Disrupted lymph node and splenic stroma in mice with induced inflammatory melanomas is associated with impaired recruitment of T and dendritic cells

International audienceMigration of dendritic cells (DC) from the tumor environment to the T cell cortex in tumor-draining lymph nodes (TDLN) is essential for priming naïve T lymphocytes (TL) to tumor antigen (Ag). We used a mouse model of induced melanoma in which similar oncogenic events generate two phenotypically distinct melanomas to study the influence of tumor-associated inflammation on secondary lymphoid organ (SLO) organization. One tumor promotes inflammatory cytokines, leading to mobilization of immature myeloid cells (iMC) to the tumor and SLO; the other does not. We report that inflammatory tumors induced alterations of the stromal cell network of SLO, profoundly altering the distribution of TL and the capacity of skin-derived DC and TL to migrate or home to TDLN. These defects, which did not require tumor invasion, correlated with loss of fibroblastic reticular cells in T cell zones and in impaired production of CCL21. Infiltrating iMC accumulated in the TDLN medulla and the splenic red pulp. We propose that impaired function of the stromal cell network during chronic inflammation induced by some tumors renders spleens non-receptive to TL and TDLN non-receptive to TL and migratory DC, while the entry of iMC into these perturbed SLO is enhanced. This could constitute a mechanism by which inflammatory tumors escape immune control. If our results apply to inflammatory tumors in general, the demonstration that SLO are poorly receptive to CCR7-dependent migration of skin-derived DC and naïve TL may constitute an obstacle for proposed vaccination or adoptive TL therapies of their hosts

Control of CD8 T cell proliferation and terminal differentiation by active STAT5 and CDKN2A/CDKN2B.

International audienceCD8 T cells used in adoptive immunotherapy may be manipulated to optimize their effector functions, tissue-migratory properties and long-term replicative potential. We reported that antigen-stimulated CD8 T cells transduced to express an active form of the transcription factor signal transducer and activator of transcription 5 (STAT5CA) maintained these properties upon adoptive transfer. We now report on the requirements of STAT5CA-expressing CD8 T cells for cell survival and proliferation in vivo. We show that STAT5CA expression allows for greater expansion of T cells in vivo, while preserving dependency on T-cell-receptor-mediated tonic stimulation for their in vivo maintenance and return to a quiescent stage. STAT5CA expression promotes the formation of a large pool of effector memory T cells that respond upon re-exposure to antigen and present an increased sensitivity to γc receptor cytokine engagement for STAT5 phosphorylation. In addition, STAT5CA expression prolongs the survival of what would otherwise be short-lived terminally differentiated KLRG1-positive effector cells with up-regulated expression of the senescence-associated p16(INK) (4A) transcripts. However, development of a KLRG1-positive CD8 T cell population was independent of either p16(INK) (4A) or p19(ARF) expression (as shown using T cells from CDKN2A(-/-) mice) but was associated with expression of transcripts encoding p15(INK) (4B) , another protein involved in senescence induction. We conclude that T-cell-receptor- and cytokine-dependent regulation of effector T cell homeostasis, as well as mechanisms leading to senescent features of a population of CD8 T cells are maintained in STAT5CA-expressing CD8 T cells, even for cells that are genetically deficient in expression of the tumour suppressors p16(INK) (4A) and p19(ARF)

Deliverable 6.3 DEMO Insurance Value Assessment - PART 7: FRANCE - Brague

The Brague River basin is a 68 km² catchment located along the French Mediterranean coast between the cities of Cannes and Nice. On 3rd Oct. 2015, the basin was severely hit by an extreme flash flood (time return was over 100 years). The basin very flat lowlands experienced numerous damages and casualties related to this flooding event. The several campsites located in these areas were closed by State decision because of the flood risk but dozens of houses remain at risk. The closing of the campsite opened a window of opportunity to redefine the economic activities of the valley and the river corridor in order to improve its life, landscape and environmental quality and to decrease the flood risk.Within the NAIAD project, several teams of researchers and experts in forest and river management, natural hazards (flood, erosion, wildfire), vulnerability and damage assessment, economy and decision aid gathered to perform an in-depth study of the Brague River catchment. More precisely, we studied its peculiarities, the potential efficacy and efficiency of flood protections measures based on green or grey measures, as well as their co-benefit.NBS flood alleviation strategies studied for the Brague catchment are a combination of both retention measures by small natural retention areas in the upper catchment, along with a widening of the river corridor in the lowlands enhanced by floodplain reconnection. Floodplain works consist in several measures as bed and bridge widening, forest corridor and wetlands restoration, and large woods debris management. They are integrated in a so-called “giving-room-to-the-river” strategy. Two levels of ambition, namely high and very high, are considered as well as a more classical grey scenario based on huge retention dams.This report presents the assessment of the Basin state in term of flood risk and river quality. Total costs of the three protection strategies were evaluated. Damage related to historical events and to theoretical floods with known return period were computed in the current and projects’ situation, thus enabling to compute mean annual avoided damage. The co-benefit related to NBS strategies were also evaluated using two different methods: transfer of values based on a meta-regression-analysis of values provided in other catchments and a contingent valuation performed locally through the interview of more than 400 peoples in the basin. The cost-benefit analysis demonstrates that costs are higher than the main benefit, i.e., avoided damage, but when including co-benefit the balance may reach higher benefits than costs for NBS strategies, though not for the grey solution. It worth being stressed that several intangible criteria, e.g., the improvement of the natural status of the river, are poorly captured by the monetary methods and a complementary multicriteria decision framework was developed to handle both tangible and intangible criteria

