Illumina HiSeq 2500 sequencing; RNA-seq of effector (CD43+) CD8+ T cells, CD45+ hematopoietic cells and CD45- tumor/stromal cells from mice bearing bilateral AT-3 tumors following radio-immunotherapy of which one tumor was irradiated
RNA-seq of effector (CD43+) CD8+ T cells, CD45+ hematopoietic cells and CD45- tumor/stromal cells from mice bearing bilateral AT-3 tumors following radio-immunotherapy of which one tumor was irradiated
Using flow cytometry, CD43+ CD8+ T cells ('CTLs'), CD45+ hematopoietic cells, and CD45- (tumor/stromal) cells were isolated from both the irradiated and non-irradiated tumors of 9 mice per experimental group, and material from 3 mice was pooled per sample to retrieve sufficient RNA. Mice were injected with 0.5 x 106 AT-3 cells into this fat pad on one side and with 2.5 x 10^6 AT-3 cells on the contralateral flank. The latter tumor was irradiated, and the other tumor served as the non-irradiated 'abscopal' tumor. Tumor size was measured using a caliper, and treatment was initiated when the tumors reached 20-25mm2.  Therapy was done with n=9 mice per group. Radiotherapy (RT) was applied using an XRAD225-Cx system (Precision X-Ray, North Branford, CT). The tumor(s) were localized on the CT scan and targeted with RT at 0.1 mm precision using round collimators 1.0 or 1.5 cm in diameter. A single fraction of 10 Gy (225 peak kilovoltage (kVp), filtered with 0.3 mm of copper (3 Gy/min) was delivered. Immunomodulatory α-PD-1 and α-CD137 mAbs were diluted in PBS. The antibodies were administered twice weekly for 2 weeks either intraperitoneally (α-PD-1 mAb, 100 μg per injection), or intratumorally (α-CD137 mAb, 25 μg per injection), with the first dose delivered immediately after RT treatment. The flow cytometric-isolated cells were immediately collected in RLT lysis buffer (QIAGEN) and total RNA was extracted using the RNAeasy mini kit (QIAGEN) in accordance with the manufacturer's instructions. The integrity of the total RNA was assessed using a 2100 Bioanalyzer System (Agilent). Only RNA samples with an RNA Integrity Number (RIN) > 8 were used to create the library. Poly-A-selected RNA libraries were prepared using the Illumina TruSeq polyA stranded RNA kit.
Experimental Factor: cell type
hematopoietic cell
Experimental Factor: irradiate
x-ray
Experimental Factor: dose
10 Gy