TLR2 agonist PSK activates human NK cells and enhances the antitumor effect of HER2-targeted monoclonal antibody therapy

Abstract

Purpose: The therapeutic effect of trastuzumab monoclonal antibody (mAb) therapy has been shown to be partially dependent on functional natural killer (NK) cells. Novel agents that enhance NK cell function could potentially improve the antitumor effect of trastuzumab. We recently identified polysaccharide krestin (PSK), a natural product extracted from medicinal mushroom Trametes versicolor, as a potent toll-like receptor 2 (TLR2) agonist. This study was undertaken to evaluate the effect of PSK on human NK cells and the potential of using PSK to enhance HER2-targeted mAb therapy.

Experimental design: Human peripheral blood mononuclear cells were stimulated with PSK to evaluate the effect of PSK on NK cell activation, IFN-γ production, cytotoxicity, and trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). Whether the effect of PSK on NK cells is direct or indirect was also investigated. Then, in vivo experiment in neu transgenic (neu-T) mice was carried out to determine the potential of using PSK to augment the antitumor effect of HER2-targeted mAb therapy.

Results: PSK activated human NK cells to produce IFN-γ and to lyse K562 target cells. PSK also enhanced trastuzumab-mediated ADCC against SKBR3 and MDA-MB-231 breast cancer cells. Both direct and interleukin-12-dependent indirect effects seem to be involved in the effect of PSK on NK cells. Oral administration of PSK significantly potentiated the antitumor effect of anti-HER2/neu mAb therapy in neu-T mice.

Conclusion: These results showed that PSK activates human NK cells and potentiates trastuzumab-mediated ADCC. Concurrent treatment with PSK and trastuzumab may be a novel way to augment the antitumor effect of trastuzumab.

©2011 AACR

Figures

Figure 1

PSK stimulates IFN-γ production from CD56bright NK cells. (A) Dose-dependent induction of IFN-γ secretion by PSK. Shown are IFN-γ concentrations (mean±SD) in culture supernatant from duplicate culture wells of PBMC stimulated with different concentrations of PSK for 24 hours. Similar results were obtained from three different donors. (B) Representative dot plots showing the gating of CD56dim and CD56bright NK cells and IFN-γ production in PBS control or PSK-stimulated CD56dim or CD56bright NK cells. (C) Summary graph showing the mean±SD of the percentages of IFN-γ positive cells among total CD56dim and CD56bright NK cells in PBMC from 5 different donors. ns, not significant; **, p<0.01 using two-tailed Student t test. (D) Overlay histogram showing CD25 and CD69 expression on CD56dim and CD56bright NK cells. Filled histogram: NK cells from Control PBS group; Unfilled histogram: NK cells from PBMC treated with PSK (100 μg/ml). Results are representative of 3 independent experiments.

Figure 2

PSK stimulates CD107a mobilization and enhances the lysis of K562 tumor cells. (A) Representative dot plots showing expression of CD107a in NK cells in control and PSK-treated PBMC. The PBMC was stimulated with PSK (100 μg/ml) or control PBS for 24 hr. Then the cells were mixed with target K562 cells at 2:1 ratio. Anti-CD107a was added to the culture and incubated for 6 hr. (B) Summary graph showing the percentage (mean±SD) of CD107a positive NK cells from 3 different donors and treated with or without PSK stimulation. *, p<0.05 using two-tailed Student t test. (C) The lysis of K562 target tumor cells by PSK-stimulated or unstimulated PBMC. Shown are the percentages of specific lysis (mean±SD in triplicate wells) at the indicated E:T ratios. PBMC were stimulated with PSK (or control PBS) for 48 hr before the initiation of cytolytic assay. Difference between PBS and PSK group at different E:T ratio were analyzed using 2 way ANOVA. Similar results were obtained from three independent experiments using PBMC from 3 different donors.

