PI3Kδ inhibition programs T cells with stemness and metabolic fitness that favor Tpex differentiation, resist terminal exhaustion, and remodel the TME toward an inflammatory state, supporting a practical strategy to improve ACT efficacy in solid tumors.
Abstract
T cell exhaustion limits the durability of adoptive cell therapy (ACT) in solid tumors. Because PI3Kδ signaling governs T cell differentiation, we hypothesized that transient, high-dose PI3Kδ inhibition would endow T cells with durable stemness and metabolic fitness to resist terminal exhaustion and alter the immune composition of the tumor microenvironment (TME).
Mouse and human T cells were treated in vitro with the PI3Kδ inhibitor CAL-101 (30 uM) prior to chronic antigen stimulation and ACT into B16 melanoma. Metabolic fitness was assessed by mitochondrial dependence and spare respiratory capacity. Single-cell RNA sequencing and spatial transcriptomics profiled T cell states, spatial localization, and TME remodeling post ACT. Pathway analyses interrogated oxidative phosphorylation (OXPHOS), glycolysis, proliferation, inflammatory signaling, and myeloid composition.
CAL-101—treated T cells acquired enhanced stemness and metabolic fitness, with increased mitochondrial dependence and spare respiratory capacity while maintaining normal basal metabolism. Under chronic stimulation, including in human T cells, they resisted terminal exhaustion and preserved stem-like properties. In tumors, treated cells preferentially differentiated into progenitor exhausted T cells (Tpex), displayed superior infiltration, and upregulated the Cxcl10/Cxcr3 axis. Tumors harboring CAL-101—treated T cells showed reduced glycolysis, OXPHOS, and proliferation alongside increased pro-inflammatory signaling and fewer immunosuppressive tumor-associated macrophages. Single-cell analysis revealed CAL-101 treated T cells concurrently increased OXPHOS, proliferation, and immune signaling pathways.
Brief PI3Kδ inhibition programs T cells with stemness and metabolic fitness that favor Tpex differentiation, resist terminal exhaustion, and remodel the TME toward an inflammatory state, supporting a practical strategy to improve ACT efficacy in solid tumors.
NIH RO1 grant: CA257954, Dartmouth TQCR Training Grant: T32CA260626
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
There is substantial interest in developing novel engineering strategies to promote the sustained metabolic fitness of therapeutic T cells. We previously showed that overexpression of RAS homologue enriched in brain (RHEB), a positive regulator of mammalian target of rapamycin complex 1 (mTORC1), promotes aerobic glycolysis and increases the anti-tumor functions of effector CD8+ T cells. To address whether these effects are conserved in CD4+ T cells, we have now examined how enforced activation of mTORC1 activity affects CD4+ T cell differentiation and function. Rheb overexpression induced a more balanced metabolic shift in CD4+ T cells than in CD8+ T cells, with increases in both oxidative phosphorylation and aerobic glycolysis. Although Rheb overexpression initially increased CD4+ T cell activation and proliferation in vitro, the underlying population architecture was complex, involving a shift to both more proliferative, cytotoxic-like cell states as well as more quiescent cell clusters characterised by counter-regulation of mTORC1 activity. Following adoptive transfer, tumor antigen-specific Rheb-transduced CD4+ T cells showed greater persistence but were less efficient than controls in eliminating tumor. This functional deficiency could be explained by a greater propensity of persisting Rheb-transduced CD4+ T cells to develop features of immune exhaustion, as evidenced by expression of multiple co-inhibitory receptors and impaired proliferation upon tumor rechallenge. Together, these data demonstrate the dynamic population response to tuning of T cell mTORC1 and the need to separately appraise cellular outputs of therapeutic CD4+ versus CD8+ T cells when metabolic pathways are manipulated by the same method.
