Gating strategy.

<p>The gating strategy used for all the samples was to first define singlet and morphology by using forward versus side scatter, followed by the exclusion of dead cells (aqua dye negative events) and the exclusion of CD14, CD19, CD56, CD11c and CD123 positive cells (dump gate). Live cells were gated on CD3+ and CD45RO+ cells. Next, CCR9+ and/or β7+ cells were gated and defined as gut homing T-cells. Then CD4+ and CD8+ T cells were gated and finally the expression of CD161 on the gut homing CD4+ (top) and CD8+ (bottom) T-cells was gated. T cell activation was determined by the simultaneous expression of CD38 and HLADR on gut homing CD4+ and CD8+ T cells.</p

ART-naive and short-term ART experienced individuals have decreased frequency of CD161-expressing gut homing CD8+ T-cells.

<p>Frequencies (expressed as percentages) of peripheral blood (PB) memory gut homing (CD45RO+ CCR9+ B7+) CD4+ (A) and CD8+ (C) T-cells expressing CD161 were determined by flow cytometry on PBMCs of HIV-infected individuals (HIV+), antiretroviral (ART) naïve (n = 18), ART experienced individuals on short-term (n = 15) and long-term treatment (n = 32), HIV controllers (n = 6) and HIV seronegative individuals (SN, n = 5). Scatter plots were used to represent the data. Horizontal lines indicate median values. Each symbol represents one individual. The red symbols represent the females in each group. Groups were compared using Kruskal-Wallis test correcting for multiple comparisons using the Dunnett´s post-test. Only significant corrected p values are shown ***p<0.0005, **p<0.005, *p<0.05. Spearman's rank correlation between PB gut homing CD4+ CD161+ T-cells and plasma viral load (pVL) in the ART naive group (B). Gender was color-coded as follows: red dots, women and black dots, men.</p

The frequency of PB gut homing CD4+ and CD8+ T-cells is altered in HIV infection.

<p>Frequencies (expressed as percentages) of peripheral blood (PB) memory gut homing (CD45RO+ CCR9+ β7+) CD4+ (A) and CD8+ (B) T-cells was determined by flow cytometry on PBMCs of HIV-infected individuals (HIV+), antiretroviral (ART) naïve (n = 18), ART experienced individuals on short-term (n = 15) and long-term treatment (n = 32), HIV controllers (n = 6) and HIV seronegative individuals (SN, n = 5). Scatter plots were used to represent the data. Horizontal lines indicate median values. Each symbol represents one individual. The red symbols represent the females in each group. Groups were compared using Kruskal-Wallis test correcting for multiple comparisons using the Dunnett´s post-test. Only significant corrected p values are shown ***p<0.0005, **p<0.005, *p<0.05. Gender was color-coded as follows: red dots, women and black dots, men.</p

Recognition frequency of DR mutations.

<p>ELISpot responses to a panel of 140 DR-WT peptide pairs were assessed in 49 individuals. The number of peptide pairs for which, both WT and DR, only DR, or only WT peptides were recognized was assessed per individual. The scatter plot shows the result of three Wilcoxon tests comparing the paired data between groups, significant p values are indicated. Error bars show the median with interquartile range.</p

Peptide design for the evaluation of the “cornering hypothesis”.

<p><b>A.</b> Pro and RT CTL epitope map used for peptide design. Peptides were designed based on previously-described optimal CTL epitopes (blue lines) and overlapping statistically-predicted HLA-associated positions (red lines) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147571#pone.0147571.ref013" target="_blank">13</a>]. Specific changes in the WT sequence known to mediate ART resistance are indicated as blue residues. <b>B.</b> 128 peptides were designed using experimentally confirmed CTL epitopes that overlap positions associated with DR. Both the WT peptide and the DR variants were synthesized. In the example shown, two DR mutations are reflected in an HLA-A*68:01-restricted epitope and four peptides were design reflecting the different combinations of mutations. <b>C.</b> Ninety additional peptides were designed based on 5 regions containing HLA footprints and DR mutation sites reported in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147571#pone.0147571.ref013" target="_blank">13</a>]. A representative example is shown for the set of peptides designed to test the effect of DR mutation D67N, which is also associated with an HLA-B*15 footprint. Both WT and DR peptides were synthesized and tested individually.</p

Wide spectrum of differential magnitudes of response to WT and DR peptide pairs.

<p><b>A.</b> Differential magnitudes of response to each DR-WT peptide pair (Δ) are shown for each responder. Δ were calculated as the magnitude of response (SFC/million PBMC) to the DR sequence minus the magnitude of response to the WT sequence in each subject individually. A median differential magnitude of response was then calculated across all individuals responding to either the WT, the DR or both sequences. Boxes represent 50% of differential magnitude to each DR-WT peptide pair, whiskers maximum and minimum of the differential magnitude. <b>B.</b> Only median Δ responses are shown. Peptide pairs with significantly higher differential response to DR are shown in red; peptide pairs with significantly higher differential response to WT are shown in blue (p<0.05).</p

Fluid accumulation rate during hospitalization.

<p>(A) Estimated linear model including all study participants during the study period of 7 days. (B) Estimated linear model of one individual during the study period of 7 days.</p

Fluid accumulation rate per groups.

<p>(A) Estimated linear model in the group of Survivors-No AKI; the Survivors-AKI Group; and the Deceased-AKI group during the study period of 7 days. (B) The different slopes in the group of Survivors-No AKI; the Survivors-AKI group; and the Deceased-AKI group during the study period of 7 days.</p

Accumulated fluid balance per day during hospitalization.

<p>Accumulated fluid balance per day during hospitalization.</p

Phase I of fluid accumulation rate.

<p>(A) Estimated linear model in the group of Survivors-No AKI; the Survivors-AKI group; and the Deceased-AKI group during Phase I. (B) The different slopes in the group of Survivors-No AKI; the Survivors-AKI group; and the Deceased-AKI group during Phase I.</p