肿瘤微环境中的免疫抑制因素是限制T细胞免疫治疗效果的主要障碍。这些不利条件不仅削弱了T细胞的肿瘤浸润能力，还通过增加代谢压力和损害线粒体功能，导致肿瘤浸润性T淋巴细胞的代谢失衡和功能耗竭。研究表明，在免疫抑制的肿瘤微环境中存在一类TCF1⁺ T细胞亚群，被称为耗竭前体T细胞。由于其具有较高的增殖和分化潜能，在体内表现出更强的持久性和抗肿瘤活性。酸性肿瘤微环境作为实体瘤的典型特征，其不仅抑制T细胞的效应功能，还影响树突状细胞和肿瘤相关巨噬细胞的活性及分化。此外，酸性肿瘤微环境诱导的免疫抑制细胞因子如IL-10，TGF-β，同时抑制抗原呈递细胞的功能，进一步削弱T细胞的抗肿瘤活性。然而，酸性TME是否直接参与调控T细胞的肿瘤浸润能力以及浸润至肿瘤微环境中的“干性样”T细胞的产生和维持，目前尚不清楚。

为了深入探讨酸性肿瘤微环境对T细胞的免疫调控作用，我们对来自29种肿瘤患者的肿瘤组织和正常组织的测序数据进行了整合分析，发现肿瘤酸度与T细胞浸润能力呈负相关。这一结果提示酸性肿瘤微环境可能通过特定机制阻碍T细胞向肿瘤组织的浸润。进一步研究发现，细胞外酸化通过抑制RNA甲基化酶METTL3的表达，从而降低了细胞黏附分子ITGB1 mRNA的m⁶A甲基化修饰，导致Integrin β1蛋白表达减少，最终抑制了T细胞的浸润能力。而Integrin β1作为T细胞跨越内皮屏障和肿瘤基质的关键黏附分子，主要负责介导T细胞与细胞外基质的相互作用。因此，酸性肿瘤微环境通过调控Integrin β1的表达，阻碍了T细胞向肿瘤组织的迁移和定植。此外，我们通过基因改造使T细胞过表达Integrin β1，以进一步验证Integrin β1在T细胞肿瘤浸润中的作用。我们发现，过表达Integrin β1的T细胞在肿瘤组织中积累增加，并且表现出更强的肿瘤杀伤能力。同时，与对照组相比，Integrin β1过表达显著抑制B16-OVA以及HepG2-CD19荷瘤小鼠的肿瘤生长，并延长其生存期。这些结果进一步证实了Integrin β1在调控T细胞肿瘤浸润过程中发挥着关键作用。

此外，我们还探究了酸性肿瘤微环境是否参与调控T细胞分化。研究发现，长期酸处理显著促进了小鼠及人TCF1⁺ T细胞亚群的形成。长期体外酸处理不仅维持了Tpex细胞的存活，还显著提升了其增殖和分化能力。进一步的过继转移实验表明，经体外长期酸处理的TCR T细胞在体内具有更强的抗肿瘤效应。即使在酸性TME中，这些经长期酸处理的T细胞依然能够有效浸润至肿瘤组织，并维持较高的效应功能。在机制层面，体外长期的酸处理通过抑制甲硫氨酸的摄入，削弱了T细胞内的一碳代谢（甲硫氨酸循环）过程。甲硫氨酸循环是T细胞维持DNA，RNA以及组蛋白甲基化水平的重要代谢途径。体外长期酸处理导致细胞内甲基供体S-腺苷甲硫氨酸减少，从而重塑了T细胞内“干性样”相关基因的表观遗传图谱。此外，表观遗传学分析显示，体外长期酸处理显著增强了调控Tpex细胞分化的关键基因位点的表达（TCF7，LEF1，KLF2），并上调了多种与Tpex细胞命运相关的转录因子表达水平，最终促进了TCF1⁺ T细胞的分化。

综上所述，我们围绕酸性微环境对T细胞免疫的多层次调控机制进行了系统性研究，发现了酸性肿瘤微环境在调控T细胞浸润和命运决定中的新功能和新机制。此外，通过基因改造增加Integrin β1的表达以及优化T细胞在酸性肿瘤微环境中的存活和效应功能，我们开发了一种增强T细胞肿瘤浸润的新策略。这一发现为肿瘤免疫治疗提供了新的思路和靶点，并有望进一步提升实体瘤患者对免疫疗法的响应率。