F. Sillito, Elisa Armbrecht, A. O'Neill et al.· Journal of Immunology· 0 citations
The efficacy of adoptive cell transfer (ACT) therapy in solid tumors is often limited by the functional exhaustion and insufficient persistence of infused CD8+ T cells within the tumor microenvironment. Through the integrated analysis of single-cell transcriptomic data, this study identified enolase 1 (ENO1), a key rate-limiting enzyme in glycolysis, as a core gene highly correlated with the superior anti-tumor phenotype of tumor-infiltrating lymphocytes (TILs). However, in vitro functional validation demonstrated that the overexpression of Eno1 failed to substantially enhance the anti-tumor efficacy of mouse T cells, suggesting the presence of a downstream metabolic regulatory node within the glycolytic cascade that restricts the conversion of carbon flux. To overcome this limitation, we introduced the small molecule activator TEPP-46 to target a crucial downstream metabolic hub, pyruvate kinase M2 (PKM2). Transcriptome sequencing confirmed that PKM2 activation successfully induced systemic metabolic rewiring in CD8+ T cells and broadly upregulated the expression of cytotoxicity- and memory-related genes. In an in vivo B16-OVA melanoma model, OT-1 T cells subjected to In vitro TEPP-46 pretreatment exhibited significantly enhanced tumor-suppressive capabilities and effectively promoted the preferential differentiation of T cells into central memory T cells (Tcm). In summary, this study highlights the importance of targeting downstream metabolic nodes to bypass intrinsic metabolic restrictions in T cells. It demonstrates that in vitro metabolic pretreatment via PKM2 activation represents an effective translational strategy for optimizing the anti-tumor efficacy of ACT cell products.
Unknown authors· International Journal of Mol...· 0 citations
These findings identify a novel microbial SCFA as a potential metabolic-epigenetic regulator, capable of reactivating effector programs in TEX cells while blocking terminal exhaustion, and provide insights into developing new therapeutic approaches to reprogram TEX cells and enhance the efficacy of T cell immunotherapy.
Asmaa M. Yousif, Amira Yousif, Ava Lowin et al.· Journal of Immunology· 0 citations
In the Ja281 KO mouse model, we found that transferring TCRαβ+CD4⁻CD8⁻ double-negative (DN) T cells into these mice inhibited breast cancer (BC) growth. This DN T cell transfer-mediated antitumor response depends on host NK cells and IFNγ, but is independent of IFNγ and perforin produced by the donor DN T cells. The underlying mechanism is unknown. Since implanted tumor cells fail to grow in the transferred mice, it is challenging to study the associated cellular and molecular processes.
Herein, we conducted a transcriptomic analysis to identify gene signatures related to cell signaling and functional changes. RNA sequencing was combined with antibody-mediated cell depletion to investigate the dynamic cellular and molecular changes.
Results showed that 42 hours after tumor inoculation, the major biological processes and signaling pathways significantly upregulated in DN T-transferred mice compared to wild-type controls included glycolysis, fatty acid metabolism, enhanced NK cell activation and IFNγ production, and elevated CCL8 expression. By day 3 post-inoculation, IFNγ-induced signaling pathways were significantly elevated in the tumors of DN T-transferred mice. Additionally, myeloid cells were observed infiltrating the tumor site. On days 3 and 5 after tumor inoculation, genes related to antigen presentation, T cell differentiation and activation were consistently upregulated in tumors from DN T-transferred mice. Interestingly, antibody-mediated depletion of CD8+ but not CD4+ T cells partially restored BC growth in some the transferred Ja281 KO mice, although tumor development remained significantly delayed compared to non-transferred controls.
In summary, DN T cell transfer promotes glycolysis and fatty acid oxidation shortly after tumor inoculation, induces NK cell activation and type 1 myeloid cell infiltration within 2—3 days, and is followed by enhanced T cell activation. CD8+ T cells appear to play a role in the sustained control of BC growth in this model.
