Integrated HIV DNA is preferentially cleared over 2-LTR circles after coculture recreating the EC phenotype in vitro in the absence of spinoculation.

<p>EC resting CD4+T cells were inoculated without spinoculation, cultured for 3 days and cocultured with autologous CD8+T cells or cultured alone for 16 hours. DNA was then isolated. The fold changeis calculated by dividing the copies of the specific HIV DNA intermediate per resting CD4+T cell (i.e. integrated or 2-LTRs) cultured alone by the copies from the same samples cultured with autologous CD8+T cells. For cocultured samples the level measured was multiplied by 10, which is the dilution factor of effectors added to targets. The line represents the median and p value was calculated using the Student's t-test.</p

Superinfected resting CD4+T cells express HIV proteins without producing detectable viral spread.

<p>(A) Resting CD4+T cells were isolated from frozen PBMC by negative bead depletion and spinoculated with HIV-1_NL4-3. Cells were cultured for 3 days without stimulation in the presence saquinavir (SQV). Resting conditions refer to culture in RPMI+ 10% FCS. After 3 days cells were collected, labeled with a viability dye and activation markers, then fixed and permeabilized and stained for intracellular HIV Gag. (B) Resting CD4+T cells were gated by excluding HLA-DR, CD25 and CD69 and analyzed for the presence of intracellular Gag with or without addition of Raltegravir at the time of infection, using a reverse-transcriptase inhibitor-treated control to set the Gag positive gates. At time 0 and day 3 resting CD4+T were determined to be 99% negative for activation markers. (C) The frequency of cells expressing HIV Gag after <i>in vitro</i> infection and 3 day culture was compared among normal donors (ND, n = 6) and elite controllers (EC, n = 6). (D) One representative experiment in an EC. Total HIV DNA was measured in superinfected resting or activated cells and compared between +SQV and -SQV fractions. (E) Summary data from 4 different EC donors as in C, showing the ratio of total HIV DNA in the -SQV divided by the +SQV fraction. (Lines represent the median and p values were generated using the Students t-test.</p

HIV Gag is expressed <i>in vivo</i> in resting CD4+T cells.

<p>The percentage of integrated HIV expressing HIV Gag was calculated by dividing the Gag positive cells per sorted cells (column 3) by the correction for live cells (column 4). We then divided this number by the integrated copies per PBMC (column 5) and multiplied by the RU5 per cell of the sorted Gag + resting CD4+T cells (and then multiplied by 100) to get the percent of integrated expressing Gag. This calculation assumes that all of the integrated HIV DNA is in the resting CD4+T cell compartment of the PBMC, which makes the percent expressed an underestimate.</p

GPR cells can be detected <i>in vivo</i>.

<p>(A) Representative sort plots and gating strategies from a treated non-controller showing selection of resting CD4+T cells by lineage (except CD4) and activation markers stained in PE (left plot) and the selection of Gag positive and negative cells (right plot). The numbers inside the boxes represent proportion of resting CD4+T cells. Numbers outside of the boxes represent the copies of HIV DNA per cell followed by the number of events collected for each. (B) Summary data from 3 sort experiments where HIV DNA was measured in Gag negative and Gag positive sorted populations from ART-suppressed non-controllers as in (A). A paired Student's t-test was used to determine statistical significance. Lines represent median values. (C) Sort data from one individual was gated more conservatively than the 3 previous sorts for Gag positive cells. The numbers inside the boxes represent percent of resting CD4+T cells. Numbers outside of the boxed represent percent of cells positive for HIV DNA (100% =  ∼1 copy of HIV DNA per cell) followed by the number of events collected for each.</p

SIV-specific CD8⁺ T cells from LTNP/EC mediate greater lysis of SIV-infected CD4⁺ T-cell targets compared with progressors.

<p>GrB target cell activity (<b>A</b>) and infected CD4 elimination (ICE) (<b>B</b>) are shown for LTNP/EC (n = 10, GrB target cell activity; n = 11, ICE) and progressors (n = 11). Horizontal bars represent the median values. <b>C.</b> Correlation between ICE and GrB target cell activity (n = 22) was determined by the Spearman rank method. Red, blue and cyan dots represent LTNP/EC, progressors and one SIV-uninfected animal, respectively.</p

Infected CD4 Elimination (ICE) inversely correlates with plasma SIV RNA levels in SIV-infected rhesus macaques.

<p>Red dots represent LTNP/EC (n = 11). Blue dots represent progressors (n = 11). Statistical analysis was performed by the Spearman rank method.</p

SIV-specific CD8⁺ T cell cytotoxicity measured by granzyme B delivery or Infected CD4 Elimination (ICE).

<p><b>A.</b> The top panels show granzyme B (GrB) target cell activity representative of a “high responder”. The bottom panels show GrB target cell activity representative of a “low responder”. Values indicate percentages of targets with increased fluorescence due to GrB substrate cleavage. Background GrB target cell activity measured in response to uninfected targets (left column) was subtracted from responses measured against infected targets (right column) to determine net GrB target cell activity (red values). <b>B.</b> ICE values calculated based on p27 expression (sum of the upper quadrants) as described in the Methods, are shown in red for the same “high responder” (78.8%, top row) and “low responder” (22.3%, bottom row) as shown in A. Quadrant values indicate percentages of gated targets. In all experiments, CD4⁺ T cell lines were used as targets. CD8⁺ T cells that had been stimulated with SIV-infected targets for 6 days were used as effectors. GrB target cell activity and ICE were calculated after 1 hour of incubation of effectors and plated at an E∶T ratio of 25∶1.</p

SIV-specific CD8⁺ T cells of LTNP/EC mediate greater per-cell killing of SIV-infected targets than those of progressors, which is not simply due to higher true E∶T ratios.

<p><b>A.</b> The true effector to target (E∶T) ratios, determined by measurements of IFN-γ-secreting CD8⁺ T-cell effectors and p27-expressing CD4⁺ T-cell targets, respectively, as described in the Methods and shown in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003195#ppat.1003195.s001" target="_blank">Figure S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003195#ppat.1003195.s002" target="_blank">Table S1</a>, were compared between LTNP/EC (n = 11) and progressors (n = 11). Horizontal bars represent the median values. <b>B, C.</b> GrB target cell activity (<b>B</b>) or ICE (<b>C</b>) responses plotted against the true E∶T ratios are shown for LTNP/EC (n = 10, GrB target cell activity; n = 11, ICE) and progressors (n = 11). GrB target cell activity is shown after subtraction of background. The response curves were analyzed by regressing ICE and GrB on log true E∶T ratios using analysis of covariance. The standard two-tailed t test from regression analysis was used to compare estimated GrB target cell activity and ICE of LTNP/EC with that of progressors at the 5.8 E∶T ratio, the median of the combined E∶T ranges of both groups.</p

Characteristics of macaques and prediction of SIV control.

<p>ICE, Infected CD4⁺ T cell Elimination (%). LTNP/EC, Long-Term Nonprogressor/Elite Controllers. SP, Slow Progressor. N/A, Not Applicable. PBMC from more than one time point were used in the animals marked with an asterisk.</p