NextSeq 500 sequencing; Interferon gene therapy reprograms the leukemia microenvironment inducing protective immunity to multiple tumor antigens
Interferon gene therapy reprograms the leukemia microenvironment inducing protective immunity to multiple tumor antigens
C57BL/6 female mice (8 weeks old) were irradiated and transplanted with mouse HSPC transduced with a control mTie2-GFP-mirT LV. At 20 weeks of age mice were sacrificed. Spleen tissue was dissociated and cells subjected to FACS sorting. Cd11b+ cells were recovered.  IFN treatment was performed by reconstituting lethally irradiated mice with hematopoietic stem and progenitor cells (HSPCs) that were previously transduced with a lentiviral vector (LV) containing the cDNA sequence of the IFN-alpha gene under the control of the Tie2 promoter/enhancer sequences. Among differentiated hematopoietic cells, Tie2 is selectively expressed in a population of tumor-infiltrating monocytes (TEMs), thus resulting in tumor-specific IFN-alpha delivery. The LV design also incorporates into the IFN-alpha transgene multiple miR-126 and miR-130a target sequences to suppress post-transcriptionally transgene expression in HSPCs limiting potential transgene in this compartment. C57BL/6 female mice (8 weeks old) were irradiated and transplanted with mouse HSPC transduced with a mTie2-IFN-mirT LV. At 20 weeks of age mice were sacrificed. Spleen tissue was dissociated and cells subjected to FACS sorting. Cd11b+ cells were recovered. C57BL/6 female mice (8 weeks old) were irradiated and transplanted with mouse HSPC transduced with a control mTie2-GFP-mirT LV. At 16 weeks of age mice were injected intravenously with 10-100 thousand B-ALL cells. At 20 weeks of age mice were sacrificed. Spleen tissue was dissociated and cells subjected to FACS sorting. Cd11b+ cells were recovered. Droplet-based digital 3′ end scRNA-Seq was performed on a Chromium Single-Cell  Controller (10X Genomics, Pleasanton, CA) using the Chromium Single Cell 3′ Reagent Kit  v2 according to the manufacturer's instructions. Briefly, suspended single cells were  partitioned in Gel Beads in Emulsion (GEMs) and lysed, followed by RNA barcoding, reverse  transcription and PCR amplification (12-14 cycles). Sequencing-ready scRNA-Seq libraries were  prepared according to the manufacturer's instructions, checked and quantified on 2100  Bioanalyzer (Agilent Genomics, Santa Clara, CA) and Qubit 3.0 (Invitrogen, Carlsbad, CA)  instruments.