IL-2 is inactivated by the acidic pH environment of tumors enabling engineering of a pH-selective mutein (RNA-Seq). IL-2 is inactivated by the acidic pH environment of tumors enabling engineering of a pH-selective mutein (RNA-Seq)

Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin (IL)-2, a critical cytokine in T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation and anti-tumor immunity by CD8+ T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Ra with higher affinity, triggers STAT5 activation and drives CD8+ T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Finally, we find that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues. Overall design: Activated human CD8+ T cells were rested O/N, transferred in complete media pH 7.5 or 6.5 and stimulated for 4 h with 10 nM IL-2 or Switch-2

细胞因子（cytokine）在细胞外空间与受体结合，以调控免疫应答。目前对于细胞外空间的理化特性如何影响细胞因子信号转导，尚未完全阐明。
本研究显示，作为T细胞免疫中的关键细胞因子，白细胞介素-2（interleukin-2，IL-2）的活性受pH值的显著调控，其信号转导在肿瘤的酸性微环境中受到抑制。肿瘤产生的乳酸会抑制信号转导与转录激活因子5（signal transducer and activator of transcription 5，STAT5）的激活、效应细胞分化以及CD8阳性T细胞的抗肿瘤免疫功能，同时使得高剂量IL-2疗法的疗效大打折扣。
通过定向进化技术，我们成功筛选出一种pH选择性的IL-2突变蛋白（Switch-2）。Switch-2与IL-2受体α亚基（interleukin-2 receptor alpha subunit，IL-2Rα）的结合亲和力更高；相较于中性pH环境，其在酸性pH条件下更能强效激活STAT5信号通路并诱导CD8阳性T细胞的效应功能。因此，高剂量Switch-2疗法可诱导强烈的免疫激活与肿瘤排斥反应，同时降低正常组织中的靶标毒性。
最后我们发现，对pH的敏感性是多种细胞因子共有的普遍特性，这一特性在健康与病变组织中均与免疫及免疫治疗密切相关。
整体实验设计：将活化的人CD8阳性T细胞静置过夜后，转移至pH为7.5或6.5的完全培养基中，并用10 nM的IL-2或Switch-2刺激4小时。