Exposure to HIV-1 replication and PBMC HIV-1 DNA levels one year following cART.

<p>The estimated 12 month AUC was significantly positively associated with HIV-1 DNA levels at one year (r = 0.51, p = 0.004).</p

Study Cohort: Pre-therapy Viral Load, CD4%, and Therapeutic Regimens^a.

<p>Study Cohort: Pre-therapy Viral Load, CD4%, and Therapeutic Regimens<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154391#t001fn001" target="_blank">^a</a>.</p

Differential PD-1 expression on A2-BMLF-1 and A2-BRLF-1 CD8+ T cells at AIM and convalescence.

<p>PD-1 expression on A2-BMLF-1 and A2-BRLF-1 CD8+ T cells during AIM (upper panels) and convalescence (lower panels). Representative dot plots demonstrate differences in PD-1 percent positivity, and histograms showing differences in distribution and PD-1 MFI of these EBV-specific CD8+ T cells. Histograms overlay the two subsets on a background of the total CD8+ T cell population.</p

PD-1 expression on CD8+ T cells during AIM: correlations with viral load and contraction at convalescence.

<p><b>A</b>. Left panel: Viral load (log transformed) and the corresponding PD-1 MFI of CD8+ T cells are plotted (Spearman r = 0.698; p = 0.008). Right panel: Viral load (log transformed) and the corresponding PD-1 MFI of A2-BMLF-1 (solid squares, dotted line) and A2-BRLF-1 cells (open circles, solid line) are plotted (Spearman r = 0.170; p = 0.578 and r = 0.560; p = 0.046, respectively). <b>B</b>. Left panel: PD-1 MFI of CD8+ T cells during AIM and the corresponding fold-change in CD8+ T cells (log-transformed) are plotted (Spearman r = 0.955; p<0.0001). The fold-change in CD8+ T cells is the ratio of the cell #/ml during AIM over the cell #/ml during convalescence. Right panel: PD-1 MFI of A2-BMLF-1 (solid squares, dotted line) and A2-BRLF-1 cells (open circles, solid line) during AIM and the corresponding fold-change in each of these subsets (log-transformed) are plotted (Spearman r = 0.646; p = 0.037, and r = 0.764; p = 0.009, respectively). Lines of best fit are shown for each combination of variables.</p

PD-1 expression on EBV-specific CD8+ T cells at AIM and convalescence.

<p><b>A</b>. The percentages of EBV-specific CD8+ T cells that expressed PD-1 are shown. Two lytic (A2-BMLF-1 and A2-BRLF-1) and two latent (A2-EBNA-3c and B7-EBNA-3a CD8+ T cells) antigen specific CD8+ T cell subsets are shown. Lines define paired results for each individual. <b>B</b>. The median fluorescence index (MFI) of PD-1 expression for these EBV-specific CD8+ T cells is shown. All assays were performed with the PD-1 antibody conjugated to PE. The MFI was normalized as described in methods. Lines define paired results for each individual. All paired comparisons indicated by brackets differ significantly by the Wilcoxon signed rank test; * p<0.05, ** p<0.01, *** p<0.001.</p

Estimation of PBMC HIV-1 DNA Decline.

<p><b>a) Estimation of PBMC HIV-1 DNA Decline in All Children over the First Year of cART</b>. ET (closed circle and solid line) and LT children (open circle and dashed line) experienced a significant decline in PBMC HIV-1 DNA after one year of cART (-1.04±0.11 log₁₀ DNA copies per million PBMC, p<0.001 in ET and -0.74 ±0.13 log₁₀ DNA copies per million PBMC, p<0.001 in LT). <b>b) Estimation of PBMC HIV-1 DNA Decline in Children with Controlled HIV-1 Replication</b>. ET (closed circle and solid line) and LT children (open circle and dashed line) experienced a marked decline in PBMC HIV-1 DNA levels in the first year of cART (-1.03±0.12 log₁₀, p<0.001 in ET and -0.82±0.16 log₁₀, p<0.001 in LT), but the rates of decline did not differ significantly between ET and LT (p = 0.3). Similarly, in years 1 through 4 of cART, PBMC HIV-1 DNA levels continued to decline significantly in both the ET (p<0.001) and LT groups (p<0.001), but the decline slopes were not statistically significantly different between the groups.</p

High level PD-1 expression on CD8+ T cells during AIM and convalescence.

