ICAM-1, CCR4, CD4,CD8 and CD3 and analysed by flow cytometry. <b>(A)</b> Frequency of CADM1⁺CD4⁺ and CADM1⁺CD8⁺ T cells in uninfected individuals, ACs and patients with HAM. Bars denote mean ± SEM. Statistical analysis: Kruskal-Wallis test with Dunn post-test, 95% confidence interval (CI). Significant differences are highlighted, *** denotes p<0.0001. <b>(B)</b> Frequency of CADM1⁺CD4⁺ T cells versus PVL. Grey dashed line indicates median percentage of CADM1⁺CD4⁺ T cells/PBMC in 8 uninfected controls. Spearman correlation was performed for AC (p = 0.01, r_s = 0.66) and HAM (p = 0.003, r_s = 0.83). Continuous line represents linear regression of AC and dashed line that of HAM.</p

CADM1⁺CD4⁺ T cells are highly susceptible to CTL-mediated lysis.

<p>Purified CD4⁺ T cells from 6 HLA-A*02⁺ infected individuals (AC, n = 3; HAM, n = 3) were placed in culture to allow viral protein expression. After 6h incubation, cells were loaded with 0–2μM of an HLA-A*02-restricted CMV peptide, pp65 (NLVPMVATV) and co-cultured with a pp65-specific CTL clone at an E:T ratio of 1:1 for a further 12 hours. Cells were then stained for surface and intracellular antigens and analysed by flow cytometry. The median specific killing of Tax⁺ and CADM1⁺ cells was calculated after subtracting the background in the 0 μM peptide sample. Bars denote mean and SEM. Wilcoxon signed rank test was performed to compare populations. * denotes p = 0.03, Tax⁺CADM1⁺ vs. Tax⁺CADM1^–; # denotes p = 0.03, Tax^<b>–</b>CADM1⁺ vs.Tax^<b>–</b>CADM1^–.</p

CADM1⁺ cells are killed more efficiently than CADM1^– cells by autologous CTLs.

<p>CD8⁺ T cells were depleted from PBMCs from 23 HTLV-1 infected donors (AC, n = 11; HAM, n = 12) and added back at a range of CD4⁺:CD8⁺ ratios. Cells were co-cultured for 18 hours, after which the frequency of Tax and/or CADM1 expressing cells was assayed by flow cytometry. <b>(A)</b> Representative dot plots showing Tax and CADM1 expression by live CD4⁺ T cells in a single individual. Numbers indicated in dot plots refer to percentage of live CD4⁺ T cells in each respective sample. <b>(B)</b> The absolute frequency of each subset in CD8⁺ depleted fraction was used as a baseline to calculate the percentage of each subset killed at each CD4⁺:CD8⁺ ratio. <b>(C)</b> The rate of lysis of each subset / %CD8⁺ T cells/day was calculated as described in materials and methods. Bars represent mean ± SEM. Statistical analysis: Friedman test with Dunn post-test, 95% CI. *** denotes p<0.0001 <b>(D)</b> The rate of lysis of Tax⁺CADM1⁺ cells significantly correlated with that of Tax^<b>–</b>CADM1⁺ cells. Statistical analysis: Spearman correlation. Solid linear regression line, ACs; Dashed linear regression line, HAM.</p

The frequency of CRTAM⁺CD8⁺ T cells is positively correlated with the rate of lysis of Tax⁺CADM1⁺ cells.

<p>CD8⁺ T cells cultured at the physiological CD4⁺:CD8⁺ ratio in the autologous CTL lysis assay (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005560#ppat.1005560.g004" target="_blank">Fig 4</a>) were stained with antibodies specific for CADM1 and CRTAM (AC, n = 11, empty circles; HAM, n = 12, closed circles). (A) Representative dot plot of CADM1 and CRTAM on live CD8⁺ T cells. Numbers indicate percentage of cells within gate. (B) Total frequencies of CADM1⁺ and CRTAM⁺ cells within live CD8⁺ T cells. Bars represent mean and SEM. The frequency of CRTAM⁺CD8⁺ T cells was positively correlated with the rate of lysis of the Tax⁺CADM1⁺ T cells (C) and Tax^<b>–</b>CADM1⁺ T cells in HAM patients (D). Continuous line, regression line for ACs; dashed line, regression line for HAMs.</p

Tax⁺ cells are more frequent in smaller clones.

<p>Mean fraction of Tax⁺ cells in bins of increasing clonal abundance (total number of cells in each respective clone). The fraction of Tax⁺ cells was negatively correlated with clonal abundance (p<10⁻¹⁶, χ² test for trend).</p

Genomic environment at HTLV-1 proviral integration site associated with proviral expression after 18 h in culture.

<p>CD8-depleted PBMCs were placed in culture overnight and sorted by flow cytometry to isolate Tax⁺ and Tax⁻ cells, followed by integration site analysis of sorted cells. (A)–(C): proportion of observed integration sites compared to random expectation. (A) Frequency of integration in proximity to transcriptional units (RefSeq) in clones that were 100% Tax⁺ or 100% Tax⁻, according to the relative transcriptional orientation of the provirus and the host gene. The peak of integration at 1 kb mirrors that observed in vivo in unsorted cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003271#ppat-1003271-g001" target="_blank">Figure 1B</a>). However, the integration site in Tax⁻ clones was more likely than in Tax⁺ clones to possess a nearby upstream TSS in the same orientation, and less likely to lie nearby a downstream TSS in the same orientation (or any relative position in the opposite orientation). (B) The provirus in Tax⁻ clones (blue) was oriented in the same transcriptional sense as the host gene in which it was integrated more frequently than random. The orientation of Tax⁺ clones (pink) did not differ from random. (C) Frequency of integration in proximity to CpG islands in clones that were 100% Tax⁺ or 100% Tax⁻. The peak of integration at 1 kb mirrors that observed in vivo in unsorted cells and in vitro (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003271#ppat.1003271.s004" target="_blank">Figure S4</a>). (D) Mean fraction of Tax⁺ cells in clones with a TSS at a given distance (log scale) from the integration site, according to the relative transcriptional orientation of the provirus and the host TSS. The dotted line denotes the mean fraction of Tax⁺ cells across all clones. (E) Frequency distribution of clones according to the frequency of Tax⁺ cells in the respective clones. See supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003271#ppat.1003271.s007" target="_blank">Figure S7</a> for detailed frequency distribution separated according to clone abundance.</p

Influence of proximity to TFBS on Tax expression.

<p>(A) Mean fraction of Tax⁺ cells in clones with a TFBS (based on ChIP-seq experiments) at a given distance from the IS. Four representative plots are shown. (B) TFBS that were independently associated with Tax expression were identified by multivariate analysis, outcome measure . TFBS shown above the line were associated with Tax expression (OR>1); TFBS below the line were associated with Tax silencing (OR<1). Model 1 and Model 2 (carried out independently) test for TFBS within 1 kb and 100 bp of IS, respectively.</p