Sheep skin lymph CD103 and CD11b -type DC in vitro response to Canine Adenovirus vector
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ABSTRACT: Transcriptomic profiling of sheep skin lymph DC subsets after 16h in vitro culture in medium or stimulated by non replicative Canine Adenovirus serotype 2 Reference design: total enriched lymph dendritic cells- Biological replicates: 4 sheep (70958, 50274, 30066, 11261)- Samples: FACS sorted CD103 -type and CD11b type cultured in medium or with CAV2
Project description:Transcriptomic profiling of ovine skin lymph dendritic cell subsets CD26+ and CD26- compared to total enriched lymph dendritic cells. CD26+ and CD26- dendritic cells (DC) from sheep skin lymph were sorted by flow cytometry and were analysed for their transcriptomic profile. We demonstrate that the minor sheep CD26+ skin lymph DC subset shares significant transcriptomic similarities with mouse CD8a+ and human BDCA3+ DC. This allowed the identification of a common set of phenotypic characteristics for CD8a-like DC in the 3 mammalian species, i.e. SIRPlo, CADM1hi, CLEC9Ahi, DEC205hi, XCR1hi. Compared to CD26- DC, the sheep CD26+ DC show (1) potent stimulation of allogeneic naive CD8+ T cells with high selective induction of the Ifn-gamma and Il22 genes; (2) dominant efficacy in activating specific CD8+ T cells against exogenous soluble antigen; (3) selective expression of functional pathways associated to high capacity for antigen cross-presentation. Reference design (total enriched lymph dendritic cells) - Biological replicates: 2 sheep #10081 and #30066
Project description:Regulatory T cells (Tregs) play a key role in suppressing systemic effector immune responses, thereby preventing autoimmune diseases but could also potentially contribute to tumor progression. As a result, there is significant interest in the clinical manipulation of Tregs. However, the mechanisms governing induced Treg (iTreg) differentiation are not fully understood. In the present study, we phenotypically profiled human iTregs during early stages of in vitro differentiation at single-cell level using multiparametric mass cytometry. A panel of 25 metal-conjugated antibodies specific to a range of markers associated with human Tregs was used to characterize these immunomodulatory cells. We found that iTregs highly express the transcription factor Foxp3 and characteristic Treg-associated surface markers (e.g. CD25, PD1, CD137, CCR4, CCR7, CXCR3, and CD103). Expression of co-inhibitory factors (e.g. TIM3, LAG3, and TIGIT) increased slightly at late stages of iTreg differentiation. Further, CD103 was selectively upregulated in the iTreg compartment while negatively regulated by Foxp3. Overall, our study highlights that during early stages of differentiation, iTregs resemble memory-like Treg features, and opens possibilities for studying molecular mechanisms of Treg function.
Project description:Transcriptomic profiling of sheep skin lymph DC subsets after 16h in vitro culture in medium or stimulated by non replicative Canine Adenovirus serotype 2
Project description:CD8+ cytotoxic T cells are critical for viral clearance from the lungs upon influenza virus infection. The contribution of cross-presentation to the induction of anti-viral cytotoxic T cells remains debated. Here, we used a recombinant influenza virus expressing a NS1-GFP reporter gene to visualize the route of antigen presentation by lung dendritic cells (DC) upon viral infection in vivo. We found that lung CD103+ DC are the only subset to carry intact GFP protein to the draining lymph nodes. Strikingly, lung migratory CD103+ DC are not productively infected by influenza virus and thus induce virus-specific CD8+ T cells through the cross-presentation of antigens from virally infected cells. We also show that CD103+ DC resistance to infection correlates with an increased antiviral state in these cells that is dependent on the expression of IFN receptor alpha. In conclusion, these results establish that efficient cross-priming by migratory lung DC is coupled to the acquisition of an anti-viral status, which is dependent on type I IFN signaling pathway. mRNA profiles were generated by deep-sequencing in Illumina HiSeq2000 from alveolar macrophages and CD103+ dendritic cells from lungs of untreated and flu-treated mice
Project description:Afferent Lymph was collected from up to 5 pen raised, nematode naïve, outbreed Romney sheep (Tag No’s 1001, 1002, 1003, 1004, 1006) over a 12 week period. During this time a Pre-Infection sample was collected, the sheep were immunised by 3 truncated (2 week) T. colubiformis infections (40,000 Tc) followed by a Challenge Infection with 40,000 T. colubiformis L3 (Challenge Infection weeks 1-3). The Challenge Infection will enable us to determine changes in gene expression when the sheep gut rejects the parasitic nematodes. The overall design is summarised in the below table.
