Overcoming hypoxia-induced functional suppression of NK cells

Abstract

BackgroundNatural killer (NK) cells are immune cells capable of killing virally infected cells and tumor cells without the need for antigen stimulation. Tumors, however, can create a suppressive microenvironment that decreases NK function. A feature of many tumors is hypoxia (low oxygen perfusion), which has been previously shown to decrease NK function. A high affinity NK (haNK) cell has been engineered to express a high affinity CD16 receptor as well as internal interleukin (IL)-2 for increased antibody-dependent cellular cytotoxicity (ADCC) and activation, respectively. We sought to investigate the tolerance of NK cells versus haNK cells to hypoxia.MethodsWe exposed healthy donor (HD) NK and X-irradiated haNK cells to normoxia (20% oxygen) as well as hypoxia (0% oxygen) and investigated their ability to kill prostate, breast and lung tumor cell lines after 5 hours. We also used monoclonal antibodies cetuximab (anti-EGFR) or avelumab (antiprogrammed death-ligand 1) to investigate the effects of hypoxia on NK ADCC. Genomic and proteomic analyzes were done to determine the effect of hypoxia on the expression of factors important to NK cell function.ResultsWhile HD NK cell cytolytic abilities were markedly and significantly impaired under hypoxic conditions, haNK cells maintained killing capacity under hypoxic conditions. NK killing, serial killing and ADCC were maintained under hypoxia in haNK cells. IL-2 has been previously implicated in serial killing and perforin regeneration and thus the endogenous IL-2 produced by haNK cells is likely a driver of the maintained killing capacity of haNK cells under hypoxic conditions. Activation of signal transducer and activator of transcription 3 (STAT3) is not seen in haNKs under hypoxia but is significant in HD NK cells. Pharmaceutical activation of STAT3 in haNKs led to reduced killing, implicating active STAT3 in reduced NK cell function.ConclusionsIn contrast to HD NK cells, haNK cells are resistant to acute hypoxia. The potent cytolytic function of haNK cells was maintained in an environment comparable to what would be encountered in a tumor. The data presented here provide an additional mechanism of action for haNK cells that are currently being evaluated in clinical trials for several tumor types.

NK cell infiltration is associated with improved overall survival in solid cancers: A systematic review and meta-analysis

“…In addition to infiltration, the solid tumor microenvironment restricts NK cell reactivity. In tumors infiltrated with NK cells that are suppressed, interventions to support NK cell reactivity and overcome immunosuppression, such as checkpoint blockade or local cytokine production may prove efficacious [122] , [123] , [124] , [125] . NK cells isolated from patients with solid tumors including head and neck 126 , 127 , gallbladder 128 , and ovarian 129 cancers exhibit increased expression of inhibitory receptors and decreased expression of activating receptors.…”

Arrested development: suppression of NK cell function in the tumor microenvironment

“…HIF‐1α regulates gene expression programs involved in many processes, such as cell proliferation, survival, glucose metabolism, and angiogenesis 49 . It has been demonstrated that NK cells are hyporesponsive under hypoxic conditions in vitro , displaying reduced expression of NKG2D, CD107a, and granzyme B with concomitant reductions in cytotoxicity 49–52 . Similarly, human peripheral NK cells cultured under hypoxic conditions in vitro displayed reduced IFN‐γ, granzyme B, and CD107a protein levels that could be restored using a HIF‐1α inhibitor 53 .…”

“…49 It has been demonstrated that NK cells are hyporesponsive under hypoxic conditions in vitro, displaying reduced expression of NKG2D, CD107a, and granzyme B with concomitant reductions in cytotoxicity. [49][50][51][52] Similarly, human peripheral NK cells cultured under hypoxic conditions in vitro displayed reduced IFN-c, granzyme B, and CD107a protein levels that could be restored using a HIF-1a inhibitor. 53 Experiments using in vivo tumor models have proposed that HIF-1a À/À NK cells promote tumor clearance by inhibiting vascular endothelial growth factor (VEGF)-driven angiogenesis.…”

Hypoxia-Driven HIF-1α Activation Reprograms Pre-Activated NK Cells towards Highly Potent Effector Phenotypes via ERK/STAT3 Pathways

“…Hypoxic stress within the tumor microenvironment shapes the NK cell phenotype, thereby inducing tumor resistance and generating immune-suppressive cells. For example, exposure of NK cells to hypoxic conditions of 1% pO 2 resulted in drastic suppression of NK cytotoxicity against natural tumor targets [ 10 , 11 , 12 ]. Furthermore, tumor cells themselves adopt several strategies to evade NK cell-mediated killing by directly downregulating NK-activating receptors and/or indirectly releasing immunosuppressive factors such as TGFβ, IL-10, IDO, and soluble NK receptor ligands, namely ULBPs and MICA [ 12 ].…”