Figure 3

PSK enhances trastuzumab-mediated ADCC against SKBR3 and MDA-MB-231 breast cancer cells. (A) Expression of HER2 on SKBR3 and MDA-MB-231 cells. The cells were stained with PE conjugated anti-human HER2 (empty histogram) or isotype control (filled histogram). (B) Percentages of specific lysis of trastuzumab- or control IgG-coated SKBR3 and MDA-MB-231 target cells. Shown are mean±SD of triplicate wells at different E:T ratios. ■ indicates PSK-stimulated effector PBMC; ● indicates control PBS-treated effector cells. PBMC were treated with PSK (10 μg/ml) in RPMI for 72 hr before the initiation of cytolytic assay. Difference between PBS and PSK group at different E:T ratio were analyzed using ANOVA. Similar results were obtained using PBMC from 5 different donors as summarized in Supplemental Table. (C) IFN-γ levels (mean±SD) in culture supernatant from the 4 hr ADCC incubation. Control IgG: tumor targets were coated with control irrelevant IgG and incubated with PBMC with no prior PSK stimulation; trastuzumab: tumor cells were coated with trastuzumab and then incubated with PBMC with no prior PSK; trastuzumab+PSK: tumor cells were coated with trastuzumab and incubated with PSK (10 μg/ml, 72 hr)-stimulated PBMC. *, p

Figure 4

PSK has both direct and IL-12-dependent effects on NK cells. Results shown in A-C were generated using PBMC, and results in D-F were generated using purified NK cells. (A) Expression of CD25 on NK cells in PBMC treated with PBS, PSK (100 μg/ml), or PSK plus anti-IL-12 Ab (10 μg/ml) for 24 hr. (B) Percentage of IFN-γ positive NK cells among total CD56bright NK cells in PBMC treated with PBS, PSK, or PSK plus anti-IL-12 Ab. (C) The level of IFN-γ in culture supernatant from PBMC treated with PBS, PSK, or PSK plus anti-IL-12 Ab. (D) CD25 expression in purified NK cells stimulated with PBS, PSK (100 μg/ml), IL-12 (1 ng/ml), or PSK+IL-12 for 24 hr. (E) Percentage of IFN-γ positive NK cells among total CD56bright NK cells in purified NK cells stimulated with PBS, PSK, IL-12, or PSK+IL-12. (F) The level of IFN-γ in culture supernatant of purified NK cells stimulated with PBS, PSK, IL-12, or PSK+IL-12. The FACS graphs show response from a representative donor. The summary graphs show data from 4-5 different donors analyzed in independent experiments. Each dot represents an individual donor. The horizontal line represents the group average. *, p<0.05; **, p<0.01; ***, p<0.001 between two treatment groups using Student t test.

Figure 5

PSK induces the secretion of proinflammatory cytokines and chemokines by PBMC and NK cells. (A) The levels of IL-12p40, TNF-α, IL-6, IL-8, MIP-1α, MIP-1β, IL-1α, and IL-1β in culture supernatant from PBMC treated with PSK (100 μg/ml) or control PBS for 24 or 48 hr, as determined in Luminex analysis. *, pt test. Shown are mean±SD of results from 3 independent donors. (B) Shown are levels of TNF-α, MIP-1α, and MIP-1β in culture supernatant from purified NK cells treated with PSK (100 μg/ml), IL-12 (1 ng/ml), or PSK+IL-12. Each data point represents response from an individual donor (N=5). (C) Time course of TNF-α, MIP-1α, and MIP-1β induction by PSK in one donor.

Figure 6

PSK enhances the anti-tumor effect of 7.16.4 in neu transgenic mice, and the anti-tumor effect of combinatorial PSK and 7.16.4 is partially dependent on NK cells and CD8 T cells. In (A) and (B), shown are tumor growth curve (A) and overall survival (B) in mice receiving PBS (●), PSK (■), control IgG+PSK (◇;), 7.16.4 (▲), 7.16.4 plus PSK (▼). Each data point in (A) represents mean±SD in the treatment group, n=10 per group. Similar results were obtained from two independent experiments. ***, pt test. ***, p<0.001 between CD8 T cell or NK cell depletion group and no depletion group (PSK+7.16.4). There is no difference between CD4 T cell depletion group and no depletion group.