Ionic immune suppression within the tumour microenvironment limits T cell effector function. Mus musculus

Tumours progress despite being infiltrated by effector T cells. Tumour necrosis is associated with poor survival in a variety of cancers. Here, we report that that necrosis causes release of an intracellular ion, potassium, into the extracellular fluid of human and mouse tumours. Surprisingly, elevated extracellular potassium ([K+]e) was sufficient to profoundly suppress mouse and human T cell anti-tumour function. Elevations in [K+]e acted to acutely impair T cell receptor (TCR) dependent Akt-mTOR phosphorylation and effector function. Potassium mediated suppression of Akt-mTOR signalling and T cell effector function required intact activity of PP2A, a serine/threonine phosphatase. The suppressive effect mediated by elevated [K+]e required a T cell-intrinsic increase in intracellular potassium ([K+]i) and was independent of changes in plasma membrane potential (Vm). Finally, ionic reprogramming of tumour-specific T cells via over-expression of the voltage-gated potassium channel Kv1.3 lowered [K+]i and improved effector functions in vitro and in vivo, with this gain of function being dependent on intact channel function. Consequently, Kv1.3 T cell expression enhanced tumour clearance and the survival of melanoma-bearing mice. These results uncover a previously undescribed ionic checkpoint against T cell function within tumours and identify new strategies for cancer immunotherapy. Overall design: RNA expression was measured by RNA-Seq on day 5 of cultures, maintained in individual biologial triplicates which were stimulated with immobilized anti-CD3/28 antibodies or kept in complete media (no stim) - with equivalent conditions treated with isotonic media containing elevated potassium.

尽管效应T细胞（effector T cell）浸润肿瘤，肿瘤仍可发生进展。肿瘤坏死在多种癌症中均与不良预后密切相关。本研究发现，肿瘤坏死会导致细胞内离子钾离子释放进入人类和小鼠肿瘤的细胞外液。令人意外的是，升高的细胞外钾离子浓度（[K+]e）足以显著抑制小鼠与人类T细胞的抗肿瘤功能。[K+]e升高可急性损伤T细胞受体（T cell receptor, TCR）依赖的Akt-mTOR磷酸化过程及T细胞效应功能。钾离子介导的Akt-mTOR信号通路抑制与T细胞效应功能受损，均依赖于丝氨酸/苏氨酸磷酸酶PP2A的完整活性。升高的[K+]e所介导的抑制作用，需要T细胞内源性的细胞内钾离子浓度（[K+]i）升高，且不依赖于细胞膜电位（Vm）的变化。最后，通过过表达电压门控钾离子通道Kv1.3对肿瘤特异性T细胞进行离子重编程，可降低[K+]i并在体内外改善T细胞的效应功能，这一功能获得效应依赖于该通道的完整功能。因此，Kv1.3在T细胞中的表达可增强肿瘤清除能力并延长黑色素瘤荷瘤小鼠的生存期。本研究揭示了肿瘤微环境中此前未被报道的、针对T细胞功能的离子检查点，并为癌症免疫治疗提供了全新策略。整体实验设计：于培养第5天通过RNA测序（RNA-Seq）检测基因表达水平，所有样本均设置生物学重复三份，分别用固定化抗CD3/28抗体刺激或在完全培养基中培养（无刺激），同时设置等渗高钾培养基处理的平行对照组，各组实验条件保持一致。