Summary of the HIV-infected cohort.

<p>Median (IQR) shown for all parameters; n: numbers.</p><p>Summary of the HIV-infected cohort.</p

Box plots of all manual and FLOCK gated populations between HIV-infected and -uninfected subjects.

<p>Box plot representation of summary results of the two groups generated by the three methods used to investigate the same HIV immunopathogenesis dataset, manual data analysis (left), FLOCK data analysis using the single HIV reference (middle) and FLOCK data analysis using the artificial reference (right). The data is presented in a box and whisker plot where the horizontal line in the box is the median population occupation, the edges of the boxes are the 25th and 75th percentiles of the population occupation and the ‘whiskers’ represent the 10th and 90th percentiles of the population occupation, and the dots indicate outliers. The purple and grey boxes represent the HIV+ and healthy control group, respectively, where the green dots indicate outliers that are AIDS patients. Results of the multiple Mann-Whitney tests followed by Bonferroni adjustments between the HIV and healthy control group for each gated population is shown using P value significance codes found directly above: 0 *** 0.001 ** 0.01 * 0.05.</p

Flow cytometry gating and FLOCK populations.

<p>The manual gating strategy used to gate for the CD4+ T cells is shown in the top panel (A). The CD4+ T cell events were uploaded to immPort (immport.niaid.nih.gov) for FLOCK analysis. The unique populations identified by FLOCK using the single HIV reference (bottom left) (B) and artificial reference (top right) (C) is shown. The artificial reference is made-up of 2% of the CD4+ T cells from each subject in the HIV cohort, where as the single HIV reference is made-up of the CD4+ T cells from a single individual from the HIV cohort that appeared to be biologically representative of the cohort.</p

Clustering of HIV-infected and -uninfected subjects with manual and FLOCK gating principles.

<p>The top panel shows the heat map representation of the matrices containing the cell population frequencies of the manual gating results (A), the FLOCK results using one HIV infected subject that identified biologically relevant cell populations (B) and the FLOCK results using an artificial of the HIV-infected subjects as a reference (C). The bottom panel shows the principle component analysis (PCA) was performed on the matrices illustrated in A-C to investigate whether there were difference between the control, HIV-infected and AIDS subjects. The results of the PCA performed on the manually determined population frequencies is shown in (D), the results of the K-S test that compared the HIV infected individuals to the healthy controls for PC1 (P value = 0.0009, D value = 0.495) and PC2 (P value = 0.3, D value = 0.236) are shown below the biplot. The FLOCK data using one HIV infected subject that identified biologically relevant cell populations is shown in (E), the results of the K-S test that compared the HIV infected individuals to the healthy controls for PC1 (P value = 0.04, D = 0.353) and PC2 (P value = 0.02, D value = 0.378) are shown below the biplot. The FLOCK data using an artificial of the HIV-infected subjects as a reference is shown in (F), the results of the K-S test that compared the HIV infected individuals to the healthy controls for PC1 (P value = 0.02, D value = 0.384) and PC2 (P value = 0.0008, D value = 0.497) are shown below the biplot. A detailed overview of the FLOCK populations in (B) and (C) can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137635#pone.0137635.s001" target="_blank">S1 Table</a>.</p

Non-parametric Spearman rank tests correlation analysis of the manual, sFLOCK and aFLOCK immunopathological populations to the clinical parameters.

<p>^<b>a</b> Bonferroni corrections have been performed.</p><p>Non-parametric Spearman rank tests correlation analysis of the manual, sFLOCK and aFLOCK immunopathological populations to the clinical parameters.</p

Cohort characteristics.

<p>Median (IQR) is shown for all parameters except n and sex.</p>A<p> = Untreated HIV infected subjects.</p>B<p> = Long-term treated HIV infected subjects.</p>C<p>ND = Not Determined.</p>D<p>NA = Not Applicable.</p

HIV- and CMV-specific CD8+ T cell expression of inhibitory receptors and T-bet/Eomes in untreated HIV infection.

<p>(A) Gating strategy illustrating the localization within the T-bet/Eomes axis of HIV- (red) and CMV-specific CD8+ T cells (blue). (B) Distribution within the T-bet^dimEomes^hi and T-bet^hiEomes^dim population of HIV- and CMV-specific CD8+ T cell responses. All data is derived from the untreated HIV infected subjects (n = 52). Median and IQR are shown in all graphs and non-parametric Kruskal Wallis test followed by Dunn's multiple comparison test was performed to compare all pairs of columns; *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001. (C) The frequency of PD-1, CD160, 2B4 and PD-1+CD160+2B4+ on HIV- and CMV-specific CD8+ T cells. Mann-Whitney tests were performed to conclude significance between the groups (median and IQR). (D) Localization of PD-1+CD160+2B4+ expressing HIV-specific CD8+ T cells (red) within the T-bet/Eomes axis and distribution between the T-bet^dimEomes^hi and T-bet^hiEomes^dim population. Wilcoxon matched-pairs single rank test was performed to show on significant differences between the groups. (E) SPICE analysis of inhibitory receptors combinations between the T-bet^dimEomes^hi (red) and T-bet^hiEomes^dim (blue) population for HIV-specific CD8+ T cells. Median and IQR are shown for all bars and Wilcoxon matched-pairs single rank tests were performed to compare outcomes between groups; *<i>P</i><0.05. Permutation test was performed between the pie charts.</p

Phenotypic characterization of T-bet and Eomes expression in untreated HIV-infection.

