Serial killing of tumor cells by human natural killer cells--enhancement by therapeutic antibodies

Abstract

Background: Natural killer cells are an important component of the innate immune system. Anti-cancer therapies utilizing monoclonal antibodies also rely on the cytotoxicity of NK cells for their effectiveness. Here, we study the dynamics of NK cell cytotoxicity.

Methodology/principal findings: We observe that IL-2 activated human NK cells can serially hit multiple targets. Using functional assays, we demonstrate that on an average, a single IL-2 activated NK cell can kill four target cells. Data using live video microscopy suggest that an individual NK cell can make serial contacts with multiple targets and majority of contacts lead to lysis of target cells. Serial killing is associated with a loss of Perforin and Granzyme B content. A large majority of NK cells survive serial killing, and IL-2 can replenish their granular stock and restore the diminished cytotoxicity of 'exhausted' NK cells. IL-2 and IL-15 are equally effective in enhancing the killing frequency of resting NK cells. Significantly, Rituximab, a therapeutic monoclonal antibody increases the killing frequency of both resting and IL-2 activated NK cells.

Conclusion/significance: Our data suggest that NK cell-based therapies for overcoming tumors rely on their serial killing ability. Therefore, strategies augmenting the killing ability of NK cells can boost the immune system and enhance the effectiveness of monoclonal antibody-based therapies.

Figure 1

IL-2 activated human NK cells can kill multiple targets. IL-2 activated primary human NK cells were co-incubated with ⁵¹Cr labeled 221 target cells at an E∶T ratio of 0.8∶1 to 0.05∶1 for 4 and 16 hours. (A) 51 Cr release assay was used to calculate % specific lysis for 4 and 16 hours duration. (B) % specific lysis was used to calculate killing frequency by dividing the number of target cells killed by number of effector cells used for each E∶T ratio. (C) Killing frequency of IL-2 activated NK cells using four independent donors for 4 and 16 hours duration.

Figure 2

Serial killing by an NK cell. (A) A single NK cell makes multiple contacts with tumor cells resulting in lysis of tumor cells. Dye-labeled IL-2 activated NK cells (N, red) were incubated with unlabelled 221 tumor cells at an E∶T ratio of 0.2∶1 for 16 hours at 37°C and 5% CO2 and images were recorded by time lapse microscopy. Dead cells were detected using Sytox Green and numbered in the order of lysis. Time (h:mm) is indicated in the upper left corner of each image. (B) Majority of conjugate formations result in lysis. Conjugate formations between NK and 221 cells were randomly selected and lysed target cells counted. To rule out the possibility of interference of dyes in killing process, a control ⁵¹Cr release assay was performed with labeled cells. No difference was noted between the killing ability of labeled and unlabeled cells (data not shown).

Figure 3

Serial killing of tumor cells by NK cells is mediated by Perforin and Granzyme B. (A) IL-2 activated NK cells, alone (−221) or together with 221 target cells (+221) were incubated for a period of 16 hours at an E∶T ratio of 0.2∶1 and then stained intracellularly for Perforin and Granzyme B content. NK cells were identified by gating on CD56 positive cells. Fold expression shown here represents the ratio of MFI of Perforin/Granzyme B and respective isotypes. Figure is representative of multiple donors. (B) IL-2 activated NK cells were incubated alone or in combination with 221 targets at an E∶T ratio of 0.2∶1. After 16 hours, cells were harvested and stained for CD56 expression (NK cells) and annexinV (apoptotic cells). NK and 221 cells alone were used as controls. Forward and sideward scatter gating was used to gate on free NK cells, free 221 cells or conjugates. Figure is representative of multiple donors.

Figure 4

IL-2 restores the killing ability of inactivated NK cells. IL-2 activated NK cells were incubated with or without 221 targets for 16 hours at an E∶T ratio of 0.2∶1. Thereafter, NK cells were re-isolated by CD56 positive selection using MACS columns and cultured in the presence of IL-2 (100 U/ml). Killing assays against 221 target cells were performed on day 1 (A) and 2 (B) after isolation. Data from three independent donors show that NK cells regain their lost cytotoxic ability on day 2 in response to IL-2 treatment.

Figure 5

Increasing the killing frequency of fresh NK cells by cytokines and Rituximab. (A) Freshly isolated NK cells were cultured with IL-2 (10 or 100 U/ml) alone (A) or with IL-2 (100 U/ml) and IL-15 (10ng/ml), alone or in combination (B) IL-2 (100 U/ml) alone or in combination with IFN-α (1000U/ml) or poly I∶C (50 µg/ml) (C) for one week. Thereafter, stimulated NK cells were tested against 221 target cells using 4 hours Cr-⁵¹ release assays. Combining IFN-α or poly I∶C with only 10 U/ml IL-2 also showed no enhancement of cytotoxicity (data not shown). (D) 221 target cells were treated with Rituximab (10 µg/ml) and killing frequency of freshly isolated NK cells was assessed by 4 and 16 h ⁵¹Cr release assay and compared to untreated targets. Data are representative of three independent experiments using NK cells from different donors.

Figure 6

Rituximab increases killing frequency of IL-2 activated NK cells. 221 target cells were treated with Rituximab (10 µg/ml) and killing frequency of NK cells was assessed by 4 h (A) or 16 h (B) ⁵¹Cr release assay and compared to untreated or isotype treated targets. (C) Data from three independent donors show that rituximab increases the killing frequency of NK cells two fold as compared to untreated target cells.