Overcoming hypoxia-induced functional suppression of NK cells

Abstract

Background: Natural 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.

Methods: We 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.

Results: While 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.

Conclusions: In 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.

Keywords: immunology; oncology; tumors.

Figure 1

Induction of acute hypoxia of healthy donor (HD) natural killer (NK) cells or high affinity NK (haNK) cells exposed to 0% oxygen. (A) HD NK cells or haNK cells were incubated in ambient (20%) oxygen or 0% oxygen in the presence of the hypoxia indicator pimonidazole for 5 hours at 37°C. Cells were stained for Hypoxyprobe (green, 63×), DAPI nuclear stain (blue) and visualized by microscopy. Insets: average pimonidazole positive staining cells positive per high power field; (right) secondary antibody control (40×); (B) haNK cells continue to produce endogenous interleukin (IL)-2 under hypoxic conditions. Cells were incubated in 20% oxygen or 0% oxygen for 5 hours at 37°C. IL-2 expression was assessed by western blot analysis and quantified using band densitometry analysis normalizing to GAPDH (inset panels), and (C) quantified by ELISA. This experiment was repeated three times with similar results.

Figure 2

Acute hypoxia inhibits killing by healthy donor (HD) natural killer (NK) but not high affinity NK (haNK) cells. Prostate (PC3), breast (MCF-7) and lung (H460) cancer cell lines were labeled with ¹¹¹In and co-incubated with either of three HD (HD897, HD836, HD950) NK or haNK cells in 20% or 0% oxygen for 5 hours at 37°C. ¹¹¹In release was measured and used to calculate per cent lysis of target cells. Inset: killing of H460 cells was done at 1% oxygen in addition to 20% and 0% oxygen. *P≤0.05 using Student’s t-test. This experiment was repeated two or more times with similar results. ns, not significant.

Figure 3

Acute hypoxia inhibits antibody-dependent cellular cytotoxicity (ADCC) in healthy donor (HD) natural killer (NK) cells but not high affinity NK (haNK) cells. ¹¹¹In-labeled prostate (PC3), breast (MCF-7) and lung (H460) cancer cell lines were incubated in 20% or 0% oxygen at 37°C with either HD (HD867 and HD950) NK cells or haNK cells. ADCC was demonstrated using an antiprogrammed death-ligand 1 (anti-PD-L1) antibody (avelumab, hIgG1, 2 µg/mL). After 5 hours, ¹¹¹In was measured to determine per cent lysis of target cells. (A) Lysis and ADCC by HD NK and haNK cells. CD16 polymorphism is indicated for each donor. (B) Killing by NK cells from patients with cancer (CP1 and CP2). ADCC was determined using an anti-PD-L1 antibody (avelumab, hIgG1, 2 µg/mL) or an anti-EGFR antibody (cetuximab, hIgG1, 2 µg/mL). *P≤0.05 using Student’s t-test. ns, not significant.

Figure 4

Perforin expression is maintained in high affinity natural killer (haNK) cells in response to acute hypoxia. (A) Perforin expression as measured by flow cytometry in healthy donor (HD) NK and haNK cells incubated in 20% (gray) and 0% (blue) oxygen for 5 hours at 37°C. Inset: % positive cells (mean fluorescent intensity). (B) Cells were incubated as in (A) and perforin expression was measured by immunofluorescence (63×). Insets: average perforin positive staining cells positive per high power field; (right) secondary antibody control (40×). (C) ¹¹¹In-labeled prostate (PC3) cells were co-incubated with HD NK or haNK cells at 37°C for 5 hours under 20% or 0% oxygen. Lysis of target (PC3) cells was determined and used to calculate killing frequency (killing frequency=# target cells killed/# effector cells plated), a measure of the serial killing ability of NK cells. Results are normalized to HD NK at 20% oxygen. *P≤0.05 using Student’s t-test. This experiment was repeated two times with similar results.

Figure 5

Differential gene and protein expression in healthy donor (HD) NK and high affinity natural killer (haNK) cells in response to hypoxia. HD NK and haNK cells were incubated in 20% or 0% oxygen for 5 hours at 37°C. RNA from two HD NK and haNK cells was extracted and RNAseq was performed in triplicate to analyze changes in gene expression in response to hypoxia. (A) Number of genes (and per cent) changed by <20% or >20% in HD NK and haNK cells. (B) Changes in RNA expression of genes important to NK cell function are presented as the ratio of the gene RNA transcripts per million (TPM) of expression under 0% O₂ vs the 20% O₂ in log2 space. (C) Number of proteins assayed and detected for both HD NK and haNK samples. (D) Diagram of proteins exclusive to HD NK or haNK cells as well as selected proteins shared between both cell types. Quantification of selected proteins is shown in (E). Each sample was run in triplicate and results are presented as mean±%coefficients of variation.

Figure 6

Phospho-STAT3 (pSTAT3) reduces natural killer (NK) killing capacity. (A) STRING protein network depicting the connections between multiple proteins identified as having an important role in the NK and high affinity (ha)NK response to hypoxia. (B) Flow cytometry analysis of pSTAT3 in healthy donor (HD) NK cells and haNK cells incubated in 20% or 0% oxygen. (C) Capillary western blot analysis of total STAT3 and pSTAT3 in haNK cells incubated in 20% or 0% oxygen. Quantification of pSTAT3 normalized to vinculin loading control is given under each lane. (D) Western blot analysis of pSTAT3 in haNK following the addition of colivelin (100 µM), a pSTAT3 activator. (E) Per cent lysis of PC3 target cells by haNK cells with the addition of colivelin (100 µM). Cells were incubated for 5 hours at 20% or 0% oxygen at 37°C. *P≤0.05 using Student’s t-test.