Baer Alan, Colon-Moran Winston, Xiang Jinhua, Stapleton Jack T, Bhattarai Nirjal
Division of Cellular and Gene Therapies, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland.
Research Service, Iowa City Veterans Affairs Medical Center, and the Department of Internal Medicine, University of Iowa, Iowa City, IA.
PLoS One. 2017 Oct 26;12(10):e0187123. doi: 10.1371/journal.pone.0187123. eCollection 2017.
T cell signaling is required for activation of both natural and therapeutic T cells including chimeric antigen receptor (CAR) T cells. Identification of novel factors and pathways regulating T cell signaling may aid in development of effective T cell therapies. In resting human T cells, the majority of Src-family of tyrosine kinases (SFKs) are inactive due to phosphorylation of a conserved carboxy-terminal tyrosine residue. Recently, a pool of enzymatically active SFKs has been identified in resting T cells; however, the significance of these is incompletely understood. Here, we characterized the role of active SFKs in resting human T cells. Pharmacologic inhibition of active SFKs enhanced distal TCR signaling as measured by IL-2 release and CD25 surface expression following TCR-independent activation. Mechanistically, inhibition of the active pool of SFKs induced nuclear translocation of NFAT1, and enhanced NFAT1-dependent signaling in resting T cells. The negative regulation of NFAT1 signaling was in part mediated by the Src-kinase Lck as human T cells lacking Lck had increased levels of nuclear NFAT1 and demonstrated enhanced NFAT1-dependent gene expression. Inhibition of active SFKs in resting primary human T cells also increased nuclear NFAT1 and enhanced NFAT1-dependent signaling. Finally, the calcineurin inhibitor FK506 and Cyclosporin A reversed the effect of SFKs inhibition on NFAT1. Together, these data identified a novel role of SFKs in preventing aberrant NFAT1 activation in resting T cells, and suggest that maintaining this pool of active SFKs in therapeutic T cells may increase the efficacy of T cell therapies.
天然T细胞和治疗性T细胞（包括嵌合抗原受体（CAR）T细胞）的激活都需要T细胞信号传导。鉴定调节T细胞信号传导的新因子和途径可能有助于开发有效的T细胞疗法。在静息的人类T细胞中，大多数酪氨酸激酶Src家族（SFKs）由于保守的羧基末端酪氨酸残基的磷酸化而处于无活性状态。最近，在静息T细胞中发现了一批具有酶活性的SFKs；然而，其意义尚未完全明确。在此，我们对静息人类T细胞中活性SFKs的作用进行了表征。通过TCR非依赖性激活后IL-2释放和CD25表面表达来衡量，活性SFKs的药理抑制增强了远端TCR信号传导。从机制上讲，抑制活性SFK池会诱导NFAT1的核转位，并增强静息T细胞中NFAT1依赖性信号传导。NFAT1信号传导的负调节部分由Src激酶Lck介导，因为缺乏Lck的人类T细胞中核NFAT1水平增加，并表现出增强的NFAT1依赖性基因表达。抑制静息原代人类T细胞中的活性SFKs也会增加核NFAT1并增强NFAT1依赖性信号传导。最后，钙调神经磷酸酶抑制剂FK5（06）和环孢素A逆转了SFKs抑制对NFAT1的影响。总之，这些数据确定了SFKs在防止静息T细胞中异常NFAT1激活方面的新作用，并表明在治疗性T细胞中维持这一活性SFK池可能会提高T细胞疗法的疗效。
