Potentiating the anti-tumor response of tumor infiltrated T cells by NAD⁺supplementation

Abstract

Tumor immunotherapies have provided clinical benefits, yet great potential remains for optimizing therapeutic effects. Here, we show that low NAD + levels restrict the function of 20 tumor infiltrating T lymphocytes (TILs). TILs harvested from human ovarian tumor tissues showed decreased NAD + levels compared with T cells from paired peripheral blood samples.The combination of whole-genome CRISPR and large-scale metabolic inhibitor screens implicated the NAD + biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT) is required for T cell activation. Further isotopic labeling and LC-MS studies confirmed that NAD + 25 depletion suppressed mitochondrial energy biosynthesis in T cells. Excitingly, NAD + supplementation significantly enhanced the tumor cell-killing efficacy of CAR-T cells ex vivo, and extended animal survive in both adoptive CAR-T model and immune checkpoint blockade treatment models in vivo. This study demonstrates an over-the-counter nutrient supplement NAD + could robustly boost the efficacy of T cell-based immunotherapy and provides insights 30 into the cellular basis of T cell metabolic reprogramming in treating cancers. One Sentence Summary:NAD + supplementation during cancer immunotherapies significantly enhances T cell activation and tumor killing capacity.Main Text: 35 Cancer immunotherapies including adoptive transfer of naturally-occurring tumor infiltrating lymphocytes (TIL) and genetically-engineered T cells, as well as the use of immune checkpoint inhibitors to boost the function of T cells have emerged as promising approaches to achieve durable clinical responses of otherwise treatment-refractory cancers 1-5 . Although cancer immunotherapies have been successfully utilized in the clinic for subsets of patients, there are 40 several limitations which prevent the broad use of these therapies for entire patient populations 6,7 . Given the function of T cells as key mediators for tumor destruction, their characteristics (e.g., durability, longevity, and killing efficiency, etc.) substantially determine the clinical outcomes of many immunotherapies [8][9][10][11] . Studies have established that successful clearance of tumors mediated by infiltrated T cells can be limited by physical barriers generated by stroma 45 3 cells 12 , immuno-suppressive networks 13 , and nutrient limitations within the microenvironment 14,15 . Thus, efforts to promote the stemness, proliferation and activation capacity of T cells should enable improvements to the efficacy of cancer immunotherapies.Recently, cellular metabolic processes have been reported to shape T cell differentiation and functional activity [16][17][18] . Modulation of metabolic process such as fatty acid catabolism can 50 improve T cell activation and therapeutic function [19][20][21][22][23][24] . It was also reported that there is a strong link between metabolic activity in tumor infiltrated T cells (TIL) and their effector function 25,26 , and stimulation mitochondrial biogenesis via enforced expression of PGC1α resulted in...

The Key Role of NAD+ in Anti-Tumor Immune Response: An Update

“…Interestingly, NAD + intracellular levels were restored by adding NAD + to the culture medium, thus suggesting that T cells are able to uptake NAD + from the environment. This was able to restore T cell activation, confirming that high NAD + levels are essential for T cell activation (31). Appealing results were obtained by comparing the NAD + levels of tumor infiltrating lymphocytes (TIL) and peripheral blood lymphocytes (PBL), in samples from ovarian cancer patients and from melanoma preclinical models.…”

“…A novel study analyzed the role of NAD + in the activation of the anti-tumor T cell response (31). Genetic and metabolic analysis of the Jurkat T-ALL cell line allowed the genes involved in T cell activation to be identified.…”

“…Genetic and metabolic analysis of the Jurkat T-ALL cell line allowed the genes involved in T cell activation to be identified. Nampt was proved as a key factor for T cell activation in both analysis, and three compounds known to target Nampt (FK866, STF-118804, and GMX1178) as the most disruptive to T cell activation (31). Accordingly, FK866 was able to inhibit T cell activation, as witnessed by the downregulation of CD69, CD25 and ICOS activation markers, inhibition of calcium flux and phosphorylation of signaling proteins.…”

Metabolic optimization of adoptive T cell transfer cancer immunotherapy: A historical overview

“…Eventually, a G0S9 product was identified as NAMPT, an enzyme mediating salvage synthesis of NAD + . 29,30 This indicated an early need for NAD + , a conclusion recently confirmed by Yuetong Wang et al 31 Unaware of our G0S9 work, from their search 'to identify genes involved in the regulation of T cell activation', Wang et al 31 concluded that 'the NAD + biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT) is required for T cell activation'. Indeed, specific inhibitors of NAMPT (eg FK866) were 'ranked among the most disruptive compounds to T cell activation', with CD8 T cells being more sensitive than CD4 T cells.…”

“…Wang et al 31 went further. Rather than lectin, they employed anti-CD3 and anti-CD28 antibodies to polyclonally activate human T cells in vitro.…”