Tumor-initiated inflammation overrides protective adaptive immunity in an induced melanoma model in mice

We studied the effect of the immune system on two differentially aggressive melanomas developing in mice on conditional deletion of the INK4A/ARF tumor suppressor gene, with concomitant expression of oncogene H-Ras(G12V) and a natural cancer-germline tumor antigen (TA). "Slow progressor" melanomas contained no activated T lymphocytes (TL). In contrast, "aggressive" melanomas were infiltrated by activated TLs lacking effector molecules and expressing high levels of PD-1, indicating an exhausted phenotype. Aggressive melanomas were also infiltrated by immature myeloid cells (IMC). Infiltration was associated with local inflammation and systemic Th2/Th17-oriented chronic inflammation that seemed to impair further activation of TLs, as tumor-specific T cells adoptively transferred into mice bearing aggressive melanomas were poorly activated and failed to infiltrate the melanoma. This immunosuppression also led to the incapacity of these mice to reject inoculated TA-positive tumors, in contrast to slow-progressing melanoma-bearing mice, which were responsive. To test the role of adaptive immunity in tumor progression, we induced melanomas in immunodeficient RagKO compound mice. These mice developed aggressive but not slow-progressing melanomas at a higher frequency and with a shorter latency than immunocompetent mice. Immunodeficient mice also developed abnormal inflammation and infiltration of IMCs in a manner similar to immunocompetent mice, indicating that this phenotype was not dependent on adaptive immunity. Therefore, tumor-intrinsic factors distinguishing the two melanoma types control the initiation of inflammation, which was independent of adaptive immunity. The latter delayed development of aggressive melanomas but was overridden by inflammation. Cancer Res; 70(9); 3515-25. (c)2010 AACR

Evaluation de la contamination en 2,4,6-tribromophenol d'eau et d'organismes marins exposés à des rejets de chloration

International audience2,4,6-tribromophenol (TBP) is implied in the production of brominated flame retardants but is also a major chlorination by-product in seawater. A growing number of studies indicate that TBP is highly toxic to the marine biota, but the contribution of anthropogenic sources among natural production is still under question concerning its bioaccumulation in marine organisms. Here, several water sampling campaigns were carried out in the industrialized Gulf of Fos and clearly showed the predominant incidence of industrial chlorination discharges on the TBP levels in water, at the 1-10 ng L-1 level in average and reaching up to 580 ng L-1 near the outlets. The bioaccumulation of TBP was measured in 90 biota samples of the Gulf of Fos. The concentrations found in European conger muscle tissues (140 to 1000 ng g-1 lipid weight, in average), purple sea urchin gonads (830 to 880 ng g-1 lipid weight, in average), and Mediterranean mussel body (1500 to 2000 ng g-1 lipid weight, in average) were above all published references. Significant correlations with fish length (European conger) and gonad somatic index (purple sea urchin) were also identified. Comparatively, fish, urchins and mussels from other Mediterranean sites analyzed within this study showed a lower bioaccumulation level of TBP, consistently with what found elsewhere. Industrial outflows were thus identified as hotspots for TBP in seawater and marine organisms. The environmental risk assessment indicated a high potential toxicity in the industrial Gulf of Fos, in particular near the outlets, and a limited threat to human but toxicological references are lacking

Genes up-regulated or down-regulated in Amela tumors involved in EMT.

