It is noteworthy to highlight that some of the immune response mechanisms that we found overrepresented in the present work have been previously identified in rainbow trout RBCs transfected with GVHSV in vitro. On the other hand, GVHSV-transfected RBCs induce specific antibodies against VHSV in the RG7800 serum of rainbow trout which shows that RBCs expressing a DNA vaccine are able to elicit a humoral response. These results open a new direction in the research of vaccination strategies for fish since rainbow trout RBCs actively participate in the innate and adaptive immune response in DNA vaccination. Based on our findings, we suggest the use of RBCs as target cells or service providers for the future design of novel vaccine strategies. gene transcripts expression monitorization in blood and head kidney (data not shown) and in the bibliography [3,28]. For the GVHSV-transfected RBC reinfusion assay, adult rainbow trout (20C25 cm) were anesthetized with 40 mg/L tricaine and reinfused intravenously (iv) with previously extracted autologous peripheral blood RBCs (PB-RBCs) (15 106 cells) that were GVHSV-transfected in vitro as previously explained [14]. For the in vitro transfection of RBCs, Ficoll-purified PB-RBCs were transfected by electroporation with 4 g of GVHSV plasmid per 106 cells using the Neon? Transfection System (Life Technologies, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fish were immunized with im or iv injection of 4 g GVHSV for immunization controls. At 30 dpi, blood was drawn from your caudal vein and left immediately at 4C to separate the serum from your cell pellet. 2.4. Purification of Head Kidney and Peripheral Blood RBCs for Transcriptome Analysis, by Means of FACS Single-Cell Sorting Head kidney and peripheral blood from individuals immunized with GVHSV or TFP1 were extracted at 14 dpi, and sampled in RPMI-1640 medium (Dutch modification) (Gibco, Thermo Fischer Scientific Inc., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) gamma irradiated (Cultek, Madrid, Spain), 1 mM pyruvate (Gibco), 2 mM L-glutamine (Gibco), 50 g/mL gentamicin (Gibco), 2 g/mL fungizone (Gibco), 100 U/mL penicillin (Sigma-Aldrich), and 100 g/mL RG7800 streptomycin (Sigma-Aldrich). Head kidney tissue was disaggregated with a Pasteur pipette and exceeded through a 40 m Falcon nylon cell strainer (BD Biosciencies, Madrid, Spain) using a plunger of a 1 mL syringe. Then, cells from head kidney and peripheral blood were stained with 500 nM SYTO RNA Select (Molecular Probes, Thermo Fisher Scientific, Inc.) for 20 min at room temperature as recommended by the manufacturer. Head kidney RBCs (HK-RBCs) and peripheral blood RBCs (PB-RBCs) were FACS Rabbit polyclonal to dr5 single-cell sorted using a BD FACSJazz? cell sorter (BD Biosciences, Madrid, Spain) using SYTO RNA Select staining, which staining RNA and allows to sort populations based on cell RNA quantity. RBCs populace for sorting was selected based on SYTO RNA select staining (FITC) and side scatter light (SSC), RG7800 as indicated in Supplementary Physique S1, using the mask 1.0 drop single. Then, samples were visualized in the IN Cell Analyzer 6000 Cell Imaging system (GE Healthcare, Little Chalfont, UK) to verify a purity of 99.99% (Supplementary Figure S1). 2.5. Transcriptome Analysis of RBCs Thirty-two individuals (16 for TFP1 injection and 16 for GVHSV injection) were immunized as explained above. At 14 dpi, approximately 102 HK-RBCs and 106 PB-RBCs of each individual were sorted as explained in the previous section. Each sample was resuspended in lysis buffer (Clontech, Takara Bio, Mountain View, CA, USA) and RNase RG7800 Inhibitor (Invitrogen, ThermoFisher Scientific Inc.) as indicated in Reference [14] and then grouped in 2 pools of 8 individuals for each condition (TFP1 or GVHSV) and tissue (HK-RBCs or PB-RBCs) (Physique 1). Samples were preserved at ?80 C until cDNA library construction. cDNA was directly produced from pooled lysed cells using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech, Takara Bio). RNA-Seq library preparation and sequencing were carried out by STABVida Lda (Caparica, Portugal) as previously explained [29]. Sequence reads are available at SRA-NCBI, SRA-NCBI Accession SRP133501. Open in a separate window Physique 1 The general workflow of experimental actions RG7800 from sample collection, 14 dpi, to data analysis. 2.6. Proteome Analysis of RBCs Thirty-two individuals (16 for TFP1 injection and 16 for GVHSV injection) were immunized as explained above. Peripheral blood was extracted at 14 dpi, and PB-RBCs were purified by 2 density gradient centrifugations (1600 rpm, Ficoll 1.007; Lymphoprep, Reactiva, Sigma-Aldrich) as previously explained [9]. The 99.9% purity of RBCs was estimated by optical microscopy (Supplementary.
It is noteworthy to highlight that some of the immune response mechanisms that we found overrepresented in the present work have been previously identified in rainbow trout RBCs transfected with GVHSV in vitro