Nicotinamide, a Form of Vitamin B₃, Promotes Expansion of Natural Killer Cells That Display Increased In Vivo Survival and Cytotoxic Activity,

Abstract 4035

NK cells are cytotoxic lymphocytes that have drawn considerable attention in recent years as a promising tool for immunotherapy in patients with various refractory hematological malignancies and metastatic solid tumors. Clinical results of experimental protocols have shown only a partial response attributed mainly to the relatively low number of NK cells infused and their short in vivo persistence.

An important challenge, therefore, in advancing the clinical applicability of NK cells is to expand ex vivo NK cells that display increased functionality upon in vivo infusion. In efforts to induce NK cell expansion, different combinations of cytokines have been studied. However, most reports show a modest expansion and demonstrate a need for additional stimuli.

Nicotinamide (NAM) is a form of Vitamin B3 and a potent inhibitor of enzymes that use NAD for their activity. Hence, NAM is directly involved in the control of redox sensitive enzymes, mitochondrial functions, cell metabolism, the production of energy, and cell motility.

Here we show that NAM (2.5–5 mM) enhances expansion (60-80 fold) of functional NK cells in feeder-free cultures stimulated with IL-2 and IL-15 for two weeks. This effect was observed in cultures initiated with purified CD56+ (CD56 enriched/CD3 depleted) or with CD3 depleted, peripheral blood and cord blood cells.

Immunophenotyping of the cultured NK cells has so far revealed that NAM substantially modulates three cell surface receptors. CD200R and programmed death receptor-1 (PD-1) expressed on NK cells interact with their ligands on tumor cells which leads to a suppression in NK cell anti-tumor activity and tumor immunoevasion. These two receptors are down-regulated by NAM. CD62L (L-selectin) defines an NK subset with increased self-renewal capacity and its expression was reported to be pivotal for NK cells trafficking to lymphoid organs and their homeostatic proliferation. Following expansion in culture with IL-2, CD62L is down-regulated, whilst NAM increased its expression with a dose-dependent effect.

Using a CFSE-based cytotoxicity assay we have demonstrated that NK cells cultured with NAM display higher cytotoxic activity against K562, BL2, NK-resistant COLO 205 cell lines and primary leukemia cells. In a Transwell migration assay, NK cells cultured with NAM demonstrated increased migration towards the CXCR4 ligand SDF-1. To test in vivo homing and retention, irradiated (350 RAD) NOD/SCID mice were transplanted with a similar number of cells (15–20×10⁶ /mouse) derived from two week cultures treated with or without NAM. Mice were infused with 50μg/mouse IL-2 and 5μg/mouse IL-15 every other day. To test homing, mice were sacrificed 24 hour post infusion. Number of human NK cells (CD45+CD56+) detected in the spleen and BM were significantly (p< 0.05) higher in the cohort of mice infused with NK cells cultured with NAM (7.9 and 1.39 respectively) compared to mice infused with NK cells cultured without NAM (4.13 and 0, respectively). In a different set of experiments, persistence of human NK cells was analyzed 4 and 12 days post infusion. Four days post infusion, the percentage of human NK cells in the spleen, BM, lung and liver were substantially higher in mice infused with NK cells cultured with NAM compared to mice infused with NK cells cultured without NAM (Fig 1). Even though 12 days post infusion, a decrease in the number of human NK cells was observed in comparison to day 4, still cell retention in the spleen, liver and lung was significantly greater in the cohort infused with NK cells cultured with NAM (13.45, 0.6, 9.21% Vs. 1.26, 0.12, 2.85%, (p<0.05), respectively). The calculated decrease in the number of human NK cells from day 4 to 12 was 50% less in the NAM cohort, suggesting enhanced in vivo survival of NK cells cultured with NAM.

Table 1:

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In vivo persistence of ex vivo expanded NK cells

Table 1:

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In vivo persistence of ex vivo expanded NK cells

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In conclusion, expansion of NK cells with NAM was found to increase in vivo homing and survival and to augment tumor cytotoxic effect of NK cells. This suggests a potential for enhancing the clinical efficacy of adoptively transferred NK cells. Based on these intriguing findings we are developing a cell product for adoptive cell-mediated immune therapy.