(19)F-MRI for monitoring human NK cells in vivo

Abstract

The availability of clinical-grade cytokines and artificial antigen-presenting cells has accelerated interest in using natural killer (NK) cells as adoptive cellular therapy (ACT) for cancer. One of the technological shortcomings of translating therapies from animal models to clinical application is the inability to effectively and non-invasively track these cells after infusion in patients. We have optimized the nonradioactive isotope fluorine-19 ((19)F) as a means to label and track NK cells in preclinical models using magnetic resonance imaging (MRI). Human NK cells were expanded with interleukin (IL)-2 and labeled in vitro with increasing concentrations of (19)F. Doses as low as 2 mg/mL (19)F were detected by MRI. NK cell viability was only decreased at 8 mg/mL (19)F. No effects on NK cell cytotoxicity against K562 leukemia cells were observed with 2, 4 or 8 mg/mL (19)F. Higher doses of (19)F, 4 mg/mL and 8 mg/mL, led to an improved (19)F signal by MRI with 3 × 10(11) (19)F atoms per NK cell. The 4 mg/mL (19)F labeling had no effect on NK cell function via secretion of granzyme B or interferon gamma (IFNγ), compared to NK cells exposed to vehicle alone. (19)F-labeled NK cells were detectable immediately by MRI after intratumoral injection in NSG mice and up to day 8. When (19)F-labeled NK cells were injected subcutaneously, we observed a loss of signal through time at the site of injection suggesting NK cell migration to distant organs. The (19)F perfluorocarbon is a safe and effective reagent for monitoring the persistence and trafficking of NK cell infusions in vivo, and may have potential for developing novel imaging techniques to monitor ACT for cancer.

Keywords: Fluorine 19 (19F); in vivo imaging and adoptive cell therapy (ACT); magnetic resonance imaging (MRI); natural killer cells (NKs).

Figure 1.

High uptake of ¹⁹F nanoemulsions by human NK cells do not affect their viability. Human NK cells were expanded for 12 d from PBMCs of a healthy donor and sorted on day 12 of expansion by magnetic bead isolation. Sorted NK cells (CD3⁻ CD56⁺) were co-incubated with or without ¹⁹F (Cell Sense) for 24 h then analyzed for (A) Percent viability using Trypan blue and determined before (0 hour) and after (24 h) co-culture of NK cells with 2 mg/mL, 4 mg/mL or 8 mg/mL ¹⁹F. Data presented for 11 compiled experiments. (B) Representative NMR spectra of TFA (control) or NK cells labeled with 2 mg/mL, 4 mg/mL or 8 mg/mL PFPE show ¹⁹F signal increasing with the concentration of PFPE in cell media. (C) Concentration of ¹⁹F/NK cell determined by NMR for NK cells exposed to different concentration of PFPE for 24 h. Three replicates were set up per group. Bar graph values represent the mean ± SEM tested by a one-way ANOVA. Data representative of at least three experiments with reproducible results.

Figure 2.

¹⁹F labeling of human NK cells does not alter their surface expression of activating natural cytotoxic receptors and chemokine receptors. (A–G) Human NK cells unlabeled or labeled with 4 mg/mL ¹⁹F (Cell Sense) for 24 h analyzed by flow cytometry for: (A–B) The percent expression in the NK cytotoxic receptors (NCRs) NKp30, NKp46 and NKp44 vs. IgG1 control stains. (A) Illustrates representative dot plots for each NCR and their isotype controls (IgG1) for unlabeled NK cells or NK cells labeled with ¹⁹F. (B) Shows percentage of NKp30, NKp46 and NKp44 on the NK cells (CD3 negative CD56⁺) in five healthy donors. (C–E) The percent expression or (F–G) mean fluorescent intensity (MFI) in the activating receptors DNAM-1 (DNAX Accessory Molecule-1) and NKG2D and in chemokine receptors CX3CR1 and CXCR4 compared to isotype controls in ¹⁹F-labeled or unlabeled NK cells. (D) Percent expression or (G) MFI in the chemokine receptors CX3CR1, CXCR4 or isotype controls after gating on the of CD3neg CD56⁺ NK cells. MFI numbers are indicated within the histograms with color-coded MFIs indicated in the legend and corresponding to the histograms. (E) Shows the percent and (F) MFI in CX3CR1, CXCR4, DNAM-1 and NKG2D on NK cells from five healthy donors labeled or not with ¹⁹F. All gates and histograms are pre-gated on CD3⁻ CD56⁺ NK cells. Bar graph values represent the mean ± SEM tested by two-way ANOVA. Data representative of at least four independent experiments with reproducible results. n.s.= not significant.