The immunosuppressive factors within the tumor microenvironment represent a major barrier to the effectiveness of T-cell-based immunotherapy. These adverse conditions not only impair the tumor infiltration capacity of T cells but also induce metabolic stress and mitochondrial dysfunction, leading to metabolic imbalance and functional exhaustion of tumor-infiltrating lymphocytes. Studies have shown the presence of a progenitor-exhausted TCF1⁺ T cell subset within the immunosuppressive tumor microenvironment. Due to their high proliferative and differentiation potential, progenitor-exhausted T cells exhibit greater persistence and enhanced antitumor efficacy in vivo. The existence of progenitor-exhausted T cells is crucial for maintaining long-term immune responses, as they possess self-renewal capability and can differentiate into terminally exhausted T cells to sustain antitumor effects. Acidic tumor microenvironment, a hallmark of solid tumors, not only suppresses the effector function of T cells but also significantly affects the activity and differentiation of dendritic cells and tumor-associated macrophages. Additionally, the acidic environment promotes the release of immunosuppressive cytokines while inhibiting the function of antigen-presenting cells, further diminishing T-cell antitumor activity. However, it remains unclear whether the acidic tumor microenvironment directly regulates T-cell tumor infiltration and the generation and maintenance of “stem-like” TILs.

To explore the role of the acidic tumor microenvironment in T-cell immune regulation, we performed an integrated analysis of sequencing data from tumor and normal tissues of 29 types of cancer patients. The results revealed a significant negative correlation between tumor acidity and T-cell infiltration capacity, suggesting that the acidic environment may impede T-cell infiltration through specific mechanisms. Further investigation demonstrated that extracellular acidification suppressed the expression of the RNA methyltransferase METTL3, reducing the m6A methylation of the ITGB1 mRNA, a cell adhesion molecule. Consequently, the protein level of Integrin β1 was decreased, ultimately inhibiting T-cell infiltration. Integrin β1, a key adhesion molecule, mediates T-cell interactions with the extracellular matrix, enabling them to traverse endothelial barriers and tumor stroma. Therefore, the acidic tumor microenvironment blocks T-cell migration and settlement within tumor tissues by downregulating Integrin β1 expression. To validate the role of Integrin β1 in T-cell tumor infiltration, we genetically engineered T cells to overexpress Integrin β1, which significantly enhanced their infiltration capability. In a B16-OVA tumor-bearing mouse model, T cells overexpressing Integrin β1 showed markedly increased accumulation in tumor tissues and exhibited enhanced tumor-killing capacity. Compared to the control group, mice receiving Integrin β1-overexpressing T cells displayed significant tumor volume reduction and prolonged survival. These results further confirmed the critical role of Integrin β1 in mediating T-cell tumor infiltration.

Furthermore, we explored whether the acidic microenvironment regulates T-cell fate determination. Our results showed that long-term acid treatment significantly promoted the formation of TCF1⁺ T-cell subsets in both mice and humans. In vitro, prolonged exposure to an acidic environment not only preserved the survival of progenitor exhausted T cells but also enhanced their proliferative and differentiation potential. Adoptive transfer experiments further demonstrated that acid-treated TCR-engineered T cells exhibited stronger antitumor effects in vivo. Remarkably, these acid-treated T cells retained their infiltration capacity and maintained high effector functions within the acidic tumor microenvironment. Mechanistically, extracellular acid treatment was found to inhibit methionine uptake, thereby impairing the one-carbon metabolism, specifically methionine cycle, in T cells. The methionine cycle is a critical metabolic pathway for maintaining DNA and RNA methylation in T cells. Acid treatment led to reduced production of the intracellular methyl donor S-adenosylmethionine, which in turn reshaped the epigenetic landscape of T cells associated with a “stem-like” phenotype. Epigenomic analysis revealed that acid treatment significantly increased the chromatin accessibility of key gene loci regulating progenitor-exhausted T cell differentiation, resulting in the upregulation of multiple transcription factors associated with Tpex cell fate, ultimately promoting TCF1⁺ T-cell differentiation. 

In conclusion, we conducted a comprehensive investigation into the multifaceted regulatory mechanisms of acidic tumor microenvironment on T-cell immunity, uncovering novel functions and mechanisms through which the acidic tumor microenvironment influences T-cell infiltration and fate determination. Additionally, by genetically enhancing Integrin β1 expression and optimizing T-cell survival and function in acidic environments, we developed a new strategy to improve T-cell tumor infiltration. This discovery offers new insights and potential therapeutic targets for enhancing the efficacy of immunotherapy in solid tumor patients.