Interferon-gamma release assay conversion after M. tuberculosis exposure specifically associates with greater risk of progression to tuberculosis: a prospective cohort study in Leicester (UK)

ObjectivesWe investigated whether quantifying the serial QuantiFERON-TB Gold (QFT) response improves tuberculosis (TB) risk stratification in pulmonary TB (PTB) contacts.Methods297 untreated adult household PTB contacts, QFT tested at baseline and 3 months after index notification, were prospectively observed (median 1460 days). Normal variance of serial QFT responses was established in 46 extra-pulmonary TB contacts. This informed categorisation of the response in QFT-positive PTB contacts as: converters; persistently QFT-positive with significant increase (PPincrease); and without significant increase (PPno-increase).ResultsEight co-prevalent TB (disease ≤ 3 months after index notification) and 12 incident TB (>3 months after index notification) cases were diagnosed. Genetic linkage to the index strain was confirmed in all culture-positive progressors. Cumulative 2-year incident TB risk in QFT-positive contacts was 8.4% (95% CI, 3.0% - 13.6%); stratifying by serial QFT response, significantly higher risk was observed in QFT-converters (28%), compared with PPno-increase (4.8%) and PPincrease (3.7%). Converters were characterised by exposure to index cases with a shorter interval from symptom onset to diagnosis (median reduction 50.0 days, p=0.013).ConclusionQFT conversion rather than quantitative changes of a persistently positive serial QFT response, associates with greater TB risk and exposure to rapidly progressive TB

Lack of IL-10 increases cellular influx into the lungs and delays recovery during RSV infection.

<p>BALB/c mice and IL-10^−/− mice were infected with 10⁶ PFU RSV i.n. (day 0). (A) Illness was monitored daily by changes in weight for 14 days after RSV infection; percentage of original weight (day 0) is shown. (B) Copies of the RSV L gene were quantified in the lung on day 4 post infection using qPCR. (C) Total numbers of cells in the lung and BAL were enumerated on day 4 and 8 from naïve or RSV infected mice. (D) Total numbers of neutrophils in the BAL were quantified using differential cell counting of H&E stained cytospins slides on day 4 and 8 post infection. (E) Total numbers of NK cells (CD3⁻ NKp46⁺) and CD3 gated CD4⁺Foxp3⁻ and CD8⁺ T cells in the lung were quantified using flow cytometry on day 4 and 8 post RSV infection. Error bars indicate the SEM. The data is representative of three independent experiments with n = 4–5 mice per group.</p

Increased frequency of IFN-γ-expressing T cells in IL-10^−/− mice after RSV infection.

<p>BALB/c mice were infected with 10⁶ PFU RSV i.n. (day 0). CD3⁺ gated, CD4⁺ and CD8⁺ T cells from lungs on day 8 post RSV infection were analyzed by flow cytometry. Representative plots of intracellular IFN-γ expression after a 3 hr PMA/ionomycin restimulation are shown. In addition, quantifications of the frequencies of IFN-γ expression in CD4⁺ T cells and CD8⁺ T cells are shown. The data are representative of three independent experiments with n = 4–5 mice per group.</p

Lack of IL-10 increases chemokine and cytokine production in the airways during RSV infection.

<p>BAL samples from BALB/c and IL-10^−/− mice were analyzed for indicated cytokines and chemokines on day 4 and 8 after RSV infection using Luminex. A group of naive BALB/c controls are included in the day 4 data. Error bars indicate the SEM. The data are pooled from two independent experiments with n = 4–5 mice per group.</p

Blocking IL-10R signalling increases cellular influx into the lungs and delays recovery during RSV infection.

<p>BALB/c mice were infected with 10⁶ PFU RSV i.n. (day 0). Where indicated, mice were injected with anti-IL-10R antibody on day -1 (i.p.), 3 (i.p. and i.n) and 6 (i.p.) post RSV infection. Control groups were injected with rat IgG. (A) Illness was monitored daily by changes in weight for 8 days after RSV infection; the percentage of original weight is shown. (B) Viral titer was measured in the lung on day 4 post infection by quantifying RSV L gene copies by qPCR. (C) Total numbers of cells in the lung and BAL were enumerated on day 4 and 8 from naïve or RSV infected mice. (D) Total numbers of neutrophils in the BAL were quantified using differential cell counting of H&E stained cytospins slides on day 4 and 8 post infection. (E) Total numbers of NK cells (CD3⁻ NKp46⁺) and CD3-gated, CD4⁺Foxp3⁻ and CD8⁺ T cells in the lung were quantified using flow cytometry on day 4 and 8 post RSV infection. Error bars indicate the SEM. The data are representative of two independent experiments with n = 4–5 mice per group.</p

Dynamics of Antigen-Engaged B Cell–Helper T Cell Interactions

<div><p>(A) Time-lapse images of Ig-tg B cells interacting with TCR7 CD4⁺ T cells ~30 h after immunization with HEL in adjuvant, showing T cells moving along behind B cells. The pathways of a B cell (pink dotted line) and a T cell (blue dotted line) remaining bound to each other for more than 1 h are shown (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030150#sv005" target="_blank">Video S5</a>).</p> <p>(B) Time-lapse images showing the dynamics of a B-T conjugate.</p> <p>(C) The <i>t-x, t-y, t-z</i> plots of the interacting B (red line) and T (green line) cells traced in (A). Note the B cell makes turns before the T cell (arrows).</p> <p>(D and E) Velocity measurements of unpaired (D) and paired (E) B and T cells, showing that paired T cells slow to the velocity of the B cell. Velocity data are from 16 cells of each type.</p> <p>(F) Time-lapse images of a B cell interacting with two T cells.</p> <p>(G) Time-lapse images showing an encounter of a B cell and a T cell to form a conjugate.</p></div

