GSE135377
Novel roles for GZMA and RASGRP1 in suppressing dissemination of both Theileria annulata-transformed macrophages and human B-lymphoma cells
Theileria annulata is a tick-transmitted apicomplexan parasite that infects and transforms bovine leukocytes into disseminating tumors that cause a disease called tropical theileriosis. Using comparative transcriptomics we identified genes transcriptionally perturbed during Theileria-induced transformation and highlighted a small set of genes associated with leukocyte dissemination. CRISPR/Cas9-mediated knock-down of GZMA and RASGRP1 in macrophages attenuated for dissemination led to a regain in their dissemination in Rag2/gC mice confirming their suppressor roles in vivo. Comparing the transcriptomes of 934 human cancer cell lines to that of Theileria-transformed bovine B cells again highlighted GZMA and RASGRP1 and CRISPR-mediated overexpression of GZMA and RASGRP1 dampened the dissemination potential of human B-lymphomas. The ensemble provide evidence for a novel suppressor function in the dissemination of both T. annulata-transformed bovine leukocytes and human B-lymphomas. Overall design: Bovine BL3, TBL3, BL20, TBL20  B lymphocytes, and Ode macrophages were cultured in RPMI 1640 medium supplemented with 2 mM of L-glutamine (Lonza, catalogue number 12-702F) and 10 mM Hepes (Lonza, catalogue number 17-737E), 10 % heat-inactivated FBS (Gibco, catalogue number 10082147), 100 units/ml of Penicillin and 100 µg/ml of streptomycin (Lonza, catalogue number 17-602E) and 10mM beta-mercaptoethanol (Sigma-Aldrich, catalogue number M6250) for BL3/TBL3 and BL20/TBL20. The virulent (Vir) hyper-disseminating Ode cell line was used at low passage (53-71), while its attenuated (Att) poorly disseminating vaccine counterpart corresponded to passages 309-317. The OCI-LY19 cell line (DSMZ, ACC 528) was cultured in Minimum Essential Medium Eagle - Alpha Modification (Gibco, catalog number 12000063) supplemented with 2.2g/L of sodium bicarbonate (Thermofisher Scientific, catalog number 25080094), 20% FBS, 10 mM Hepes and 100 units/ml of Penicillin and 100 µg/ml of streptomycin. The RI-1 cell line (DSMZ, ACC 585) was cultured in RPMI1640 and supplemented with 10% FBS, 100 units/ml of Penicillin and 100 µg/ml of streptomycin and 10 mM Hepes. All cell lines were incubated at 37°C with 5% CO2 and were regularly tested for mycoplasma contamination.  Cells were seeded in 3 biological replicates at a density of 2.5x105 cell/ml. RNA extraction was performed using the PureLink RNA Mini Kit (Life technologies, catalogue number 12183018A) following the manufacturer's instructions. Strand-specific RNA-sequencing (ssRNA-seq) libraries were prepared using the Illumina Truseq Stranded mRNA Sample Preparation Kit (Illumina, catalogue number RS-122-2101) following the manufacturer's instructions. Transcriptome data was analyzed further.