<i>Brucella</i>-infected DCs induce Vγ9Vδ2 T cells to produce IFN-γ even when they are added 24 h p.i.

<p><i>Brucella</i>-infected DCs (MOI = 20) were cultured alone or in the presence of untreated or stimulated (0.2 nM HMB-PP) Vγ9Vδ2 T cells. Vγ9Vδ2 T cells were added to DCs 1 h or 24 h p.i. At 24 h, 48 h and 72 h p.i., supernatants were collected. IFN-γ was assessed by ELISA test. Data shown are the mean +/− SD of triplicates and representative of three independent experiments performed with cells from different donor. A significant difference between infected DCs in the presence or not of Vγ9Vδ2 T cells was calculated by using Student’s t test and is indicated by (**) where p<0.01 and (***) where p<0.001.</p

Full Restoration of <em>Brucella</em>-Infected Dendritic Cell Functionality through Vγ9Vδ2 T Helper Type 1 Crosstalk

<div><p>Vγ9Vδ2 T cells play an important role in the immune response to infectious agents but the mechanisms contributing to this immune process remain to be better characterized. Following their activation, Vγ9Vδ2 T cells develop cytotoxic activity against infected cells, secrete large amounts of cytokines and influence the function of other effectors of immunity, notably cells playing a key role in the initiation of the adaptive immune response such as dendritic cells. <em>Brucella</em> infection dramatically impairs dendritic cell maturation and their capacity to present antigens to T cells. Herein, we investigated whether V T cells have the ability to restore the full functional capacities of <em>Brucella</em>-infected dendritic cells. Using an in vitro multicellular infection model, we showed that: 1/<em>Brucella</em>-infected dendritic cells activate Vγ9Vδ2 T cells through contact-dependent mechanisms, 2/activated Vγ9Vδ2 T cells induce full differentiation into IL-12 producing cells of <em>Brucella</em>-infected dendritic cells with functional antigen presentation activity. Furthermore, phosphoantigen-activated Vγ9Vδ2 T cells also play a role in triggering the maturation process of dendritic cells already infected for 24 h. This suggests that activated Vγ9Vδ2 T cells could be used to modulate the outcome of infectious diseases by promoting an adjuvant effect in dendritic cell-based cellular therapies.</p> </div

Activated Vγ9Vδ2 T cells are still able to induce maturation of <i>Brucella</i>-infected DCs even when they are added 24 h p.i.

<p>A/<i>Brucella</i>-infected DCs (MOI = 20) were cultured alone or in the presence of stimulated (0.2 nM HMB-PP) Vγ9Vδ2 T cells. Vγ9Vδ2 T cells were added to DCs 1 h or 24 h p.i. At 24 h, 48 h and 72 h p.i., supernatants were collected and cells were stained. CD83 and CD86 expression analysis were realized on CD1a+ cells. IL-12 was assessed by ELISA test in the collected supernatants. Data shown are the mean +/− SD of triplicates and representative of three independent experiments performed with cells from different donor. A significant difference between infected DCs in the presence or not of Vγ9Vδ2 T cells was calculated by using Student’s t test and is indicated by (*) where p<0. 05 and (**) where p<0.01. (B) <i>Brucella</i>-infected DCs (MOI = 20) were cultured alone or in the presence of stimulated (0.2 nM HMB-PP) Vγ9Vδ2 T cells. Vγ9Vδ2 T cells were added either 1 h or 24 h p.i. In all cases, CFSE-stained naive CD4+ T cells were added 24 h p.i. with DC/T cell ratio 0.1. After 5 days, CD4+ T cell proliferation was analyzed by flow cytometry and the proliferation index was calculated by comparison with non-infected cells. Data shown are the mean of duplicates +/− SD of three independent experiments performed with cells from different donors. Statistical difference is calculated by comparison with <i>Brucella</i>-infected DCs by using wilcoxon rank test and is indicated by (*) where p<0. 05.</p

Cell-cell contacts between Vγ9Vδ2 T cells and infected DCs are required for maturation process.

<p><i>Brucella</i>-infected DCs (MOI = 20) were cocultured in contact (black bars) or separated by a trans-well chambers (insert, white bars) with Vγ9Vδ2 T cells in the presence or not of HMB-PP (0.2 nM). At 48 h p.i., supernatants were collected and cells were stained. CD83 (A, left panel) and CD86 (B, left panel) expression analyses were realized on CD1a+ cells by flow cytometry. Data shown are the mean +/− SD of three independent experiments performed with cells from different donors. Representative flow cytometry histograms show the expression of CD83 (A, right panel) and CD86 (B, right panel) of infected DCs alone (filled grey), cocultured in contact (grey line) or separated (black line) with HMB- or not- stimulated Vγ9Vδ2 T cells. IL-12 was assessed by ELISA test in the collected supernatants (C). Data shown are the mean +/− SD of triplicates and are representative of the three experiments. A significant difference between infected DCs in the presence or not of Vγ9Vδ2 T cells was calculated by using Student’s t test and is indicated by (*) where p<0.05 and (**) where p<0.01. Significant differences between infected DCs in contact or not with Vγ9Vδ2 T cells and between HMB- or not- stimulated Vγ9Vδ2 T cells are indicated directly on the graph.</p

Role of IFN-γ and TNF-α on DC maturation induced by Vγ9Vδ2 T cells.