Antigen-Engaged CCR7^−/− B Cells Fail to Show Directional Migration toward the Follicle–T Zone Boundary

<div><p>(A) Time-lapse images of HEL-engaged CCR7^−/− (green) and CCR7^+/+ (red) Ig-tg B cells in an inguinal lymph node. The 0-min image is ~3.5 h after antigen exposure (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030150#sv004" target="_blank">Video S4</a>). Square boxes indicate regions used for directionality analysis, shown in (B). A CCR7^−/− Ig-tg B cell (light blue circle and line) and a CCR7^+/+ Ig-tg B cell (pink circle and line) are traced as examples.</p> <p>(B and C) Tracks of antigen-engaged CCR7^+/+ (B) and CCR7^−/− (C) Ig-tg B cells originating from 30-μm follicular cubes, analyzed as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030150#pbio-0030150-g002" target="_blank">Figure 2</a>. Tracks cover 3–17 min for CCR7^+/+ cells and 3–22 min for CCR7^−/− cells.</p> <p>(D) Dot plot showing the percentage of cells that moved across the solid or dashed sides of the cubes. Filled symbols correspond to the data shown in (A–C) and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030150#sv004" target="_blank">Video S4</a>, open symbols correspond to data obtained in a second time-lapse movie collected 2.5–3.5 h after antigen injection. The cubes were located approximately 20–80 μm from the site of accumulation of CCR7^+/+ Ig-tg B cells.</p> <p>(E) The dot plots show ratios of the displacement to the path length of 8-min tracks of antigen-engaged CCR7^+/+ or CCR7^−/− Ig-tg B cells originating from the cubes described in (A–D). The left and right plots for each cell graph are the data of tracks that cross the solid and dashed sides of the cubes, respectively. A total of 74% of wild-type B cells and 81% of CCR7^−/− B cells that crossed the sides of the cubes could be tracked for 8 min. The means of each data group are shown as red bars. *, <i>p</i> < 0.01.</p> <p>(F) Velocity distribution data for CCR7^−/− Ig-tg B cells (green, <i>n</i> = 27) and CCR7^+/+ Ig-tg B cells (red, <i>n</i> = 40) tracked 2.5–4.2 h after antigen injection. The data are pooled from two time-lapse movies. Medians are indicated by arrows.</p></div

The HAM/TSP transcriptional signature comprises IFN-stimulated genes and is not present in multiple sclerosis.

<p>(A) The 80-gene transcriptional signature discovered in HAM/TSP was not present in patients with multiple sclerosis (MS). Datasets were normalized to their own controls (training set: HAM/TSP n = 10, Ctrl n = 9; test set: HAM/TSP n = 10, Ctrl n = 8; multiple sclerosis data set <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002480#ppat.1002480-Gandhi1" target="_blank">[27]</a>: patients n = 99, Ctrl n = 45). (B) Modular analysis approach. Gene expression levels were compared between HTLV-1-positive study groups and healthy control subjects on a module-by-module basis <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002480#ppat.1002480-Chaussabel1" target="_blank">[28]</a>. Modules correspond to previously identified co-regulated gene clusters that were assigned biological functions by unbiased literature profiling (see legend grid on the right). Over-abundance of transcripts in a module is depicted in red, under-abundance in blue. The intensity of the dot corresponds to the percentage of genes in the respective module that are significantly differentially expressed between the study groups. (C) Canonical pathway analysis of the 80-gene HAM/TSP signature (Ingenuity Systems Pathway Analysis software). Orange squares represent the ratio of the number of genes present in the transcriptional signature versus all genes in the pathway. Solid blue bars correspond to the P-value representing the probability that the association between the genes and the identified pathway is due to chance alone (Fisher's exact test with Benjamini-Hochberg multiple testing correction, given as log P-value).</p

Contact Times of Antigen-Engaged B cell–Helper T Cell Conjugates

<div><p>(A) The histogram shows contact time distribution for Ig-tg B cells and TCR7 CD4⁺ T cells 30 to 50 h after immunization with antigen with adjuvant. Open bars show conjugates that were tracked for the duration of contact and shaded bars show conjugates that could not be tracked for the entire period of contact because the cells entered the field as a conjugate, left the field as a conjugate, or both.</p> <p>(B) Contact time distribution for Ig-tg B cells and OT-II CD4⁺ T cells 1 to 2 d after antigen priming. Open and shaded bars as in (A).</p></div

Cellular and viral responses to IFN in HTLV-1 carriers.

<p>STAT1-P(Y701) phosphorylation in response to (A) IFN-α and (B) IFN-γ was quantified in whole blood by flow cytometry. Bar graphs represent the mean ± SEM. P-values were calculated using a two-tailed Mann-Whitney test with *P<0.05 versus controls, ^$P<0.05 versus AC; Ctrl: n = 6, AC: n = 6, HAM/TSP: n = 6. Effect of exogenous IFN-α on the expression of viral transcriptional transactivator (C) Tax (P = 0.4263), (D) the structural protein Gag (P = 0.0112) and (E) HBZ mRNA (P = 0.0742). Tax and Gag protein levels were assessed by flow cytometry, HBZ mRNA was quantified by real-time PCR. P-values were calculated using a paired Wilcoxon signed-rank test; Tax and Gag protein: HAM/TSP: n = 11; HBZ mRNA: AC: n = 2, HAM/TSP: n = 7.</p