Single-cell RNA-seq uncovers unique gene expression profiles in the spleen of hypoxic mice

Using a mouse model (Foxp3tm9(EGFP/cre/ERT2)Ayr/J x Ai14-tdTomato) that allows the study of lineage stability and genetic mapping of T regulatory cells (Tregs), together with single-cell RNA sequencing, we aimed to characterize how hypoxia impacts the transcriptome of Tregs and, in addition, all cell populations present in the spleen. In these mice, Foxp3, the signature transcription factor of Tregs, drives the expression of tamoxifen-inducible Cre, leading to the expression of tdTomato marking cells on the Treg lineage. The tdTomato mRNA contains a WPRE sequence, which was used to identify the cell clusters with potential enrichment of Tregs. Hypoxia resulted in a gene expression profile consistent with exTregs or Tregs with less suppressive activity, such as downregulation of Il2ra and upregulation of Tnfrsf4 within CD4 memory T cell cluster.Four six-weeks-old male Foxp3EGFP-cre-ERT2 (Strain #016961) crossed with Ai14- tdTomato (Strain #:007914) reporter mice received 75 mg tamoxifen/g body wt/day for 5 days. After two weeks of recovery, two mice were exposed to hypobaric hypoxia (380 mmHg) and two were housed at ambient barometric pressure (normoxia, ~630 mmHg, Albuquerque, NM) for 5 days. Then mice were anesthetized with isoflurane/O2 mix, injected with heparin (50 U), spleen harvested, and mice euthanized by removing lungs and heart. A suspension of splenocytes was generated by mincing the spleens and disaggregating them through a 70 um mesh filter. Red blood cells were lysed before centrifugation at 300 RCF for 5 minutes. The cell pellet was rewashed with 20 mL DPBS. Cells were centrifuged again at 300 RCF for 5 minutes before the resuspension of the cell pellet in 10 mL DPBS for counting and viability assessment. The Invitrogen Countess II automated cell counter was used to determine cell counting and viability. The chamber slide was loaded with 10 uL of 1:1 cell suspension in 0.4% trypan blue stain according to Countess II specifications. After cell count was determined, a 5 mL aliquot of each sample was made at a concentration of 2*105 viable cells/mL in DPBS according to 10x Genomics Single Cell Seq specifications for optimal input concentration of 200 viable cells/uL.