肿瘤微环境的酸性是一种可以通过质子泵抑制剂克服的免疫逃逸机制
The acidity of the tumor microenvironment is a mechanism of immune escape that can be overcome by proton pump inhibitors
我们最近报道，将pH值降至肿瘤中经常检测到的值，会在体外对人及小鼠CD8+ T淋巴细胞造成可逆的无能。同样的情况也发生在体内，在肿瘤微环境使用质子泵抑制剂（PPI），缓冲肿瘤酸度，可以恢复T细胞的功能和提高免疫治疗的疗效。
关键词:无反应，T细胞，黑色素瘤，前列腺癌，过继免疫治疗，主动免疫治疗
肿瘤微环境是动态的，随着肿瘤细胞的进化和功能的调整，以满足肿瘤细胞生存和生长的迫切需要。因此，由微环境改变(通常由致癌驱动突变引起)引起的细胞代谢的改变有助于肿瘤环境的形成。
例如，癌细胞渴求能量、比正常细胞消耗更多的葡萄糖，主要是通过有氧糖酵解来处理，也就是所谓的“Warburg效应”。
这种变化的代谢模式与乳酸的产生、质子的积累和细胞内外pH的反向梯度有关，导致细胞外pH的下降。低pH值已被证明可以有利于选择更具有攻击性的后代，并有利于肿瘤侵袭，对于酸性肿瘤微环境如何影响T细胞免疫，我们知之甚少。最近我们已经证明，将pH值降低到肿瘤肿块中最常检测到的值(pH 6-6.5)，会导致体外培养的人类和小鼠肿瘤特异性CD8+ T淋巴细胞出现无能状态。
这种情况的特点是显著损伤细胞溶解的活动和细胞因子分泌, 耦合降低表达的白介素2受体的α-链 (IL-2Rα)和T细胞受体(TCR),以及减少STAT5和ERK对TCR信号的活化反应。我们还发现，从B16黑色素瘤中获得的肿瘤浸润淋巴细胞(TILs)，其细胞外pH约为6.5(具体通过体内磁共振波谱测定)，表现出类似的无能表型。
因此，在人类和小鼠的实验环境中，肿瘤酸性对影响肿瘤特异性效应T细胞的负调控(图1A)，可能确实有助于抗肿瘤免疫功能的紊乱。
虽然之前有报道说缺氧和/或癌细胞代谢改变可能有助于免疫抑制，我们的研究结果表明，酸度本身代表了一种免疫逃逸机制。进一步的研究将有助于揭示酸度降低T细胞适应性的机制。由于穿孔素脱颗粒(degranulation)、细胞因子释放和增殖在pH 6.5时明显受损，我们的假设是酸度改变了调节生理活动的生化平衡，包括胞吐、分泌和增殖。
图1所示。质子泵抑制剂对肿瘤细胞和肿瘤浸润淋巴细胞的影响。
(A)肿瘤细胞碱性的细胞内pH由特定的调控机制维持，包括由液泡H+-ATPases (V-ATPases)介导的调控机制。这些泵用于将H+离子挤出细胞外空间，降低肿瘤微环境中的pH值。根据我们的数据，局部酸度有利于在浸润CD8+效应细胞中T细胞无能的产生。(B)质子泵抑制剂(PPIs)是亲脂性和弱碱性的前药，容易穿透细胞膜，集中在酸性细胞室，在那里它们非常不稳定，并转化为生物活性抑制剂。因此，在PPIs存在的情况下，肿瘤细胞不再能够控制细胞内pH并发生凋亡。PPIs还能增加细胞外pH值，使CD8+ T细胞克服无能并恢复肿瘤杀伤活性。因此，PPI治疗提高了T细胞免疫治疗的疗效。
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有趣的是，在pH 6.5时培养的淋巴细胞中观察到的无能状态在pH缓冲作用下是可逆的，尽管长时间暴露在这样的酸性环境或较低的pH值下会导致永久性损伤和T细胞凋亡。
因此，我们研究了肿瘤pH缓冲是否能改善体内CD8+细胞功能。质子泵，如空泡H+-ATPases (V-ATPases)，在癌细胞中上调，保护细胞内酸性和凋亡，参与酸性肿瘤微环境的建立。
在使用高剂量esomeprazole (12.5 mg/Kg)治疗黑素瘤小鼠后，采用质子泵抑制剂PPI (PPI)减少胃酸分泌。
我们发现瘤内pH值迅速增加，在60分钟内达到7.0左右，并持续数小时。在体内pH与PPIs的缓冲作用与TIL效应功能改善有关(图1B)。此外,我们发现一个更高效的生产干扰素γ(IFNγ)在每个细胞的基础上。
从PPI治疗的小鼠中收集的TILs显示pERK表达的增加，证实了我们的体外数据，并证明了低pH诱导的反应力可以通过PPI治疗来克服。
PPIs是一种亲脂弱碱性的前药，很容易穿透细胞膜，集中在酸性细胞室中，在酸性细胞室中非常不稳定，转化为磺胺类药物，代表活性抑制剂。
PPIs是一种具有肿瘤选择性的免疫调节剂，因为它们不会影响(至少在我们手中)缺乏原药物激活所需的酸性条件的器官的T细胞，如脾脏、肺和肾。
PPIs对pH值较低的组织的选择性作用可能也解释了为什么这些药物可以在非常大的剂量下使用而没有明显的毒性，因为它发生在Zollinger-Ellison综合征患者的治疗中。
最重要的是，我们已经证明，用PPIs治疗荷瘤小鼠不仅可以提高过继细胞免疫治疗的疗效，还可以提高抗癌疫苗的疗效。
我们工作的一个局限是，我们没有研究PPIs在体内对其他白细胞的效应功能的影响，如调节性T细胞、B细胞、自然杀伤(NK)细胞、NK T细胞、巨噬细胞和骨髓来源的抑制细胞。显然，PPIs的应用并不有利于这些细胞群的瘤内积累，
另一些研究报告称，给予荷瘤小鼠的泮托拉唑(一种埃索美拉唑类似物)能增强巨噬细胞的抗肿瘤活性。
总之，我们的研究结果表明，肿瘤微环境酸化是一种新的免疫逃逸机制，可以通过靶向pH调节通路的药物来克服，如PPIs，它可以增加基于T细胞的癌症免疫治疗的临床潜力。
