title: GSM8654927: Spleen_Eff_CD4_Env126, replicate 1, scRNASeq; Mus musculus; RNA-Seq
library_name: GSM8654927
library_construction_protocol: In selected experiments, WT C57BL/6 mice were immunized subcutaneously with 100μg of Env126-138 peptide and 50μg of polyI:C immune adjuvant at three time points every 7 days. Isolation of CD8+ T cells from spleens of Cor93 or Env28 mice and CD4+ T cells from spleens of Env126 transgenic mice have been performed as previously described6. Isolation of CD4+ T cells from spleens of Env126 transgenic mice was performed by negative immunomagnetic selection taking advantage of the EasySepTM Mouse CD4+ T Cell Isolation Kit (StemCell Technologies). Mice were injected i.v. with 106 HBV-specific (Cor93 or Env28) naïve CD8+ T cells and/or HBV-specific effector CD4+ T cells (Env126 TEFF). In indicated experiments, mice were splenectomized and treated i.v. with 200 μg of anti-CD62L mAb (clone MEL-14, BioXcell) 48 and 4 hours prior to cell injection and 24 hours after cell transfer. Splenectomy was performed according to standard procedures72 and as previously described 6. In selected experiments, mice were treated intraperitoneally (i.p.) with Busulfan and reconstituted i.v. with bone marrow from CD11c-iDTR-GFP mice (107). In selected experiments, dendritic cells and Kupffer cells were depleted by injecting i.p. 25 ng/g of diphtheria toxin (Sigma-Aldrich) 3 days and 1 day prior to T cell transfer and 1 day and 3 days after cell transfer. In indicated experiments, Kupffer cells were depleted by i.v. injection of clodronate-containing liposomes (10 μL/g) (Liposoma B'V) 2 days prior to T cell injection73. In indicated experiments, mice were treated i.p. with 250 μg of anti-CD40L mAb (clone MR-1), 250 μg of anti-IL12 p40 mAb (clone C17.8), 250 μg of anti-IL27 p28 mAb (clone MM27.7B1) and 2,5 μg of recombinant IL27 (Fc: LALA-PG-KIH heterodimer, Adipogen). All mAbs were purchased from BioXcell, unless otherwise indicated. Env126 CD4+ TEFF and Env126 CD4+ TN cells were fixed according to the Evercode Fixation (Parse Biosciences) protocol and single-cell RNA libraries were prepared using the Evercode split-pool combinatorial barcoding technology approach using the ParseBio Evercode WT v2 kit. Sequencing was performed on the Illumina NovaSeq 6000 sequencer with the S4 flow cell 200 cycle kit according to the sequencing protocol provided by the Evercode kit (v1.5) at a desired depth of 30,000 reads/cell.