Unknown,Transcriptomics,Genomics,Proteomics

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BCR repertoire analysis by Next-generation sequencing of lymphocytes harvested from mice vaccinated with liposomal TLR4 and TLR7 agonists

ABSTRACT: BALB/c mice were vaccinated with inactivated influenza A virus adjuvanted with liposomal TLR4 (2B182C) and TLR7 agonists (2B182C) on days 0 and 21. To examine whether the liposomal combined adjuvant increase BCR diversity, lymphocytes in the draining lymph nodes harvested on day 28 were analyzed using next-generation RNA sequencing technology.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Mus musculus

SUBMITTER: Tomoko Hayashi

PROVIDER: E-MTAB-8870 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Many modern vaccines use defined adjuvants to stimulate the innate immune system and shape the adaptive immune response. The exact nature of these innate signals and whether immune differentiation can originate within the periphery is not known. In the present study we used an ovine lymphatic cannulation model to characterise the cellular and transcriptomic profile of the afferent lymph following injection of a liposomal vaccine formulation incorporating diphtheria toxoid and the innate stimulator poly (I:C) over a 78 h period. The response to this vaccine featured an early activation of broad proinflammatory pathways (e.g. TLR signalling and inflammasome pathways) and the transient recruitment of granulocytes into the lymph. At 24hrs a more monocytic cellular profile arose coinciding with a transition to a specific antiviral response characterised by the up-regulation of genes associated with the receptors typical for the viral mimic, poly (I:C) (e.g. TLR3, RIG-I and MDA5). At the latest time points the up-regulation of IL-17A and IL-17F suggested that Th17 cells may participate in the earliest adaptive response to this vaccine. Together these data provide the most comprehensive picture of the cellular and molecular mechanisms that link the periphery to the draining lymph node following vaccination and indicate that the immune response is capable of specialising within the periphery. This study employed an ovine model of pseudoafferent lymphatic cannulation (see de Veer et al. 2010, Vaccine) to characterise the innate immune response within the afferent lymph to vaccination with liposomes+poly (I:C)+ diptheria toxoid. The sheep used in this study had both their pre-femoral lymph nodes removed at 1 year of age. Approximately 1 year after the lymph node removal, a second surgery was performed to insert a 0.96_0.58mm heparin-coated polyvinyl chloride cannula into the pseudoafferent (previous efferent) lymphatic duct of both sides. At least seven days were allowed for healing to occur after surgery before vaccinations were administered and experimental lymph samples were collected. Handling of animals and experimental procedures were all approved by the Monash University Animal Ethics Committee in accordance with the relevant licensing agreement. Vaccinations were injected subcutaneously in the area drained by the prefemoral lymph node. Afferent lymphatic samples were collected prior to vaccination as a control (PRE) and at 4-6, 26-28, 51-53 and 76-78 hours post vaccination with liposomes+poly (I:C)+ diptheria toxoid. Lymphocytes were removed from the afferent lymph using immunomagnetic separation. Next-generation sequencing was performed on RNA derived from the remaining innate immune cells. Samples from three sheep were used at all time points except 4-6 and 26-28 hours, where only two samples yielded sufficient RNA for sequencing.

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BCR repertoire analysis by Next-generation sequencing of lymphocytes harvested from mice vaccinated with liposomal TLR4 and TLR7 agonists

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