(a)<p>Genes known to be involved in EMT that show higher expression in Amela versus Mela primary tumors and are expressed at similar level in Amela primary tumors and Amela lines in culture.</p>(b)<p>Ratio of gene expression as log2 primary Amela/primary Mela > 1 with p values < 0.05;</p>*<p>Ratio of gene expression as log2 primary Amela/primary Mela between 0 and 1 with p values < 0.05;</p>(c)<p>Ratio of gene expression as Log2 (primary Amela-/cultured Amela line) < 1 and/or with p value > 0.05 (ns). Log2 (primary Amela /cultured Amela line) < - 1 corresponds to higher expression in cultured Amela line than in primary Amela.</p>(d)<p>Genes known to be involved in EMT that show lower expression in primary Amela versus Mela tumors.</p

Effect of MAPK pathway inhibitors on the expression of EMT hallmark genes in Amela tumor lines.

<p>QRT-PCR analysis for expression of 6 transcripts encoding mesenchymal and epithelial markers in Amela tumor cells in the absence (value set to 1) or presence of inhibitors, as indicated. Data are represented as mean ± s.e.m of three independent experiments in which 5 samples were analyzed. ***p value < 0.001; **p value < 0.01; *p value < 0.05.</p

EMT and TGFβ pathway signatures in Amela melanomas. A.

<p>Representative gene set enrichment analysis <b>(</b>GSEA) plots of EMT (right graph) and TGFβ pathway (left graph) gene signatures. Each plot is divided into two sections. The first section (class A) shows results for gene sets that have a positive enrichment score (gene sets that show enrichment at the top of the ranked list here associated with Amela samples). The second section (class B) shows the results for gene sets that have a negative enrichment score (gene sets that show enrichment at the bottom of the ranked list here associated with Mela samples). Data provided as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049419#pone.0049419.s002" target="_blank">Fig. S2</a>. <b>B.</b> QRT-PCR analysis for expression of transcripts encoding mesenchymal and epithelial markers in Amela (white dotted bars) and Mela (black dotted bars) tumors. Values were normalized to those for skin from control mice. Data are represented as mean ± s.e.m of three independent experiments in which 7 samples of Amela and 7 samples of Mela tumors were analyzed. **p value < 0.01; *p value < 0.05.</p

Specific expression of Ccl2 in Amela tumors is controlled by Ras signalling pathways.

<p>(A-B) QRT-PCR analysis of Ccl2 transcript expression in induced Mela and Amela primary tumors (A) and in the corresponding cell lines in vitro (B). (C) Concentration of secreted Ccl2 in the supernatant of 24 h cultures of Amela and Mela cell lines as measured by ELISA. (D) Expression of Ccl2 transcript by QRT-PCR and (E) concentration of secreted protein by ELISA, in the absence (value set to 1) or presence of inhibitors in 24 h cultures, as indicated. (A–C) Amela tumors are represented by white dotted bars and Mela tumors by black dotted bars. For primary tumor analysis (A) 6 samples of each tumor were analyzed. For <i>in vitro</i> analysis (B), values were normalized to those for B16F10 cells. In (C), supernatants from 24 h cell cultures were tested and values are expressed as ng/ml. In D–E, culture conditions were as described in Methods. Experiments involved 10 Amela lines in (D) and 4 in (E). ****p value < 0.0001; ***p value < 0.001; **p value < 0.01; *p value < 0.05.</p

Analysis of the TGFβ pathway in melanoma lines and tumors. A.

<p>Supernatants from Amela cell lines incubated in serum-free DMEM were either acid treated (acid) or not (no treatment) and were tested for TGFβ content using reporter line MFBF11 (see Methods). Bars represent means ± s.d. of triplicate wells in one representative experiment. Serum-free DMEM (no TGFβ) was used for baseline measurement. B. Flow cytometry analysis of GFP expression in SBE-GFP-transduced Amela and B16F10 cell lines preincubated in serum-free DMEM without addition (red lines) or in the presence of TGFβ (blue lines). Non-transduced Amela cell lines were used as control (gray filled). C. Comparasion of the expression of 3 genes associated with EMT by Amela^C and Amela^I cell lines by QRT-PCR (see text). Results are represented as fold change in relative expression where the value for Amela^C is set to 1 for each gene. Data are represented as mean ± s.e.m of two independent experiments in which 4 different lines of each type were tested. D-E. Flow cytometry analysis of GFP expression in SBE-GFP-transduced Amela^C cell lines as in (B). The effect of various inhibitors was assessed on Amela^C cells during the incubation in serum-free DMEM without (-) or with addition (+) of TGFβ. GFP expression in one Amela^C line before and after treatment with various inhibitors in the absence of TGFβ is shown in (D). In (E)results show fold change of mean GFP fluorescence intensity in Amela^C without (panel at left) or with (panel at right) addition of TGFβ. The value for Amela^I in the absence of TGFβ is set to 1. Data are represented as mean ± s.e.m of four independent experiments. ***p value < 0.001; **p value < 0.01; *p value < 0.05.</p