NK Cell Proliferation and Cytolytic Function Are Compromised In the Hypoxic Tumor Microenvironment

Abstract

4291 Natural Killer (NK) cells have important and potent innate immunoregulatory and immune surveillance functions against tumor. The paradoxical coexistence of tumors and anti-tumor immune cells (“Hellstrom Paradox”) may in part be explained by the pathophysiology of the “hostile” tumor microenvironment which suppress immune-cell function, such as hypoxia, low pH, low tissue glucose, and the presence of immunosuppressive cytokines and metabolites. However, the effect of the malignant environment on the ability of NK cells to infiltrate tumor and exhibit effector function is largely unknown. Therefore, we investigated the ability of NK cells to operate under conditions of hypoxia. Importantly, NK cells showed a 1,000-fold reduction in proliferative capacity when grown under chronic hypoxia (4 weeks of 1% O2). In addition, there was a corresponding decrease in cytotoxicity as revealed by chromium release assay. This was in contrast to autologous T cells which could numerically expand under corresponding growth conditions. Expression profiling uncovers profound upregulation of hypoxia-inducible genes such as EGLN1(9.9x), EGLN3(52x), LDHA(11.5x), SLC2A1(30.5x), PDK1(16.8x), VEGFA(286x) and BNIP3(138x) in hypoxic NK cells. Protein expression confirmed these changes, as NK cells under normoxic culture produced 520 nmoles/million cells of ATP, while those under hypoxic culture managed only 100 nmoles/million cells. This is consistent with a bioenergetic switch from oxidative phosphorylation to glycosis resulting from PDK1 upregulation. NK cells in hypoxia produced 61 pg/mL of VEGF compared to 1480 pg/mL for NK cells in normoxia (20% O2), as determined by ELISA. The inability of NK cells to propagate under conditions of hypoxia may be due to a drop in mitochondrial content we observed when cells were exposed to chronic hypoxia, a potential mitophagic effects of BNIP3 upregulation. In addition to the poor proliferative capacity of NK cells under hypoxia, we also noted the loss of CD56 expression on hypoxic NK cells which is associated with loss of cytotoxicity. Sequence analysis reveals that miR-210 can bind to the 3′UTR of CD56 mRNA, targeting it for degradation. Therefore, we investigated whether miR-210 levels are upregulated in hypoxic NK cells and found that increased presence correlated with loss of CD56 expression. This leads to the conclusion that NK-cell immunotherapy may be improved by downregulating miR-210 levels. Indeed, our findings help shape strategies for obtaining robust and sustained NK-cell effector function for adoptive immunotherapy in the hypoxic tumor microenvironment. Disclosures: No relevant conflicts of interest to declare.

Horses for Courses in the Era of CARs: Advancing CAR T and CAR NK Cell Therapies

“…CAR NK cells in hypoxic conditions have been performed. Yet, NK cells appear to be significantly more sensitive to hypoxia, as they display slower proliferation and impaired cytotoxicity under these conditions [55]. No obvious expression differences between T and NK cells were observed for the genes encoding oxygen sensors (PHD1, PHD2, PHD3, FIH) (Figure 3).…”

“…Therefore, it remains to be explored why T and NK cells are so profoundly different in terms of their response to hypoxia. Two approaches to mitigate the detrimental effects of oxygen deprivation on NK cells were proposed and include pre-activation with IL-2 [56] and the downregulation of miR-210 levels [55].…”

Immunometabolic Responses of Natural Killer Cells to Inhibitory Tumor Microenvironment Checkpoints

“…The responses of NK cells to hypoxic environments have been discussed previously, yet despite the available knowledge, the specific role of hypoxia on NK cells is still not fully clear. Hypoxia was shown to affect functions of NK cells by impairing their cytotoxicity [134][135][136] and altering their metabolic signatures [137]. Expression of activating receptors NKp46, NKp30, NKp44, and NKG2D [138] as well as perforin and granzyme B [138] are all downregulated on NK cells in hypoxia.…”

Immunometabolic Dysfunction of Natural Killer Cells Mediated by the Hypoxia-CD73 Axis in Solid Tumors

“…NK cells, a subset of innate lymphocytes, are sensitive to conditions of hypoxia (Ang et al, 2010; Velásquez et al, 2016). In the tumor microenvironment (TME), NK cells undergo significant metabolic reprogramming events which alter their phenotypic and functional responses (Chambers et al, 2018a; Dao and Matosevic, 2019).…”