Nicotinamide (NAM) Increases Surface Expression of CD62L On in Vitro Expanded NK Cells
Authors: Mamcarz Ewelina K. , Berg Maria , Peled Tony , Frei Gabi M , Reger Robert , Tian Xin , Childs Richard 
Published: October 2019

Abstract
Abstract 4355
Natural killer (NK) cells comprise a critical component of the innate immune system that controls infections and tumor transformation. Although proof of principle supporting the antitumor effects of adoptively infused autologous and allogeneic NK cells has been established, overall response rates appear low, possibly related to their inability to traffic to lymphoid tissues and the bone marrow where hematological malignancies such as AML, lymphomas, and multiple myeloma reside. CD62L receptors play a critical role in the recruitment of NK cells from the circulation to the bone marrow and lymph nodes. Recently, investigators have developed a number of approaches to expand NK cells ex vivo for adoptive infusion in patients with cancer. Although NK cells cultured with irradiated EBV transformed lymphoblastoid cell lines (EBV-LCL) can be expanded by more than 1000 fold and are more cytotoxic to tumor cells compared to resting or IL-2 activated NK cells, these expanded cells undergo a substantial reduction in CD62L surface expression which could hinder their ability to be recruited into the tumor microenvironment. NAM, a specific inhibitor of NAD (+) dependent enzymes, was shown to up-regulate surface expression of CD62L on freshly isolated NK cells cultured in feeder cell-free cytokine-containing media. Based on this observation, we hypothesized that CD62L expression would be increased on NK cells expanded ex vivo using EBV-LCL feeders by adding NAM to the culture media.

Methods:
Human NK cells were isolated from PBMCs of 5 healthy volunteers by depleting CD3+ T cells and subsequently selecting CD56+ cells using immuno-magnetic beads. NK cells were expanded ex vivo in flasks over 21 days by co-culturing with an irradiated EBV-LCL feeder cell line at 20:1 LCL to NK cell ratio in media containing 500 IU/mL of IL-2. NAM was added to the media on culture day 7 at a 5 mM or 7.5 mM concentration and expanded NK cells were evaluated every 2–3 days by FACS to assess their phenotype and viability and by cytotoxicity assays to assess their cytotoxicity against K562 and renal cell carcinoma targets. The effects of NAM on the proliferative capacity of ex vivo expanded NK cultures were likewise assessed.

Results:
NK cell expansion cultures containing NAM had substantially higher CD62L surface expression compared to cultures without NAM, with this effect being most pronounced in NK cell cultures containing the higher concentration (7.5 mM) of NAM (see figure). On NK cell culture days 12, 14, 16, and 21, the surface MFI of CD62L was a median 1.62, 2.44, 2.03 and 3.65 fold higher on NK cells cultured in 7.5 mM of NAM compared to cultures without NAM (p =0.017, 0.012, 0.037 and 0.046 respectively; paired t-test). No significant difference in surface expression of NK cell TRAIL, NKG2D, NKG2A, NKp30, NKp46, KIR2DL1, KIR3DL1, KIR2DL2/DL3, CD44, CD95, and CD200R was observed between NAM containing cultures and controls although NKp44 expression was slightly higher in cultures that did not contain NAM. NAM-treated and untreated NK cells had similar cytotoxic function against K562 and renal cell carcinoma (RCC) tumor cell lines. The viability of NK cells, assessed by Trypan Blue and Annexin V staining, was unaffected by NAM. The addition of NAM to cell culture media did result in a dose-dependent reduction in ex vivo NK cell expansion: on days 14 and 16, NK cells expanded a median 926 and 2963 fold in control media compared to a median 443 and 1151 fold in media containing 5 mM NAM (p=0.0029 and 0.0074 respectively) and a median 359 and 732 fold in media containing 7.5 mM NAM (p=0.0019 and 0.0003 respectively). Although the proliferation of NK cells was reduced, ex vivo expansion of NK cells still occurred in NAM containing media with NK cells expanding a median 2785 and 1360 fold by day 18 in cultures containing 5 and 7.5 mM of NAM, respectively.

Conclusion:
The addition of NAM to NK cell expansion cultures substantially increased surface expression of CD62L on NK cells without any deleterious effect on their cytotoxic function. Although NAM reduced ex vivo NK cell proliferation, NK cells still expanded more than 1000 fold in NAM containing media. These data suggest NAM-induced increases in CD62L surface expression could be used as a novel method to improve the homing capacity of ex vivo expanded NK cells to the bone marrow and the lymphoid organs, potentially enhancing their antitumor effects when adoptively infused in patients with hematological malignancies.

Disclosures:
Peled: Gamida Cell Ltd. Cell Therapy Technologies: Employment. Frei:Gamida Cell Ltd. Cell Therapy Technologies: Employment.