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The acidity of the tumor microenvironment is a mechanism of immune escape that can be overcome by proton pump inhibitors
We have recently reported that lowering the pH to values that are frequently detected in tumors causes reversible anergy in both human and mouse CD8+ T lymphocytes in vitro. The same occurs in vivo, in the tumor microenvironment and the administration of proton pump inhibitors, which buffer tumor acidity, can revert T-cell anergy and increase the efficacy of immunotherapy.
Keywords: : anergy, T cell, melanoma, prostate cancer, adoptive immunotherapy, active immunotherapy
The tumor microenvironment is dynamic, as it evolves and adjusts its functions to cope with the compelling need of tumor cells to survive and grow. Thus, modifications of the cellular metabolism imposed by the altered microenvironment (often induced by oncogenic driver mutations), contribute to the shaping of the tumor milieu.
As an example, cancer cells craving for energy take up much more glucose than normal cells and mainly process it through aerobic glycolysis, the so-called “Warburg effect.”
Such an altered metabolic pattern associates with an elevated production of lactate, proton accumulation and a reversed intra-extracellular pH gradient, causing a drop in extracellular pH2. While low pH values have been shown to select for more aggressive acid-resistant clones and to favor tumor invasion,
little is known about how an acidic tumor microenvironment affects T-cell immunity. We have recently shown that lowering the pH to values most frequently detected within the tumor mass (pH 6–6.5) causes the establishment of a state of anergy in both human and mouse tumor-specific CD8+ T lymphocytes in vitro.
This condition is characterized by a significant impairment in cytolytic activity and cytokine secretion, coupled to a reduced expression of both the α-chain of the interleukin-2 receptor (IL-2Rα) and the T-cell receptor (TCR), as well as to a diminished activation of STAT5 and ERK in response to TCR signaling. We have also found that tumor-infiltrating lymphocytes (TILs) obtained from B16 melanomas, whose extracellular pH is approximately 6.5 (as specifically measured by in vivo magnetic resonance spectroscopy), display a similar anergic phenotype.
