Peritoneal NK cells are responsive to IL-15 and percentages are correlated with outcome in advanced ovarian cancer patients

Abstract

The demonstration that ovarian carcinoma (OC) is an immunogenic disease, opens opportunities to explore immunotherapeutic interventions to improve clinical outcome. In this regard, NK cell based immunotherapy could be promising as it has been demonstrated that OC cells are susceptible to killing by cytokine-stimulated NK cells. Here, we evaluated whether percentage, phenotype, function and IL-15 responsiveness of ascites-derived natural killer (NK) cells is related to progression-free survival (PFS) and overall survival (OS) of advanced stage OC patients. Generally, a lower percentage of NK cells within the lymphocyte fraction was seen in OC ascites (mean 17.4 ± 2.7%) versus benign peritoneal fluids (48.1 ± 6.8%; p < 0.0001). Importantly, a higher CD56+ NK cell percentage in ascites was associated with a better PFS (p = 0.01) and OS (p = 0.002) in OC patients. Furthermore, the functionality of ascites-derived NK cells in terms of CD107a/IFN-γ activity was comparable to that of healthy donor peripheral blood NK cells, and stimulation with monomeric IL-15 or IL-15 superagonist ALT-803 potently improved their reactivity towards tumor cells. By showing that a higher NK cell percentage is related to better outcome in OC patients and NK cell functionality can be boosted by IL-15 receptor stimulation, a part of NK cell immunity in OC is further deciphered to exploit NK cell based immunotherapy.

Keywords: IL15; ascites; natural killer cells; ovarian cancer; survival.

Figure 1. NK, NKT and T cell percentage in benign ascites and ascites from ovarian cancer patients

(A) Fraction of CD45+ lymphocytes (white), CD45+ non-lymphocytes (grey) and CD45- cells (black) cell populations within peritoneal fluid of benign compared to malignant ovarian cancer patients, based on flow cytometric analysis of CD45 expression and forward/side scatter. (B) Percentage of NK cells, T cells, NKT cells and other lymphocytes within benign and malignant ascites. The percentage NK cells within the lymphocyte population is significantly different, two tailed T-test p < 0.0001. (C) Within the lymphocyte population the percentage of NK cells is depicted. The group of malignant ovarian carcinoma ascites patients is divided into good and poor survival based on the median survival of the analyzed patient cohort (n = 20). (D) The NK cell population is subdivided based on CD56 bright and CD56 dim cells. (E) Overall survival curve of OC patients groups with low and high CD56+ NK cell percentages in ascites. (F) Overall survival curve of OC patients groups with low and high CD3+ T cell percentages in ascites. (G–H) Progression free survival curves for low and high CD56+ NK cell and CD3+ T cell percentages in ascites. Error bars represent mean + SEM. When 2 groups were compared the Student T-test was used whereas a one-way ANOVA with Bonferroni correction was performed when comparing 3 groups. ^***p = 0.001.

Figure 2. Expression of activating receptors on CD45+CD3-CD56+ NK cells

Percentage positive 2B4, NKG2D, NKp46, NKp30, DNAM-1 and NKG2A NK cells of CD56+ NK cells in benign and malignant peritoneal fluid. The group of malignant ovarian carcinoma ascites patients is divided into lower than median overall survival and higher than median overall survival. Error bars represent mean + SEM. One way ANOVA with Bonferroni correction was performed when comparing the groups. ^*= p < 0.05, ^***= p < 0.001.

Figure 3. Degranulation assay comparing NK cells in healthy donor (HD) peripheral blood mononuclear cells (PBMCs) with ascites mononuclear cells (MNCs)

Percentage CD56+ NK cells positive for (A) CD69, (B) TRAIL, (C) CD107a, (D) IFN-γ, after 4 h stimulation with no target cells, K562 or SKOV-3 tumor cells. Error bars represent mean + SEM. Open symbols depict HD PBMCs, closed symbols depict ascites MNCs. One way ANOVA with Bonferroni correction was performed when comparing the groups.

Figure 4. Degranulation assay comparing ascites CD56+ NK cells with and without monomeric IL-15 or ALT-803 stimulation

(A–C) Percentage CD56+ NK cells positive for CD107a after 4 h co-culture with no stimulation (A), K562 cells (B) or SKOV-3 cells (C). (D–F) Percentage CD56+ NK cells positive for IFN-γ after 4 h co-culture with no stimulation (D), K562 cells (E) or SKOV-3 cells (F). Error bars represent mean + SEM. One way ANOVA with Bonferroni correction was performed when comparing the groups. ^* = p < 0.05, ^** = p < 0.01, ^*** = p < 0.001.