Demographics of subjects with successfully obtained HIV-1 <i>pol</i> sequences.

<p>Demographics of subjects with successfully obtained HIV-1 <i>pol</i> sequences.</p

Success probability of future regimen by the EuResist prediction engine.

<p>Prediction is based on drug resistance mutation profile and data on previous drug exposure, age, gender and CD4 at failure. Results are presented in ordered of increasing difference between a scenario where all approved NRTIs, NNRTIs and PIs are available versus currently available drugs in Tanzania. A) subjects failing first line therapy; B) subjects failing second line therapy (viral load included in prediction model).</p

Genotypic sensitivity scores for all reverse transcriptase and protease inhibitors.

<p>Percentage (%) of patient strains classified as susceptible, intermediate or resistant as per the Rega V.9.1.0 algorithm; first line failure (n = 43) and second line virological failure cases (n = 26) presented with separate paired bars (first line left bar, second line right bar).</p

Antiretroviral drug resistance mutations in Tanzanian subjects at first line clinico-immunological ART failure.

<p>A) stratified by treatment duration (n = 38); B) stratified by CD4 cells at failure (n = 36).</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

Aberrant MAIT cell transcription factor expression in chronic HIV-1 infection correlates with MAIT cell effector dysfunction.

<p>(A) Unstimulated PBMCs from 10 healthy controls and 17 HIV-1 infected, ART-untreated patients were stained for transcription factors, and their MAIT cell T-bet and Eomes expression profile was determined. The effect of long-term ART on MAIT cell T-bet and Eomes expression profile was also determined in paired samples from 14 HIV-infected patients. (B) Comparison of PLZF, RORγt, and Helios levels between the two MAIT cell populations in the same set of HIV-1 infected, ART-untreated patients. (C, D, E, F) Correlations between the levels of T-bet^negEomes^neg MAIT cells in ART-untreated HIV-1 infected patients with MAIT cell numbers <i>ex vivo</i> and effector functions after <i>E</i>. <i>coli</i> stimulation (MOI 10) were calculated using Spearman’s test. (G) T-bet^negEomes^neg MAIT cells were generated following a six day incubation of PBMCs from healthy controls with PFA-fixed <i>E</i>. <i>coli</i> (MOI 10). The expression levels of PLZF, RORγt, and GrzB, as well as measurement of proliferation using a Cell Trace Violet (CTV) dilution method, were determined in both T-bet^dimEomes^hi and T-bet^negEomes^neg MAIT cells (n = 3). Box and whisker plot shows 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-1 infected, ART-untreated patients, and the Wilcoxon test for paired samples.</p

IL-7 restores MAIT cell effector dysfunction.

<p>(A) MAIT cell arming following IL-7 treatment <i>in vitro</i> for 48 h in healthy controls (n = 8), and HIV-1 infected ART-untreated patients (n = 6). GrzA and Prf upregulation was determined as fold-change of geometric MFI to that of IL-7-untreated cells, whereas GrzB upregulation was determined as fold change of the percentage GrzB-expressing cells. (B) PBMCs from six HIV-1 infected ART-untreated patients were either left untreated or treated with IL-7 for 24 h, then stimulated with mildly PFA-fixed <i>E</i>. <i>coli</i> (MOI 10) for a further 24 h before measurements of MAIT cell effector functions. (C) PBMCs from nine HIV-1 infected ART-untreated patients were incubated in the presence or absence of 10 ng/ml IL-7 for 48 h and then stained for transcription factor expression as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005072#ppat.1005072.g006" target="_blank">Fig 6</a>. Correlation between plasma levels of IL-7 and MAIT cell frequency and numbers from 31 HIV-1 infected ART-untreated patients (D) or MAIT cell effector function from 14 patients (E) were calculated using Spearman’s test. Box and whisker plots show median, IQR and the 10^th to the 90^th percentile. Representative FACS plots are shown. Significance between independent samples was determined using Mann-Whitney’s test, and paired samples using Wilcoxon’s signed rank test (B) or paired t-test (C).</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

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

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