Vg1+ gd T cells promote DC activation and CD8 T cell expansion via IL-4
Vg1+ gd T cells promote DC activation and CD8 T cell expansion via IL-4
Dendritic cells were isolated from spleens of B16.Flt3L tumour-bearing mice. Briefly, spleens were finely minced in 1mg/ml Collagenase Type 3, 20 µg/ml DNase I and continuously agitated for 15 minutes. DC-T cell complexes were disrupted with the addition of 0.1M EDTA (pH7.2). Undigested fragments were removed by filtering through a 70 µM mesh before light-density separation with 1.077g/cm3 Nycodenz medium via centrifugation at 1700rpm at 4°C for 12min. To increase the purity of cDC1 precursors, the light density fraction was collected, and cDC1s/pre-cDC1s were negatively enriched for through incubation with rat mAb against CD3ε (KT3), Thy1.1 (T24/31.7), Gr-1 (RB6-8C5), B220 (RA3-6B2), Ter-119 and CD11b (M1/70) prior to incubation with BioMag goat anti-rat IgG coupled magnetic beads (Qiagen) and magnetic separation. Target cells were characterised as CD11c+, MHC-IIint/+, CD11b−, CD24hi, CD8a+ with purities from 60-80%. CD11c+, MHC-IIint/+, CD11b−, CD24hi, B220−, CD172− cells were then sorted using a BD FACSAriaTM III Cell Sorter into filtered RPMI supplemented with 50% heat-inactivated FCS and pelleted. In a 24-well plate, 5 x 105 cells were plated in complete Kenneth D Shortman RPMI (KDS-RPMI 1640, 10% FCS, 2mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 50 mM 2-mercaptoethanol). To assess cDC1 gene expression in response to IL-4, cells were incubated in the presence of -CD40 (10g/ml), IL-4 (60ng/mL), IFN- (20ng/mL), or LPS (1g/ml). Plates were then incubated at 37°C and 5% CO2 for 4 hours. For RNA isolation, cells were washed and resuspended using TRIzol (Life Technologies), snap frozen and stored at -80°C. See treatment CD11c+, MHC-IIint/+, CD11b−, CD24hi, B220−, CD172a− cells were then sorted using a BD FACSAriaTM III Cell Sorter into filtered RPMI supplemented with 50% heat-inactivated FCS and pelleted. In a 24-well plate, 5 x 105 cells were plated in complete Kenneth D Shortman RPMI (KDS-RPMI 1640, 10% FCS, 2mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 50 mM 2-mercaptoethanol). To assess cDC1 gene expression in response to IL-4, cells were incubated in the presence of a-CD40 (10mg/ml), IL-4 (60ng/mL), IFN-g (20ng/mL), or LPS (1mg/ml). Plates were then incubated at 37°C and 5% CO2 for 4 hours. For RNA isolation, cells were washed and resuspended using TRIzol (Life Technologies), snap frozen and stored at -80°C. To determine gene expression in cDC1s, mRNA was extracted using a Direct-zol RNA MicroPrep kit (Zymo Research) as per manufacturer's protocol.  Libraries were prepared using Illumina stranded mRNA library kits and the outputs sequenced to a depth of 20M reads per sample on a 150PE on Illumina NovaSeq X Plus 10B flow cell. Library preparation and sequencing was conducted at the Australian Genome Research Facility.