Acute exercise mobilizes NKT-like cells with a cytotoxic transcriptomic profile but does not augment the potency of cytokine-induced killer (CIK) cells

CD3+/CD56+ Natural killer (NK) cell-like T-cells (NKT-like cells) represent <5% of blood lymphocytes, display a cytotoxic phenotype, and can kill various cancers. NKT-like cells can be expanded ex vivo into cytokine-induced killer (CIK) cells, however this therapeutic cell product has had mixed results against hematological malignancies in clinical trials. The aim of this study was to determine if NKT-like cells mobilized during acute cycling exercise could be used to generate more potent anti-tumor CIK cells from healthy donors. An acute exercise bout increased NKT-like cell numbers in blood 2-fold. Single cell RNA sequencing revealed that exercise mobilized NKT-like cells have an upregulation of genes and transcriptomic programs associated with enhanced anti-tumor activity, including cytotoxicity, cytokine responsiveness, and migration. Exercise, however, did not augment the ex vivo expansion of CIK cells or alter their surface phenotypes after 21-days of culture. CIK cells expanded at rest, during exercise (at 60% and 80% VO2max) or after (1h post) were equally capable of killing leukemia, lymphoma, and multiple myeloma target cells with and without cytokine (IL-2) and antibody (OKT3) priming in vitro. We conclude that acute exercise in healthy donors mobilizes NKT-like cells with an upregulation of transcriptomic programs involved in anti-tumor activity, but does not augment the ex vivo expansion of CIK cells. Blood samples were collected from 3 healthy volunteers (age 27-41 yrs) at rest, during graded exercise at 80% VO2max, and at 1hr post exercise (+1H). Isolated PBMCs underwent single cell RNA sequencing (scRNAseq) and gene expression analysis was performed on NKT-like cell clusters from resting samples

CD3+/CD56+ 自然杀伤（Natural Killer, NK）细胞样T细胞（NKT-like cells）在血液淋巴细胞中占比不足5%，具有细胞毒性表型，可杀伤多种恶性肿瘤细胞。NKT样细胞可在体外扩增为细胞因子诱导的杀伤（cytokine-induced killer, CIK）细胞，然而该治疗性细胞产品在针对血液系统恶性肿瘤的临床试验中疗效参差不齐。本研究旨在探究急性骑行运动期间动员的NKT样细胞，是否可用于从健康供体中制备抗肿瘤活性更强的CIK细胞。一次急性骑行运动可使血液中的NKT样细胞数量增至原水平的2倍。单细胞RNA测序（single cell RNA sequencing, scRNAseq）结果显示，运动动员的NKT样细胞中，与增强抗肿瘤活性相关的基因及转录组程序均呈上调表达，包括细胞毒性、细胞因子应答能力及迁移相关程序。然而，运动并未增强CIK细胞的体外扩增能力，也未在21天培养后改变其表面表型。在静息状态、运动期间（分别达到最大摄氧量（VO2max）的60%和80%）或运动后1小时采集的样本中，经体外培养扩增的CIK细胞，无论是否经过细胞因子（白细胞介素-2, interleukin-2, IL-2）与抗体OKT3预刺激，均能同等有效地杀伤白血病、淋巴瘤及多发性骨髓瘤靶细胞。本研究结论为：健康供体接受急性骑行运动可募集到抗肿瘤活性相关转录组程序表达上调的NKT样细胞，但无法增强CIK细胞的体外扩增能力。本研究从3名年龄介于27~41岁的健康志愿者体内，分别采集了静息状态、分级运动至最大摄氧量（VO2max）的80%时以及运动后1小时（+1H）的血液样本。分离得到的外周血单个核细胞（peripheral blood mononuclear cell, PBMC）接受了单细胞RNA测序（scRNAseq），并对静息样本中的NKT样细胞簇进行了基因表达分析。