Abstract

　　High expression of programmed cell death protein 1 (PD-1), a typical immune checkpoint, results in dysfunction of T cells in tumor microenvironment. Antibodies and inhibitors against PD-1 or its ligand (PD-L1) have been widely used in various malignant tumors. However, the mechanisms by which PD-1 is regulated are not fully understood. Here, we report a mechanism of PD-1 degradation triggered by d-mannose and the universality of this mechanism in anti-tumor immunity. We show that d-mannose inactivates GSK3β via promoting phosphorylation of GSK3β at Ser9, thereby leading to TFE3 translocation to nucleus and subsequent PD-1 proteolysis induced by enhanced lysosome biogenesis. Notably, combination of d-mannose and PD-1 blockade exhibits remarkable tumor growth suppression attributed to elevated cytotoxicity activity of T cells in vivo. Furthermore, d-mannose treatment dramatically improves the therapeutic efficacy of MEK inhibitor (MEKi) trametinib in vivo. Our findings unveil a universally unrecognized anti-tumor mechanism of d-mannose by destabilizing PD-1 and provide strategies to enhance the efficacy of both immune checkpoint blockade (ICB) and MEKi -based therapies.

　　Introduction

　　The inability to destroy antigenic malignant tumor cells of the immune system is one of the main reasons why cancer is so difficult to cure [1]. Immunotherapies turn certain previously fatal or uncontrollable cancers into manageable diseases by boosting immune responses of T cells against tumor cells [2]. Fundamental discoveries elucidating the mechanism and cellular biology of T cells have conferred perspective strategies in tumor immunotherapies including immune checkpoint blockade, immunomodulatory factor therapy, cancer vaccines and T-cell therapy (especially chimeric antigen receptor (CAR) T-cell therapy) [3]. Antibodies targeting immune checkpoint proteins PD-1, PD-L1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have shown clinically significant therapeutic efficacy and durable responses even in advanced and chemo-resistant cancers of certain types [[4], [5], [6], [7], [8]].

　　PD-1, first identified in apoptosis [9], is a 288–amino acid transmembrane protein predominantly induced on the surface of antigen-experienced T cells mediating suppression of the immune response [[10], [11], [12]]. PD-1 contains four N-linked glycosylation sites at its extracellular IgV domain: N49, N58 (N54 in mouse PD-1), N74, and N116. Core fucosylation of N49 and N74 are pivotal for appropriate cell-surface expression and functional localization of PD-1 [13]. Blocking core fucosylation through inhibition of Fut8 reduced cell-surface expression of PD-1, thereby enhancing T cell activation and further promoting anti-tumor immune responses [13].

　　d-mannose, a C-2 epimer of glucose, exists naturally in various plants and fruits and its concentration in human blood is lower than glucose [14]. Uptake of d-mannose affects intracellular glucose metabolism attributed to inhibition of glucose metabolism by a derivative of d-mannose d-mannose-6-phosphate [15]. Supraphysiological levels of d-mannose suppressed immunopathology in mouse models of autoimmune diabetes and airway inflammation, and increased the proportion of Foxp3+ regulatory T cells (Treg cells) in mice [16]. Besides, d-mannose suppressed LPS-induced macrophage activation by impairing IL-1β production [17]. Although the role of glucose in T cell metabolism, diabetes and obesity is well characterized [[18], [19], [20]], the exact role of d-mannose in T cell immune responses remains unclear. In this study, we found that d-mannose destabilized PD-1 in human T cells and mouse myeloid immune cells through lysosomal degradation. In mouse models, d-mannose enhanced its own antitumor efficacy by downregulating PD-1. Mechanically, d-mannose potentially suppressed PD-1 endocytic recycling, thus promoting its subsequent lysosomal degradation. In the meantime, d-mannose induced phosphorylation of GSK3β at Ser9, thereby leading to GSK3β inactivation and TFE3 translocation to nucleus. Importantly, nuclear TFE3 was responsible for enhanced lysosome biogenesis and subsequent PD-1 proteolysis. Moreover, we showed that d-mannose reduced tumor growth and synergistically enhanced the efficacy of anti-PD-1 therapy in vivo. Notably, combination of d-mannose and MEKi trametinib could dramatically inhibit tumor growth in a KRAS-mutated CT26 mouse model.

　　This study reveals a new mechanism in which d-mannose is involved in the regulation of PD-1, which broadens our understanding of the involvement of d-mannose in tumor immune regulation, and also provides an experimental basis for further clinical applications of d-mannose.

　　Section snippets

　　Cell culture

　　293T, MDA-MB-231, B16F10, CT-26 and RAW264.7 cells were cultured in DMEM (BDBIO, HangZhou China) supplemented with 10 % FBS (Gibco, USA) and 100 μg/mL of penicillin and streptomycin. Jurkat and THP-1 were cultured in RPMI1640 medium (Meilun Biotechnology) containing 10 % FBS, 100 μg/mL of penicillin and streptomycin and 55 μM of beta-mercaptoethanol. B16F10 were from the American Type Culture Collection (ATCC), MC38 and CT-26 cell lines were kind gifts from Dr. Wei Jiang (Fudan University).

　　d-mannose decreases PD-1 protein level

　　d-mannose is reported to facilitate immunotherapy and radiotherapy in triple-negative breast cancer (TNBC) by targeting PD-L1 for proteasome-mediated degradation [25], while its effects on other cancer types and other immune checkpoint molecules are still unknown. To explore whether d-mannose affects PD-1 expression level,we evaluated the effects of different hexoses on PD-1 in Jurkat cells ectopically expressing Flag-tagged PD-1. We observed that d-mannose, but not other hexoses, significantly

　　Discussion

　　Antibody drugs have disadvantages such as more complex molecular structure, higher production cost, and usually need to be administered by injection, and they usually only work with proteins on the surface of cell membranes or outside the cell, which makes their application somewhat limited [45]. Therefore, the search for efficacious alternative drugs, especially small molecule drugs, remains potentially of great application. As the main source of cellular energy, a growing number of studies

　　Funding

　　This work was supported by the grants from the National Key R&D Program of China (2020YFA0803400/2020YFA0803402, 2022YFA0807100), the National Natural Science Foundation of China (82073128, 82372754, 82373402, 82172936 and 82121004), and the Fundamental Research Funds for the Central Universities.

　　CRediT authorship contribution statement

　　Wenjing Dong: Writing – original draft, Project administration, Methodology, Investigation, Formal analysis, Data curation. Mingen Lin: Writing – review & editing, Project administration, Investigation, Formal analysis, Data curation. Ruonan Zhang: Methodology, Investigation. Xue Sun: Methodology, Investigation. Hongchen Li: Methodology, Investigation. Tianshu Liu: Funding acquisition, Writing – review & editing. Yanping Xu: Writing – review & editing, Supervision, Funding acquisition. Lei Lv:

　　Declaration of competing interest

　　The authors declare no competing interests.

　　Acknowledgements

　　We thank all the laboratory members for the discussion and suggestions for this study. We thank PETCC for kindly providing us with cell lines for testing. This work was supported by the grants from the National Key R&D Program of China (2020YFA0803400/2020YFA0803402, 2022YFA0807100), the National Natural Science Foundation of China (82073128, 82372754, 82373402, 82172936 and 82121004), and the Fundamental Research Funds for the Central Universities.

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