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

Summary of the HIV-infected cohort.

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

Arming of MAIT Cell Cytolytic Antimicrobial Activity Is Induced by IL-7 and Defective in HIV-1 Infection

<div><p>Mucosa-associated invariant T (MAIT) cells represent a large innate-like evolutionarily conserved antimicrobial T-cell subset in humans. MAIT cells recognize microbial riboflavin metabolites from a range of microbes presented by MR1 molecules. MAIT cells are impaired in several chronic diseases including HIV-1 infection, where they show signs of exhaustion and decline numerically. Here, we examined the broader effector functions of MAIT cells in this context and strategies to rescue their functions. Residual MAIT cells from HIV-infected patients displayed aberrant baseline levels of cytolytic proteins, and failed to mobilize cytolytic molecules in response to bacterial antigen. In particular, the induction of granzyme B (GrzB) expression was profoundly defective. The functionally impaired MAIT cell population exhibited abnormal T-bet and Eomes expression patterns that correlated with the deficiency in cytotoxic capacity and cytokine production. Effective antiretroviral therapy (ART) did not fully restore these aberrations. Interestingly, IL-7 was capable of arming resting MAIT cells from healthy donors into cytotoxic GrzB+ effector T cells capable of killing bacteria-infected cells and producing high levels of pro-inflammatory cytokines in an MR1-dependent fashion. Furthermore, IL-7 treatment enhanced the sensitivity of MAIT cells to detect low levels of bacteria. In HIV-infected patients, plasma IL-7 levels were positively correlated with MAIT cell numbers and function, and IL-7 treatment <i>in vitro</i> significantly restored MAIT cell effector functions even in the absence of ART. These results indicate that the cytolytic capacity in MAIT cells is severely defective in HIV-1 infected patients, and that the broad-based functional defect in these cells is associated with deficiency in critical transcription factors. Furthermore, IL-7 induces the arming of effector functions and enhances the sensitivity of MAIT cells, and may be considered in immunotherapeutic approaches to restore MAIT cells.</p></div

IL-7 potently enhances MAIT cell killing of bacteria-exposed cells.

<p>(A) The effect of IL-7 on MAIT cell killing of bacteria-exposed cells was determined using 293T-hMR1 cells that were pulsed with PFA-fixed <i>E</i>. <i>coli</i> (MOI 10) and co-cultured with purified MAIT cells that were previously untreated or treated with IL-7 for 72 h. Anti-MR1 or IgG2a isotype controls were added to determine MR1-dependency of MAIT cell killing of bacteria-exposed cells. 293T-hMR1 cell death was defined as cells that were positive for both poly-caspases activities (FLICA⁺) and amine-reactive live/dead cell marker (DCM⁺). MAIT cell degranulation, and expression of GrzB and Prf were simultaneously assessed. (B) The effector to target cells (E:T) ratio curve for MAIT cell killing of PFA-fixed <i>E</i>. <i>coli</i>-pulsed 293T-hMR1 cells was determined in 3 independent donors. FLICA⁺ cells denote total apoptotic cells (left panel), whereas FLICA⁺DCM⁺ cells denote dead cells (right panel). Representative FACS plots are shown. Error bars represent mean and standard deviation.</p

IL-7 arming of MAIT cells and MAIT cell cytotoxic capacity are impaired in chronic HIV-1 infection.

<p>(A) The expression of GrzA, GrzB, and Prf at resting state was determined in 20 healthy controls and 25 HIV-infected, untreated patients, as well as in 18 paired patient samples before and after effective ART. (B,C) MAIT cell cytotoxic capacity and IFNγ production after 24 h of stimulation with PFA-fixed <i>E</i>. <i>coli</i> (MOI 10) was determined in 20 healthy controls and 25 HIV-infected, untreated patients, as well as in 18 paired patient samples before and after effective ART as described in (A). Box and whisker plots show median, IQR and the 10^th to the 90^th percentile. Representative FACS plots are shown. The Mann-Whitney test was used to determine significance between healthy controls and HIV-infected, untreated patients, and the Wilcoxon test for paired samples.</p

MAIT cells express a distinct transcription factor profile.

<p>(A) Freshly isolated PBMCs from 10 HIV-uninfected, healthy controls were stained for the transcription factors PLZF, RORγt, T-bet, Eomes, and Helios. The expression levels for these transcription factors were determined and compared in MAIT cells (red), and in conventional CD4⁺ (blue) and CD8⁺ (green) T cells. (B) MAIT cells (red) T-bet:Eomes expression ratio when compared to conventional T cells (black) and CD3^-CD161⁺ lymphocytes (brown). MAIT cells express a low T-bet: high Eomes ratio (T-bet^dimEomes^hi). (C) The transcription factor expression levels among the CD4^-CD8^- DN (blue) and CD8⁺ MAIT cell populations (green) that comprised the vast majority of total MAIT cells (typically >95%; red) were similar. Variable expression levels within the small minority CD4⁺ MAIT cells (orange) were observed. Representative FACS plots are shown. Box and whisker plots show median, IQR and the 10^th to the 90^th percentile. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (the Friedman test followed by Dunn’s post-hoc test).</p

Characteristics of HIV-1 infected patients and healthy controls.

<p>ND, not done; NA, not applicable; M, male;</p><p>*comparisons were done on uninfected versus HIV-infected patients;</p><p>^#comparisons were done on HIV-infected patients in cohort 1 versus cohort 2;</p><p>^§significance was determined using the Mann-Whitney test;</p><p>^&significance was determined using Fisher’s exact test; median (IQR) is shown for all parameters unless otherwise specified.</p><p>Characteristics of HIV-1 infected patients and healthy controls.</p