<p><b>A</b>. Representative CD8+ T cell plots of a sample obtained during AIM demonstrating the gating strategy to define PD-1 high, dim and negative populations. <b>B</b>. Percentages of PD-1 high, dim, or negative CD8+ T cells in AIM and convalescence. <b>C</b>. Percentages of PD-1 high, dim, or negative A2-BMLF-1 and A2-BRLF-1 CD8+ T cells in AIM and convalescence. Lines define paired results for each individual. Brackets indicate that differences between selected comparisons are significant (Wilcoxon signed rank test; *p<0.05, ** p<0.01, *** p<0.001).</p

Proliferation and PD-1 expression on CD8+ T cells responding to peptide stimulation.

<p><b>A</b>. Three representative examples of proliferation upon stimulation with HLA-A*0201-restricted BMLF-1 and BRLF-1 peptides. Upper and middle rows: PBMC from an individual at presentation with AIM and in convalescence; lower row: PBMC from an individual with chronic infection. Left panels (baseline prior to peptide stimulation) show PD-1 expression of the indicated tetramer positive cells (A2 BMLF-1 black line; A2 BRLF-1 grey line) superimposed on that of CD8+ T cells (grey solid). Middle panels show CD8+ T cells that proliferate (CFSE dilution) in response to the indicated peptides (A2 BMLF-1 black line; A2 BRLF-1 grey line) superimposed on unstimulated controls (grey solid). Right panels show the upregulation of PD-1 on CD8+ T cells responding to the indicated peptides superimposed on unstimulated CD8+ T cells. Marked regions on histograms depicting PD-1 expression indicate high levels of expression with corresponding percentages of A2 BMLF-1 and A2-BRLF-1 CD8+ T cells. Numbers in text boxes are the percentages of CD8+ T cells that are positive for each tetramer at baseline (left panels), or responding to peptide post-stimulation (CFSE low; middle panels). <b>B</b>. Upregulation of PD-1 on cells responding to stimulation with A2-BMLF-1 and A2-BRLF-1 peptides. Results shown are a mixture of chronic and convalescent samples in three separate assays. To demonstrate the extent of PD-1 upregulation observed, PD-1 MFI is expressed as a ratio of the MFI of cells that responded to peptide, relative to the MFI in control assays without peptide. Median PD-1 MFI ratio values for A2-BMLF-1 and A2-BRLF-1 were 8.1 and 2.8, respectively (Wilcoxon signed rank test; p = 0.06). Solid and open symbols demonstrate paired values for A2-BMLF-1 and A2-BRLF-1 for each sample.</p

EBV-specific CD8+ T cells and viral load during AIM and convalescence.

<p><b>A</b>. Absolute values (cells per ml) of CD8+ T cells, and three different EBV-specific subsets (lytic and latent antigen specific) measured in samples from individuals during AIM (within the first two weeks after presentation with symptoms) and convalescence (CONV; 6–12 months after presentation with symptoms). A2-BMLF-1 CD8+ T cells: specific for the HLA-A*0201 restricted epitope of BMLF-1, GLCTLVAML; A2-BRLF-1 CD8+ T cells: specific for the HLA-A*0201 restricted epitope of BRLF-1, YVLDHLIVV; A2-EBNA-3c CD8+ T cells: specific for the HLA-A*0201 restricted epitope of EBNA-3c, LLDFVRFMGV. When no EBV-specific CD8+ T cells were detected with these reagents, a value of 100 cells per milliliter was assigned (less than half the lowest detected value). <b>B</b>. Viral load (EBV copies per million B cells) during AIM and convalescence. Lines define paired results for each individual. All paired comparisons indicated by brackets differ significantly by the Wilcoxon signed rank test; * p<0.05, ** p<0.01, *** p<0.001.</p

Analysis of CD127 and PD-1 expression on CD8+ T cells.

<p><b>A</b>. Percentages of A2-BMLF-1, A2-BRLF-1, and total CD8+ T cells that are CD127+ in AIM and convalescence (mean and SEM). <b>B</b>. Percentages of A2-BMLF-1, A2-BRLF-1, and total CD8+ T cells subsets that are PD-1 high or PD-1 negative that are also CD127+ (in AIM and convalescence). Brackets indicate paired comparisons that differ significantly, Wilcoxon signed rank test, *p<0.05.</p