Project description:Hundreds of immune cell types work in coordination to maintain tissue homeostasis. Upon infection, dramatic changes occur with the localization, migration and proliferation of the immune cells to first alert the body of the danger, confine it to limit spreading, and finally extinguish the threat and bring the tissue back to homeostasis. Since current technologies can follow the dynamics of only a limited number of cell types, we have yet to grasp the full complexity of global in vivo cell dynamics in normal developmental processes and disease. Here we devise a computational method, digital cell quantification (DCQ), which combines genomewide gene expression data with an immune cell compendium to infer in vivo dynamical changes in the quantities of 213 immune cell subpopulations. DCQ was applied to study global immune cell dynamics in mice lungs at ten time points during a 7-day time course of flu infection. We find dramatic changes in quantities of 70 immune cell types, including various innate, adaptive and progenitor immune cells. We focus on the previously unreported dynamics of four immune dendritic cell subtypes, and suggest a specific role for CD103+CD11b- cDCs in early stages of disease and CD8+ pDC in late stages of flu infection. To better understand the physiological role of these differential dynamic changes in the DCs, we measured the genome-wide RNA expression of all four DC subpopulations from lung of influenza infected mice at four time points following infections (two mice per time-point). For sorting dendritic cells from lungs, the lungs from infected and control uninfected C57BL/6J mice were immersed in cold PBS, cut into small pieces in 5 ml DMEM media containing 10% Bovine Fetal Serum, the cell suspensions were grinded using 1ml syringe cup on a 70 μm cell strainers (BD Falcon). The cells were washed with ice cold PBS. Remaining red blood cells were lysed using ammonium chloride solution (Sigma). Cells were harvested, immersed 1ml FACS buffer [PBS+2% FBS, 1mM EDTA], Fc receptors were blocked with anti-mouse CD16/CD32, washed with FACS buffer and divided into two tubes for sorting cDC and pDC cells.
Project description:Afferent Lymph was collected over a 8 week period from up to 5 pen raised, nematode naïve, outbreed Romney sheep (Tag No’s 1001, 1002, 1003, 1004, 1006). During this time a Pre-Infection sample was collected, the sheep were immunised by 3 truncated (2 week) T. colubiformis infections (40,000 Tc L3, Immunising Infections 1-3). The treatment regime (shown below) will enable us to determine changes in gene expression during the development of immunity The overall design is summarised in the below table.
Project description:Pharmaceuticals are pseudo persistent aquatic pollutants with unknown effects at environmentally relevant concentrations. Atlantic salmon (Salmo salar) were exposed to Acetaminophen: 54.77 ± 34.67; Atenolol: 11.08 ± 7.98 and Carbamazepine: 7.85 ± 0.13 µg•L-1 for 5 days. After Acetaminophen treatment, 19 proteins were differently expressed, of which 11 were significant with respect to the control group (eight up-regulated and three down-regulated). After Atenolol treatment, 7 differently expressed proteins were obtained in comparison with the control, of which 6 could be identified (four up-regulated and 2 down-regulated). Carbamazepine exposure resulted in 15 differently expressed proteins compared with the control, with 10 of them identified (seven up-regulated and three down-regulated). Out of these, 3 features were common between Acetaminophen and Carbamazepine and one between Carbamazepine and Atenolol. One feature was common across all treatments. Principal component analysis and heat map clustering showed a clear grouping of the variability due to the applied treatments. The obtained data suggest (1) that exposure to environmentally relevant concentrations of the pharmaceuticals alters the hepatic protein expression profile of the Atlantic salmon; and (2) the existence of treatment specific processes that may be useful for biomarker development.
Project description:This experiment compared gene expression in the duodenum of [1] weaned genetically resistant sheep and weaned genetically susceptible sheep (84 days old) [2] genetically resistant sheep and genetically susceptible sheep that have been naturally challenged once with nematodes (175 days old) and [3] genetically resistant sheep and genetically susceptible sheep that have been naturally challenged twice with nematodes (276 days old). At each time point (T = 84, 175, 276 days) a four factorial design was used with four resistant animals and four susceptible animals. Each animal in the resistant group was compared to each animal in the susceptible group incorporating dye swaps. At T = 84 the platform GPL4072 was used. At T = 175 days the platform GPL4076 was used and at T =276 days the platform GPL4077 was used.
Project description:Comparison of genomic expression profiles in the mesenteric lymph nodes of naturally scrapie infected (clinical and preclinical stages) and control sheep. Expression profiles comparison of 7 clinical and 4 preclinical naturally scrapie infected sheep and 6 controls.