Thus, tumor acidity negatively regulates tumor-specific effector T cells in both human and murine experimental settings (Fig. 1A), and might indeed contribute to the dysfunction of anti-tumor immunity.
While it has been previously reported that hypoxia and/or the metabolic alterations of cancer cells may contribute to immune suppression, our results show that acidity per se represents a mechanism of immune escape. Further studies will help unraveling the mechanisms whereby acidity reduces T-cell fitness. Since perforin degranulation, cytokine release and proliferation are significantly impaired at pH 6.5, our hypothesis is that acidity alters the biochemical equilibrium that regulates physiological activities, including exocytosis, secretion and proliferation.
Figure 1. Effects of proton pump inhibitors on tumor cells and tumor-infiltrating lymphocytes.
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Interestingly, the anergic state observed in lymphocytes cultured at pH 6.5 is reversible upon pH buffering, although prolonged exposure to such an acidic environment or lower pH values cause permanent damage and T-cell apoptosis.
Thus, we investigated whether tumor pH buffering could result in improved CD8+ cell function in vivo. Proton pumps, such as vacuolar H+-ATPases (V-ATPases), are upregulated in cancer cells, protect them from intracellular acidity and apoptosis, and participate in the establishment of an acidic tumor microenvironment
Upon treatment of melanoma-bearing mice with a high dose of esomeprazole (12.5 mg/Kg), a proton pump inhibitor (PPI) employed to reduce gastric acid secretion,8 Olbe L, Carlsson E, Lindberg P. A proton-pump inhibitor expedition: the case histories of omeprazole and esomeprazole.
we registered a rapid increase in the intratumoral pH, reaching values aroung 7.0 within 60 min and maintaining them for hours. In vivo pH buffering with PPIs was associated with improved TIL effector functions in melanoma lesions (Fig. 1B). Additionally, we detected a more efficient production of interferon γ (IFNγ) on a per cell basis.
TILs collected from PPI-treated mice showed enhanced expression of pERK,3 Calcinotto A, Filipazzi P, Grioni M, Iero M, De Milito A, Ricupito A, et al. Modulation of microenvironment acidity reverses anergy in human and murine tumor-infiltrating T lymphocytes. Cancer Res
confirming our in vitro data and demonstrating that anergy as induced by low pH can be overcome by PPI treatment.
PPIs are lipophilic and weak base prodrugs that easily penetrate cell membranes and concentrate in acidic compartments, where they are very unstable and are converted into sulfonamide forms, representing the active inhibitors.
PPIs are rather tumor-selective immunomodulators, as they did not affect (at least in our hands) the T cells of organs lacking the acidic conditions required for prodrug activation, such as spleen, lungs and kidney.
The selective effects of PPIs on tissues characterized by a low pH might also explain why these drugs can be administered at very high doses without significant toxicity, as it occurs in the treatment of patients affected by the Zollinger-Ellison syndrome.
Most importantly, we have shown that the treatment of tumor-bearing mice with PPIs increases the therapeutic efficacy not only of adoptive-cell immunotherapy but also of anticancer vaccines.
One limit of our work is that we did not investigate the effect in vivo of PPIs on the effector functions of other leukocytes such as regulatory T cells, B cells, natural killer (NK) cells, NK T cells, macrophages and myeloid-derived suppressor cells. While apparently the administration of PPIs did not favor the intratumoral accumulation of any of these cell populations,
others have reported that pantoprazole (an esomeprazole analog) given to tumor-bearing mice results in enhanced tumoricidal activity by macrophages.10 Vishvakarma NK, Singh SM. Immunopotentiating effect of proton pump inhibitor pantoprazole in a lymphoma-bearing murine host: Implication in antitumor activation of tumor-associated macrophages.
Altogether, our findings show that the acidification of the tumor microenvironment is a novel mechanism of immune escape that can be overcome by drugs targeting pH-regulatory pathways, like PPIs, which can increase the clinical potential of T cell-based cancer immunotherapy.
The acidity of the tumor microenvironment is a mechanism of immune escape that can be overcome by proton pump inhibitors: OncoImmunology: Vol 2, No 1 https://www.tandfonline.com/doi/full/10.4161/onci.22058
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