The exonuclease TREX1 constitutes an innate immune checkpoint limiting cGAS/STING-mediated anti- tumor immunity
The exonuclease TREX1 constitutes an innate immune checkpoint limiting cGAS/STING-mediated anti- tumor immunity
Tumors were collected 10 days after tumor inoculation for scRNA-seq. Tumors were weighed and homogenized using a tumor dissociation kit (Miltenyi, 130-096-730) with a GentleMACS (Miltenyi, program TDK1). Cell suspensions were incubated in Fc receptor block (BD Biosciences, 2.4G2, #553142, RRID:AB_394657) and fixable viability dye (Invitrogen, 65-0865-14) in PBS. Cells were labeled with surface antigens for 20-30min at 4°C. For transcription factor staining, cells were fixed with FoxP3 Fixation Buffer (005523, eBioscience) for 30 minutes in the dark at room temperature. For cytoplasmic protein labeling, cells were fixed with Fixation buffer (554714, BD Biosciences) for 20 min at 4°C. Then, cells were stained with intracellular antibodies for 30min at 4°C. The following antibodies were used: BST2 (clone 927, BioLegend, RRID:AB_2562477), CD4 (clone RM4-5, BD Biosciences, RRID:AB_2870166), CD8 (clone KT15, MBL, RRID:AB_10597265), CD11b (clone M1/70, BD Biosciences, RRID:AB_2874105), CD11c (clone HL3, BD Biosciences, RRID:AB_2738580), CD19 (clone 6D5, BioLegend, RRID:AB_11218994), CD44 (clone IM7, BD Biosciences, RRID:AB_2870671), CD45 (clone 30-F11, BD Biosciences, RRID:AB_2870247), CD49b (clone DX5, BD Biosciences, RRID:AB_11153857), CD90.2 (clone 30-H12, BioLegend, RRID:AB_493725), F4/80 (clone BM8, BioLegend, RRID:AB_2564588), GZMB (clone GB12, Invitrogen, RRID:AB_10373420), LY6C (clone HK1.4, BioLegend, RRID:AB_2562351), LY6G (clone 1A8, BioLegend, RRID:AB_2566317), MHC-ll (clone M5/114.15.2, BD Biosciences, RRID:AB_2738192), and SCA-1 (clone D7, BD Biosciences, RRID:AB_2871229). Data were acquired on a Symphony flow cytometer (BD Biosciences) or Cytek Aurora (Cytek Bioscience) and analyzed using FlowJo software (FlowJo LLC, RRID:SCR_008520).   For scRNA-seq, cells were incubated with Fc receptor block and labeled with TotalSeq-C hashtag antibodies (clone M1/42;30-F11, BioLegend, hashtag 1-12) together with the following surface markers: CD45 (clone 30-F11, BD Biosciences, RRID:AB_2870247), CD90.2 (clone 30-H12, BioLegend, RRID:AB_493725), CD31 (clone MEC13.3, BioLegend, RRID:AB_2572181), and PDGFRa (APA5, BioLegend, RRID:AB_2043969). Subsequently, cells were labeled with calcein blue (Invitrogen) and 7-AAD (BioLegend).  Samples were sorted into T cells (CD45+CD90.2+) and non-T immune cells (CD45+CD90.2- ) with a FACSAria instrument (BD Biosciences).  Same number of T cells and non-T immune cells from each animal per each population were pooled and duplicated for loading into a Chromium single cell sorting system (10X Genomics).   Using the 10x Genomics Chromium Single Cell 5′ V2 Reagent Kit and Chromium Chip K Single Cell Kit, 4 samples of sorted single cell suspension were loaded onto the Chromium Controller. Each sample was loaded into two channels on the chip, targeting for 30,000 cell recovery per channel, effectively recovering 60,000 cells per sample. Cells from all samples were over 90% viable, with concentrations ranging from 1430 to 1770 cells/mL. GEMs were generated and cells were lysed, mRNA were barcoded and underwent reverse transcription to produce full-length cDNA. cDNA was amplified with 13 PCR cycles. Two types of libraries were generated (Gene Expression and Cell Surface Protein) according to manufacturer instructions.