Author(s): ROSS, M., NIEMIRO, G., SMITH, K., BAKER, F., ZUNIGA, T., DIAK, D., SECKELER, M., KATSANIS, E., SIMPSON, R. , Institution: HERIOT-WATT UNIVERSITY, Country: UNITED KINGDOM, Abstract-ID: 2219

INTRODUCTION:
CD31+ T-cells are a highly vasculogenic T-cell phenotype and have demonstrated potent angiogenic capabilities, such as containing elevated VEGF-A content, promoting blood flow recovery in a hindlimb ischaemia mouse model and being essential for optimal endothelial progenitor cell growth. One mechanism by which exercise may promote tissue angiogenesis and vascular repair may be through promoting angiogenic T-cell (TANG) mobilisation, resulting in exposure of these TANG cells to the endothelium. These cells are known to ingress into the circulation in response to exercise, but new technologies, such as single cell sequencing, can provide some insight into transcriptomic changes, which may underpin any upregulation of angiogenic functions.
METHODS:
Ten healthy, non-smoking, physically active participants (26 ± 5 years, 171.2 ± 12.3cm, 66.9 ± 11.9kg) underwent a 30-minute cycling exercise bout at an intensity 15% above the pre-determined lactate threshold. Peripheral blood samples were taken by cannulation before exercise (PRE) and in the final minute prior to the completion of the exercise bout (POST). Peripheral blood mononuclear cells (PBMC) were isolated from whole blood using density gradient centrifugation and T-cells quantified using flow cytometry.
CD31+ T-cells underwent single cell sequencing using 10X Genomics platform. 5’ RNA whole transcriptome libraries were generated using the 10xGenomics Chromium Next GEM Single Cell 5’ reagents kit, following recommended guidelines. The gene expression libraries were quantified, normalized, pooled, and sequenced on an Illumina NextSeq500 sequencer. Differentially expressed genes were detected, with a log2 fold cutoff of 0. Gene set enrichment analysis was performed and annotated to both KEGG and GO terms.
T-cell number changes with exercise were analysed using paired T-tests. A p-value of <0.05 was deemed statistically significant.
RESULTS:
Exercise resulted in a significant ingress of CD3+CD31+ (PRE: 268±176 cells·μL-1; POST: 437±345 cells·μL-1, p=0.027, t=2.631), driven by the changes in CD3+CD8+CD31+ (PRE: 130±73 cells·μL-1; POST: 221±165 cells·μL-1, p=0.024, t=2.708) subset, with no changes evident in the CD3+CD4+CD31+ (PRE: 123±115 cells·μL-1; POST: 186±197 cells·μL-1, p=0.091, t=1.896) T-cells. Whilst CD31+ T-cells demonstrate significantly greater expression of angiogenic genes (VEGFA, PIK3CB, MMP-9, THBS1) the acute bout did not stimulate changes in these pathways but did result in several transcriptomic changes in these CD31+ T-cells, with ribosomal biogenesis genes and pathways largely downregulated, and lymphocyte-mediated immune functional pathways upregulated.
CONCLUSION:
CD31+ T-cells exhibit an angiogenic profile, and acute exercise non-preferentially mobilises CD31+ T-cells, which may expose the vasculature to their angiogenic supporting processes, but transcriptomic changes are largely immune-, rather than angiogenesis-related.