Adoptive T cell immunotherapy as a “living drug” has brought cures to many previous untreatable cancers, but persistence and anti-tumor efficacy are commonly compromised by the cellular differentiation during the ex vivo manufacturing process involves significant expansion to generate adequate cell numbers for therapy. Decoupling expansion from naturally linked differentiation could provide a strategy for generating T cell products with the capacity for self-renewal, persistence and enhanced efficacy. Cellular metabolic reprogramming can contribute to the preservation of T cell stemness, and interrogation of metabolic regulatory circuits governing and directing T cell fate differentiation has the potential to lead to development of effective metabolic intervention strategies for T cell immunotherapy.
Methods:
We first identified specific metabolic pathways that anti-correlated with T cell exhaustion and are shared across contexts, by applying a novel computational framework that analyzes single-cell metabolic activities onto transcriptome atlases of T cell exhaustion in human TILs, and mouse chronic infection and tumor models, which pinpointed mannose metabolism anti-correlating with exhaustion. We then used a B16-OVA melanoma mouse model and human HepG2-NY-ESO-1 liver tumor model to evaluate the effect of adoptively transferred T cells on tumor control after manufacturing with mannose supplementation. Single-cell RNA-seq, CUT&TAG, metabolomics and CRISPR-Cas9 was then used to evaluate the effect of mannose supplementation on T cells.
Results:
Single-cell metabolic analysis of >300,000 T cells representing more than 300 patients across 21 cancer types integrated with T cell exhaustion datasets from chronic infections and tumor models identified impaired mannose metabolism as a prominent feature of exhausted CD8+ T cells. Conversely, experimental augmentation 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 away from glycolysis and increased the O-GlcNAcylation of β-catenin, which preserved Tcf7 expression and epigenetic marks with open chromatin associated with stemness, and closed chromatin at gene regions associated with differentiation. Finally, in vitro expansion with mannose supplementation yielded T cell products that exhibited enhanced proliferation and function in vivo, resulting in improved anti-tumor efficacy.
Conclusions:
These findings reveal cell-intrinsic mannose metabolism as a physiological regulator of CD8+ T cell fate, decoupling proliferation/expansion from differentiation, and underscore the therapeutic potential of mannose modulation in cancer immunotherapy. It further suggests that expanding T cells in the presence of D-mannose would be a viable strategy for generation of larger quantities of stem-like T cell products.