Mannose metabolism reshapes T cell differentiation to enhance anti-tumor immunity: scCITE-seq

Cellular metabolic status profoundly influences T cell differentiation, persistence, and anti-tumor efficacy. By performing single-cell metabolic analyses of T cells from human cancer and mouse chronic infection atlases, we unveiled that diminished mannose metabolism is a prominent feature of T cell dysfunction. Conversely, experimental augmentation/restoration of mannose metabolism in adoptively transferred T cells via D-mannose supplementation enhanced anti-tumor activity and restricted exhaustion differentiation both in vitro and in vivo. Mechanistically, D-mannose treatment induced intracellular metabolic programming and increased the O-GlcNAcylation of β-catenin, which preserved Tcf7 expression and epigenetic stemness, thereby promoting stem- like programs in T cells. Finally, in vitro expansion with mannose supplementation yielded T cell products for adoptive therapy with stemness characteristics, even after extensive long-term expansion, that exhibited enhanced anti-tumor efficacy. Thus, these findings reveal cell-intrinsic mannose metabolism as a physiological regulator of CD8+ T cell fate, decoupling proliferation/expansion from differentiation, and underscored the therapeutic potential of mannose modulation in cancer immunotherapy. Wild-type female C57BL/6N (B6, CD45.2+) mice were purchased from the Vital River as recipients. Female NCG (NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt) mice were purchased from the GemPharmatech. Female C57BL/6-Tg (TcraTcrb) 1100Mjb/J transgenic mice, expressing a TCR specific for chicken egg OVA SIINFEKL epitope in the context of H2Kb, were kindly provided by Dr. Yuting Ma. All male or female mice between 6 and 12 weeks of age were used and the age-matched and sex-matched mice were randomly assigned to all animal experiments. All mice were housed in the specific-pathogen-free facilities at the Institutional Animal Care in the Suzhou Institute of Systems Medicine with a cycle of 12-h/12-h light/dark, a controlled room temperature of 22-24°C, a constant humidity of 60% and with unrestricted food and water availability. All animal studies were approved and performed in accordance with the guidelines and regulations implemented by the Institutional Animal Care and Use Committee (IACUC) of Suzhou Institute of Systems Medicine (ISM-IACUC-0151-R). The maximal tumor burden permitted by IACUC did not exceed 1500mm3 in all animal experiments. B16 mouse melanoma cells, HepG2 human hepatocellular carcinoma cells, and HEK- 293T cells from the American Type Culture Collection (ATCC) were maintained in DMEM medium (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin- streptomycin (Gibco). K562 and Jurkat cells from ATCC were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS, 0.1M HEPES (Gibco), 1% non- essential amino acids (Gibco), 1mM sodium pyruvate (Gibco), 1% penicillin- streptomycin and 50μM β-mercaptoethanol (Gibco). The B16, HepG2 and K562 cells were transduced with lentivirus to overexpress OVA, human NY-ESO-1 and human CD19 antigen respectively, and were screened with appropriate antibiotics. Murine CD8+ T cells were isolated from the spleens or lymph nodes of 6-12 weeks male or female OT-I mice and were maintained in RPMI-1640 medium supplemented with 10% FBS, 0.1M non-essential amino acids (Gibco), 1% GlutaMAX (Gibco), 0.1M HEPES (Gibco), 1mM sodium pyruvate (Gibco), 50μM β-mercaptoethanol (Gibco), 1% penicillin-streptomycin (Gibco), 20U/ml mouse IL-2 (Peprotech) in the absence or presence of mannose (20mM). To activate OT-I T cells, the media was further complemented with OVA257-264 peptide (SIINFELK, 1μg/ml, or anti-mouse CD3 (2μg/ml, Biolegend) and anti-mouse CD28 (1μg/ml, Biolegend) monoclonal antibodies, and were cultured in the cell incubator at 37°C, 5% CO2 for 48 h. To elucidate the effect of mannose on the differentiation of TILs, C57BL/6N mice were injected subcutaneously with B16-OVA cells and adoptive OT-I T cells as described above. For mannose treatment, normal drinking water was exchanged for 200ml of 20% mannose in drinking water (w/v) once every week, and mice received 20% mannose (200μl) by oral gavage three times a week. The tumors were collected after T cell transferring for 14 days, and single-cell suspensions were processed and harvested. Then, the tumor-infiltrating CD8+CD45.1+ T cells were sorted by BD FACS Aria III. Subsequently, sorted T cells were incubated with anti-mouse CD16/32 antibody (Biolegend) to block the Fc receptors and labeled with TotalSeq-B Mouse Universal Cocktail V1.0 (Biolegend) according to the manufacturer’s instructions. Next, the cell suspension was loaded into Chromium microfluidic chips with 30.v3 chemistry and barcoded with a 10×Chromium Controller (10×Genomics). RNA from the barcode was subsequently reverse-transcribed. The scRNA-seq libraries were generated following the manufacturer’s instructions of Chromium Next GEM single Cell 3’Gel Bead kit v.3.1 (10×Genomic) followed by sequencing on a Novaseq6000 at the NGS Core of the Novogene.