Human lymphocytes mobilized with exercise have an anti-tumor transcriptomic profile and exert enhanced graft-versus-leukemia effects in xenogeneic mice

Abstract

BackgroundEvery bout of exercise mobilizes and redistributes large numbers of effector lymphocytes with a cytotoxic and tissue migration phenotype. The frequent redistribution of these cells is purported to increase immune surveillance and play a mechanistic role in reducing cancer risk and slowing tumor progression in physically active cancer survivors. Our aim was to provide the first detailed single cell transcriptomic analysis of exercise-mobilized lymphocytes and test their effectiveness as a donor lymphocyte infusion (DLI) in xenogeneic mice engrafted with human leukemia.MethodsPeripheral blood mononuclear cells (PBMCs) were collected from healthy volunteers at rest and at the end of an acute bout of cycling exercise. Flow cytometry and single-cell RNA sequencing was performed to identify phenotypic and transcriptomic differences between resting and exercise-mobilized cells using a targeted gene expression panel curated for human immunology. PBMCs were injected into the tail vein of xenogeneic NSG-IL-15 mice and subsequently challenged with a luciferase tagged chronic myelogenous leukemia cell line (K562). Tumor growth (bioluminescence) and xenogeneic graft-versus-host disease (GvHD) were monitored bi-weekly for 40-days.ResultsExercise preferentially mobilized NK-cell, CD8+ T-cell and monocyte subtypes with a differentiated and effector phenotype, without significantly mobilizing CD4+ regulatory T-cells. Mobilized effector lymphocytes, particularly effector-memory CD8+ T-cells and NK-cells, displayed differentially expressed genes and enriched gene sets associated with anti-tumor activity, including cytotoxicity, migration/chemotaxis, antigen binding, cytokine responsiveness and alloreactivity (e.g. graft-versus-host/leukemia). Mice receiving exercise-mobilized PBMCs had lower tumor burden and higher overall survival (4.14E+08 photons/s and 47%, respectively) at day 40 compared to mice receiving resting PBMCs (12.1E+08 photons/s and 22%, respectively) from the same donors (p<0.05). Human immune cell engraftment was similar for resting and exercise-mobilized DLI. However, when compared to non-tumor bearing mice, K562 increased the expansion of NK-cell and CD3+/CD4-/CD8- T-cells in mice receiving exercise-mobilized but not resting lymphocytes, 1-2 weeks after DLI. No differences in GvHD or GvHD-free survival was observed between groups either with or without K562 challenge.ConclusionExercise in humans mobilizes effector lymphocytes with an anti-tumor transcriptomic profile and their use as DLI extends survival and enhances the graft-versus-leukemia (GvL) effect without exacerbating GvHD in human leukemia bearing xenogeneic mice. Exercise may serve as an effective and economical adjuvant to increase the GvL effects of allogeneic cell therapies without intensifying GvHD.

Exercise-induced β2-adrenergic Receptor Activation Enhances the Antileukemic Activity of Expanded γδ T-Cells via DNAM-1 Upregulation and PVR/Nectin-2 Recognition

“…Recent work has shown that these mobilized cells subsequently infiltrate tumors and are essential in constraining tumor growth in several murine cancer models ( 42 ). We recently introduced the innovative concept that lymphocytes mobilized by exercise enhance the graft-versus-leukemia (GvL) effects of donor lymphocyte infusions ( 35 ). In addition, an exercise session can improve the ex vivo production and effectiveness of virus-specific T-cells, γδ T-cells, and tumor antigen-specific T-cells ( 43–45 ).…”

“…Mycoplasma testing was routinely performed but we did not authenticate the cell line. K562-luc2 cells were maintained at 37°C, 5% CO 2 in Iscove's DMEM supplemented with 10% FBS and 8 µg/mL blasticidin and prepared for tail vein injection as described previously ( 35 ). Mice were irradiated with 100 cGy, using a Cesium 137 irradiator, one day (day −2) before intravenous injection of expanded Vγ9Vδ2+ T-cells on day −1, mice not receiving expanded γδ T-cells (tumor-only) were injected with an equal volume of saline on the same day.…”

“…PBMCs or expanded Vγ9Vδ2+ T-cells (>95% purity) were thawed and resuspended in PBS and RNAlater solution and processed by the University of Arizona Genetics Core for scRNA-seq analysis using the 10x Genomics platform and bulk RNA-seq using the Illumina NextSeq500 platform. The scRNA-seq library generation, sequencing, and analysis were performed as described previously ( 35, 36 ). For bulk RNA-seq, RNA samples’ quality was assessed with a high sensitivity RNA Fragment Analyzer Kit (Advanced Analytics) and quantity with an RNA HS assay kit (Qubit).…”

Anticancer effects of exercise: Insights from single-cell analysis

“… 7 Additionally, 3 genes ( PMEL, TYRP1 , and EDNRB ) overexpressed in the exhausted subpopulations were associated with poor prognosis. 7 It is conceivable that the information provided by scRNA-seq could also help to characterize the molecular mechanisms underlying the effects of physical exercise on anticancer immune function as well as on the complex tumor microenvironment, as recently illustrated by 2 relevant studies 8 , 9 ( Fig. 1 ).…”

“…1 Main findings from single-cell analysis. (A) Acute and regular exercise improve cancer immunosurveillance; (B) Findings from Savage et al 8 ; (C) Findings from Batatinha et al 9 This figure was created using BioRender (https://biorender.com/). APC = antigen presenting cells; IFN = interferon; MHC = major histocompatibility complex; OXPHOS = oxidative phosphorylation; PBMCs = peripheral blood mononuclear cells; scRNA-seq = single-cell RNA sequencing; TAM = tumor-associated macrophages.…”