NIH/NCI grant R01CA276284
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Hui Zhang, Anil K. Singh· Journal of Immunology· 0 citations
Abstract Background Nasopharyngeal carcinoma (NPC) is considered a typical “hot” tumor, yet its immune evasion mechanisms remain poorly defined, and immunotherapy efficacy is suboptimal. Attenuation of intrinsic tumor cell immunogenicity to evade T cell recognition and cytotoxicity represents a major route of tumor immune evasion, whose mechanistic basis is largely unelucidated. Methods Multiplex immunofluorescence and immunohistochemistry were used to assess correlations between mitochondrial leucyl-transfer RNA synthetase 2 (LARS2) expression, CD8+ T cell infiltration and immunotherapy response in NPC tissues. In vitro CD8+ T cell co-cultures, immunodeficient nude mice and immunocompetent murine models were used to explore LARS2-dependent CD8+-mediated antitumor immunity. Blue native polyacrylamide gel electrophoresis (BN-PAGE), extracellular flux and other mitochondrial functional assays were performed to characterize the roles of LARS2 and leucine in electron transport chain (ETC) translation and oxidative phosphorylation (OXPHOS) activity. Chromatin immunoprecipitation-quantitative PCR, ELISA and flow cytometry analyzed epigenetic modifications, major histocompatibility complex class I (MHC-I) expression and antigen presentation. Leucine supplementation combined with anti-programmed cell death protein-1 (PD-1) therapy was tested in mouse models to evaluate in vivo therapeutic efficacy against NPC immune escape. Results We demonstrated that LARS2, a gene mapped to the 3p21 chromosomal region, was significantly downregulated in NPC, and its expression is positively correlated with patient prognosis and response to immunotherapy. Through in vitro and in vivo experiments, we demonstrated that restoration of LARS2 expression accelerates the translation of mitochondrial ETC subunits, enhances OXPHOS, and upregulates MHC-I expression via an epigenetic mechanism. These effects work together to enhance tumor antigen presentation and augment CD8+ T cell-mediated cytotoxicity. Furthermore, we demonstrated that a high-leucine diet increases LARS2 expression and improves the effectiveness of anti-PD-1 immunotherapy. Conclusions This study uncovers a novel mechanism by which the tumor suppressor LARS2 inhibits immune evasion in NPC through upregulation of MHC-I, and highlights a high-leucine diet as a promising strategy to sensitize tumors to immunotherapy.
Mei Yang, Yongzhen Mo, Daixi Ren et al.· Journal for ImmunoTherapy of...· 0 citations
The tumor microenvironment (TME) critically regulates cancer progression by providing biochemical and biophysical cues that shape cellular behavior. However, how defined physical microenvironments govern cancer stemness and chemoresistance through mechanotransduction remains poorly understood. Here, we systematically engineered eight tumor-mimetic microenvironments by integrating serum, oxygen, and 3D compacted culture to investigate their effects on A549 non-small cell lung cancer cells. Among all conditions, cells cultured under 3D culture (PM4C) exhibited reduced cellular stiffness, enhanced expression of cancer stemness markers (EpCAM and CD44), and significantly increased resistance to cisplatin in both in vitro and nude mouse xenograft models. Transcriptomic analysis revealed that differentially expressed genes in the PM4C group were predominantly enriched in cell adhesion, mechanotransduction, stemness, and cisplatin resistance pathways. Metabolomic profiling further revealed a substantial accumulation of anaerobic metabolites associated with the maintenance of stemness. Mechanistically, the PM4C microenvironment remodeled matrix production, cell-ECM interactions, and cytoskeletal organization while inducing epigenetic reprogramming (reduced H3K9 acetylation), collectively promoting a stem-like and chemoresistant phenotype. These findings establish a direct mechanistic link between TME and cancer cell stemness, demonstrating that TME can reprogram stemness and drug responsiveness through mechano-epigenetic regulation. This work provides a mechanobiological framework for engineering physiologically relevant tumor organoids and offers new strategies for developing TME-targeted drugs and therapies.
Duoduo Zhang, Yung-Chiang Liu, Chunchang Li et al.· ACS Applied Materials and In...· 0 citations
