Clinical details of HIV-1 infected patients used in this study.

1<p>3TC: lamivudine, ABC: abacavir, ATV: atazanavir, AZT: zidovudine, D4T: stavudine, DRV: darunavir, EFV: efavirenz, ETV: etravirine, FPV: fosamprenavir, FTC: emtricitabine, LPV: lopinavir, NVP: nevirapine, RGV: raltegravir, RTV: ritonavir, T20: enfuvirtide, TDF: tenofovir.</p

Correlation of NK cell proportions with FcRγ expression in HIV-infected individuals.

<p>(A) Correlation between NK cell FcRγ protein expression and the proportion of NK cells in patient PBMC (donors 19–27) measured by flow cytometry. (B) Proportion of NK cells in PBMC (individuals 19–27, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009643#pone-0009643-t001" target="_blank">Table 1</a>) measured by flow cytometry. The Mann-Whitney U test was used to assess statistical significance. Horizontal bars represent median values.</p

FcRγ expression in monocytes of HIV-infected and uninfected individuals.

<p>FcRγ mRNA (A) and protein (B) were determined by Q-PCR and quantitative infrared immunoblotting in extracts from monocytes purified by FACS from blood of HIV-1 infected (individuals 1–18, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009643#pone-0009643-t001" target="_blank">Table 1</a>) and control subjects. Fluorescence from FcRγ immunoblots measured at 680 nm was normalised to fluorescence of GAPDH in the same immunoblots, measured at 800 nm. FcRγ mRNA was determined from real-time PCR measurements using the comparative threshold method with GAPDH mRNA serving as internal control. Differences between groups were tested using the Mann-Whitney U test for non-parametric data, with a value &lt;0.05 assumed to be significant. Horizontal bars represent median values.</p

FcRγ expression in CD56⁺/CD94⁺ lymphocytes of HIV-infected and uninfected individuals.

<p>FcRγ mRNA (A) and protein (B) were measured in FACS-sorted CD56⁺/CD94⁺ lymphocytes from blood of HIV-1 infected (individuals 1–18, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009643#pone-0009643-t001" target="_blank">Table 1</a>) and control subjects by Q-PCR using the comparative threshold and quantitative immunoblotting method as described. The Mann-Whitney U test was used to assess statistical significance. Horizontal bars represent median values.</p

FcRγ and TCRζ expression in NK cells of HIV-infected and uninfected individuals.

<p>NK cells purified from blood of an additional 9 HIV-infected (individuals 19–27, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009643#pone-0009643-t001" target="_blank">Table 1</a>) and 8 control subjects. FcRγ protein (A) and mRNA (B) and TCRζ mRNA (C) were measured in FACS-sorted CD3⁻ CD56⁺/CD94⁺ NK cells using quantitative immunoblotting and Q-PCR as described. The Mann-Whitney U test was used to assess statistical significance. Horizontal bars represent median values.</p

TCRζ expression in CD56+/CD94+ and CD3+ T lymphocyte populations of HIV-infected and uninfected individuals.

<p>TCRζ mRNA in T lymphocytes (A) and CD56⁺/CD94⁺ lymphocytes (B) purified by FACS from blood of HIV-1 infected (individuals 1–18, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009643#pone-0009643-t001" target="_blank">Table 1</a>) and control subjects was measured by Q-PCR using the comparative threshold method as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009643#pone-0009643-g002" target="_blank">Figure 2A</a>. The Mann-Whitney U test was used to assess statistical significance. Horizontal bars represent median values.</p

Baseline characteristics, telomere length and telomerase activity by treatment arm.

<p>T/S ratio: TL was expressed as a ratio to a single (S) copy housekeeping gene 36B4 (T/S ratio). All parameters are shown as mean (SD), unless otherwise stated.</p><p>Baseline characteristics, telomere length and telomerase activity by treatment arm.</p

MONET trial- Telomere sub-study patient disposition.

<p>MONET trial- Telomere sub-study patient disposition.</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 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