<p>DCs (DC), infected DCs (inf DC) and infected DCs in the presence of Vγ9Vδ2 T cells (infDCs+γδT) were cultured with neutralizing mAbs to IFN-γ (white bars), TNF-α (striped white bars) or with isotype control Ab (black bars). At 48 h p.i., supernatants were collected and cells were stained with FITC-conjugated mAbs to CD83 (A), CD86 (B). CD83 and CD86 expression analysis were realized on CD1a+ cells by flow cytometry. IL-12 was assessed by ELISA in the collected supernatants (C). Data shown are the mean +/− SD of triplicates and are representative of the three experiments. The Student’s <i>t</i> test was used to calculate significant differences between: - 1: infected DCs in the presence or not of Vγ9Vδ2 T cells and was indicated by (*) where <i>p</i><0. 05 and (**) where <i>p</i><0.01 and 2: infected DCs cocultured with Vγ9Vδ2 T cells in the presence of neutralizing mAb or isotype control and is directly indicated on the graph.</p

Pre-Disposition and Epigenetics Govern Variation in Bacterial Survival upon Stress

<div><p>Bacteria suffer various stresses in their unpredictable environment. In response, clonal populations may exhibit cell-to-cell variation, hypothetically to maximize their survival. The origins, propagation, and consequences of this variability remain poorly understood. Variability persists through cell division events, yet detailed lineage information for individual stress-response phenotypes is scarce. This work combines time-lapse microscopy and microfluidics to uniformly manipulate the environmental changes experienced by clonal bacteria. We quantify the growth rates and RpoH-driven heat-shock responses of individual <em>Escherichia coli</em> within their lineage context, stressed by low streptomycin concentrations. We observe an increased variation in phenotypes, as different as survival from death, that can be traced to asymmetric division events occurring prior to stress induction. Epigenetic inheritance contributes to the propagation of the observed phenotypic variation, resulting in three-fold increase of the RpoH-driven expression autocorrelation time following stress induction. We propose that the increased permeability of streptomycin-stressed cells serves as a positive feedback loop underlying this epigenetic effect. Our results suggest that stochasticity, pre-disposition, and epigenetic effects are at the source of stress-induced variability. Unlike in a bet-hedging strategy, we observe that cells with a higher investment in maintenance, measured as the basal RpoH transcriptional activity prior to antibiotic treatment, are more likely to give rise to stressed, frail progeny.</p> </div

Vγ9Vδ2 T cells induce full maturation of <i>Brucella</i>-infected DCs. Non- or <i>Brucella</i>-infected DCs (MOI = 2, 5, 20, and 50) were cocultured with Vγ9Vδ2 T cells.

<p>At 48 h p.i., supernatants were collected and cells were stained with FITC-conjugated mAbs to CD83 (A), CD86 (B). CD83 and CD86 expression analyses were realized on CD1a+ cells by flow cytometry and the results were expressed as percentage of positive cells for CD83 and mean fluorescence intensity (MFI) for CD86. Data shown are the mean +/− SD of three independent experiments performed with cells from different donors. IL-12 was assessed by ELISA test in the collected supernatants (C). Data shown are the mean +/− SD of triplicates and are representative of three experiments. A significant difference between infected DCs in the presence or not of Vγ9Vδ2 T cells was calculated by using Student’s t test and is indicated by (*) where p<0.05 and (**) where p<0.01.</p

Growth inhibition and clonal cell death correlate with a high stress response.

<p>(A) Correlation between reporter intensity and growth rate in response to stress. The growth rate and fluorescence intensity of single cells are measured 130 minutes after Streptomycin treatment. Red indicates dead cells; blue, live cells. (B) Histogram of the growth rate distribution from the same data set as (A). The dashed line indicates the threshold we chose to distinguish alive from dead cells. (C) Life history of a sub-lineage from the micro-colony (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003148#pgen-1003148-g003" target="_blank">Figure 3</a> for the full lineage tree and the legend therein). The colour code represents the fluorescence intensity. Dead cells are indicated by a red dot at the end of the lineage tree. In addition, the cellular growth rate is represented inversely by line width (e.g., bold line the slow growers). The dashed line indicates the time of induction by streptomycin. Data correspond to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003148#pgen.1003148.s022" target="_blank">Video S2</a>.</p

Increased cellular membrane permeability following stress induction.

<p>Exponential phase cells of a strain carrying both p<i>^terR</i> - and p<i>^ibpAB</i> -driven fluorescence reporters are plated on LB-agar pads with or without streptomycin (3 µg/ml) and ATC (25 ng/ml). After 2–3 hours of colony growth, the reporter intensity is quantified by fluorescence microscopy. The intensity is normalized to that of the non-induced state ([streptomycin] = [ATC] = 0). The black dot in the middle of each data cloud shows the mean value of both fluorescence channels. R² and k are the coefficient of determination and slope for linear fitting. For each condition, at least 5 micro-colonies are quantified.</p

Polysulfone Membranes Coated with Polymerized 3,4-Dihydroxy‑l‑phenylalanine are a Versatile and Cost-Effective Synthetic Substrate for Defined Long-Term Cultures of Human Pluripotent Stem Cells

Clinical and industrial applications of human pluripotent stem cells (hPSC) require large amounts of cells that have been expanded under defined conditions. Labor-intensive techniques and ill-defined or expensive compounds and substrates are not applicable. Here we describe a chemically defined synthetic substrate consisting of polysulfone (PSF) membranes coated with polymerized 3,4-dihydroxy-l-phenylalanine (DOPA). DOPA/PSF is inexpensive and can be easily produced at various shapes and sizes. DOPA/PSF supports long-term self-renewal of undifferentiated human embryonic (hESC) and human induced pluripotent stem cells (hiPSC) under defined conditions. Pluripotency is maintained for at least 10 passages. Adhesion of hPSC to DOPA/PSF is mainly mediated by a specific integrin heterodimer. Proliferation and gene expression patterns on DOPA/PSF and control substrates are comparable. Labor-intensive cultivation methods and use of serum or coating with proteins are not required. Together, these features make DOPA/PSF attractive for applications where large-scale expansion of human pluripotent stem cells under defined conditions is essential