Figure 3.

Human NK cells labeled with ¹⁹F maintain their cytotoxic function in vitro. (A) Sorted human NK cells were cultured with ¹⁹F for 24 h. Extra ¹⁹F not taken up by NK cells was washed out, then NK cells (E: effectors) were cultured with ⁵¹Cr-labeled K562 tumor (T: target) for 4 h at 37°C at different E:T ratios to determine their percent lysis of tumor cells, n = 5. Values represent the mean ± SEM of one of four independent experiments tested by two-way ANOVA, with no significant difference seen. (B–D) NK cells labeled with 4 mg/mL ¹⁹F or unlabeled were stained for flow cytometry intracellularly for IFNγ and granzyme B or isotype control, n = 5 donors. (B) Compiled percentage or (C) representative flow plots or (D) MFI in isotype control, IFNγ and Granzyme B by unlabeled and ¹⁹F-labeled NK cells is illustrated. (E) Human IFNγ production was determined by ELISA in NK cells unlabeled or labeled with ¹⁹F at 2 mg/mL or 4 mg/mL ¹⁹F for 24 h or 48 h, n = 3, no difference was observed. Dot plots are representative of one of at least five experiments with reproducible results. Bar graph values represent the mean ± SEM of one of five independent experiments tested by two-way ANOVA. p < 0.05, n.s = not significant.

Figure 4.

MRI in vivo detection of ¹⁹F-labeled human NK cells in NSG mice bearing xenograft human tumors. (A–B) 10 × 10^{6 19}F-labeled (4 mg/mL PFPE for 24 h) human NK cells were injected intratumor into one NSG mouse bearing a human neuroblastoma (CHLA-20) on the right flank (T: Tumor). ¹⁹F intensity from NK cells or from the reference (Ref: Reference vial of ¹⁹F) vial is displayed on a “hot-iron” scale. Mouse was imaged for ¹H and ¹⁹F by MRI using a volumetric coil and anesthetized using ketamine and xylazine. (A) Composite ¹⁹F/¹H images at 0 or 2 d post NK cell injection are shown with 32 min and 42 min of scan time for each day respectively. (B) The number (black bars corresponds to left y-axis) and percentage (red line graph corresponds to the right y-axis) of NK detected in the tumor is denoted and was determined based on the efficiency of ¹⁹F uptake by NK cells from NMR analysis. (C–D) 7 × 10^{6 19}F-labeled (4 mg/mL PFPE for 24 h) NK cells were injected intratumor into one NSG mouse bearing a human mantle cell lymphoma (Z138) on the right flank. (C) Imaging for ¹H and ¹⁹F was established as described in (A) with scan time of 42 min for each time point. (D) Number (black bars corresponds to left y-axis) and percentage (red line graph corresponds to the right y-axis) of NK cells detected at the tumor site is denoted for each imaging time point. Values represent the mean ± SEM of one single experiment. The uncertainty in the ¹⁹F reference mean signal (${\displaystyle {\sigma }_{{\overline{S}}_R}}$) was estimated as the standard deviation of the ROI drawn on the reference (see methods for quantification of ¹⁹F signal).

Figure 5.

¹⁹F-labeled NK cell migration in NSG mice bearing human GD2⁺ melanoma and hu14.18-IL-2 treatment. 10 × 10^{6 19}F-labeled (4 mg/mL PFPE for 24 h) human NK cells were injected on the left flank subcutaneously into NSG mice (n = 2) bearing human melanoma (M21) tumor on the right flank (T: Tumor). On days 0–2 and 7–9, 50 µg/50 µL hu.14.18-IL-2 immunocytokine was injected i.t. On days 0 and 7, 1 × 10⁶ IU/0.2 mL rh-IL-2 was injected i.p. (A) Mice were imaged for ¹H and ¹⁹F by MRI for 42 min at different time points. Here, a T2-weighted ¹H image was acquired for enhanced tumor visualization. Composite ¹⁹F/¹H images are depicted for 1 mouse. (B) Illustrates the number of NK cells remaining at the site of injection at each time point for both mice. Day 0 refers to the day of implantation of ¹⁹F-labeled human NK cells. Values represent the mean ± SEM of one single experiment, n = 2.