<p>(A) Representative plots of an untreated HIV infected patient showing the distribution of total PD-1+CD160+2B4+ CD8+ T cells (orange) within different memory phenotype compartments, based on CD45RO, CD27 and CCR7 expression. The distribution of total PD-1+CD160+2B4+ CD8+ T cells was determined in all chronic untreated HIV infected subjects (B) FACS plots from an HIV infected subject showing the distribution of total T-bet^dimEomes^hi (green) cells within the different memory phenotype compartments. Also, the phenotypic distribution of total T-bet^dimEomes^hi CD8+ T cells within different memory compartments for all untreated HIV infected subjects. Median and IQR are shown for all populations. (C) The % of TM and EM compartmentalization for Gag/SL9-tet+ and pp65/NV9-tet+ cells. Paired t-tests were used to compare differences between the groups. (D) Correlations between the MFI of Eomes and TM or EM compartmentalization of Gag/SL9-tet+ and pp65/NV9-tet+ cells. Spearman non-parametric test was used to test for correlations.</p

Polyfunctional characterization of virus-specific CD8+ T cells in untreated HIV-infected individuals.

<p>(A) Distribution of PD-1+ (red) and PD-1− (blue) HIV-specific CD8+ T cells within the T-bet/Eomes axis and linkage to IFNγ and TNF, CD107a and Granzyme B expression. (B) Boolean combinations of all functional and inhibitory markers (n = 256) were combined using PCA, generating a HIV- (orange) and CMV-specific (green) PC1 and PC2 score for each HIV infected subject (n = 23). (C) Correlations between the MFI of Eomes and IFNγ, TNF, CD107a and Granzyme B or single CD107a secretion. (D) SPICE analysis of all functional combinations between the T-bet^dimEomes^hi (red) and T-bet^hiEomes^dim (blue) population for HIV-specific CD8+ T cells. Median and IQR are shown for all bars and Wilcoxon matched-pairs single rank tests were performed to compare outcomes between groups; *<i>P</i><0.05. Functional combinations where the IQR did not exceed 1% are not depicted in the graph. Permutation test was performed between the pie charts.</p

Longitudinal characterization of CD8+ T cell exhaustion and T-bet/Eomes expression following ART initiation.

<p>(A) Longitudinal analysis from baseline (same day as ART initiation) and 6 months post ART for total PD-1+CD160+2B4+ (black), T-bet^dimEomes^hi (red) and T-bet^hiEomes^dim (blue) expression on CD8+ T cells (n = 24). (B) The decay of HIV-specific CD8+ T cell magnitude (grey) and frequency T-bet^dimEomes^hi (red) post ART. (C) The frequency of naïve (asparagus) and T-bet^dimEomes^hi (red) expressing CD8+ T cells longitudinally post ART and Spearman correlation analysis between the area-under-curve (AUC) longitudinally for naïve and T-bet^dimEomes^hi expressing cells. Graphs illustrating the changes in frequency of (D) MFI of Eomes (red), PD-1 (green), CD160 (orange) and 2B4 (pink) expression, and (E) % of PD-1+CD160+2B4+ (black) and T-bet^dimEomes^hi (red) expression from baseline and longitudinally after 6 months on ART. All time-points represents the mean and 95% CI. (F) Distribution of HIV-specific CD8+ T cells within the T-bet^dimEomes^hi and T-bet^hiEomes^dim population after >10 years on ART (n = 12). (G) Frequency of HIV-specific PD-1+CD160+2B4+ CD8+ T cells at baseline (red), 6 months (green), >10 years on ART (blue) and CMV-specific PD-1+CD160+2B4+ CD8+ T cells >10 years on ART (orange). (H) Memory phenotype distribution of HIV-specific CD8+ T cells at baseline (red), 6 months (green) and >10 years on ART (blue). (I) Frequency of each individual function (of total HIV-specific response) before (red) and 6 months after ART (green) (n = 12). For G–I, median and IQR are provided for the bars, where (G–H) Kruskal Wallis test followed by Dunn's multiple comparison test was performed to compare 3 or more columns (***<i>P</i><0.001, *<i>P</i><0.05) and (I) Wilcoxon single rank tests (*<i>P</i><0.05) were used for paired comparisons.</p