Angiotensin ii infusion caused expansion of activated (sic) natural killer t cells in mesenteric vessel perivascular adipose tissue of mice

Introduction: The innate-like γδ T cells play a role in angiotensin II (AngII)-induced hypertension, vascular injury and T cell activation in perivascular adipose tissue (PVAT). Hypothesis: We hypothesized that single cell RNA sequencing (scRNA-seq) will reveal γδ T cell subpopulations in PVAT involved in hypertension, vascular injury and T cell activation. Methods: Male C57BL/6J mice were infused SC or not with 490 ng/kg/min AngII for 14 days (n=3). Hypertension was confirmed by tail cuff blood pressure measurement. Mesenteric vessels (MV) with PVAT were collected, lymph nodes removed, single cell suspension obtained and labeled with hashtag antibodies, T cells isolated by fluorescence-activated cell sorting, the 6 samples pooled in one tube, and scRNA libraries prepared with a Chromium Next GEM Single Cell 3’ Reagent Kit. Sequencing was done on an Illumina Novaseq 6000, and data analysed using Cell Ranger pipeline and Seurat tools. Cell subpopulations were validated by flow cytometry. Results: ScRNA-seq yielded 5,030 cells with 137,990 reads/cell and identified 11 T cell clusters. Subclustering of T cells expressing the T cell receptor (TCR) δ constant chain revealed 3 δ natural killer T (δNKT) (δNKT0, δNKT1 and activated δNKT cells) and 3 γδ T cell subpopulations ( Trgc1 low effector memory, TCR δ variable 4 + and apoptotic γδ T cells). AngII increased >2-fold the frequency of activated δNKT cells, and decreased by 74% δNKT0 cells. Gene expression profiling revealed that activated δNKT and δNKT0 cells could be identified using unique markers, Cd28 and Sell , respectively. Flow cytometry showed that TCRδ + CD28 + Sell - T cells were increased (393±57.2 vs 227±44.4 cells/MV-PVAT) and TCRδ + CD28 - Sell + T cells decreased (21.6±4.1 vs 45.3±4.10 cells/MV-PVAT) in MV-PVAT of AngII vs sham-treated mice. Conclusion: This study identified an activated δNKT cell subpopulation in MV-PVAT that may play a role in AngII-induced hypertension, vascular injury and T cell activation.