The Toll-like receptor 7/8 agonist resiquimod greatly increases the immunostimulatory capacity of human acute myeloid leukemia cells

Abstract

Immunotherapy for leukemia is a promising targeted strategy to eradicate residual leukemic cells after standard therapy, in order to prevent relapse and to prolong the survival of leukemia patients. However, effective anti-leukemia immune responses are hampered by the weak immunogenicity of leukemic cells. Therefore, much effort is made to identify agents that could increase the immunogenicity of leukemic cells and activate the immune system. Synthetic agonists of Toll-like receptor (TLR)7 and TLR8 are already in use as anticancer treatment, because of their ability to activate several immune pathways simultaneously, resulting in effective antitumor immunity. However, for leukemic cells little is known about the expression of TLR7/8 and the direct effects of their agonists. We hypothesized that TLR7/8 agonist treatment of human acute myeloid leukemia (AML) cells would lead to an increased immunogenicity of AML cells. We observed expression of TLR7 and TLR8 in primary human AML cells and AML cell lines. Passive pulsing of primary AML cells with the TLR7/8 agonist R-848 resulted in increased expression of MHC molecules, production of proinflammatory cytokines, and enhanced allogeneic naïve T cell-stimulatory capacity. These effects were absent or suboptimal if R-848 was administered intracellularly by electroporation. Furthermore, when AML cells were cocultured with allogeneic PBMC in the presence of R-848, interferon (IFN)-gamma was produced by allogeneic NK and NKT cells and AML cells were killed. In conclusion, the immunostimulatory effect of the TLR7/8 agonist R-848 on human AML cells could prove useful for the design of TLR-based immunotherapy for leukemia.

Fig. 1

Expression of TLR7/8 by primary AML cells. a Expression of TLR7 and TLR8 mRNA in AML cells, determined by RT-PCR. Representative results are shown for 3 patients out of 11 tested. Right side of the gel: DNA ladder. ABL1, c-abl oncogene 1 (used as housekeeping gene to check cDNA quality); AML P, AML patient. b Expression of TLR7 and TLR8 protein, determined by intracellular flow cytometry. Histogram overlays show the level of TLR7 and TLR8 expression (filled gray histograms) compared to isotype control staining (open black histograms) (n = 2 per AML cell type tested)

Fig. 2

Effect of R-848 treatment on primary AML cells: viability, phenotype, and cytokine production. a Effect of R-848 treatment on cell viability of primary AML cells. The chart shows the mean percentage ± SD of viable cells (n = 5). Annexin V- and PI-negative cells were defined as viable. b Effect of R-848 treatment on the membrane expression of MHC molecules on primary AML cells. Flow cytometry results are expressed as mean fluorescence intensity (MFI; Y-axis) (n = 8). c Effect of R-848 treatment on cytokine production by primary AML cells. The concentration of the cytokines IL-6, IL-1β, and TNF-α was measured in the supernatant 24 h after addition of R-848 to AML cells (n = 10). * P < 0.05. Unmod, unmodified AML cells; R-848, AML cells pulsed with 5 μg/mL R-848

Fig. 3

Effect of R-848 treatment on the immunogenicity of primary AML cells. The concentration of IFN-γ detected in the supernatant taken after 5 days of coculturing allogeneic naïve T cells and primary AML cells. The AML cells were treated with or without R-848 prior to coculture with allogeneic T cells. The results shown are representative of 2 AML patients. AML Unmod, unmodified AML cells; AML EP Mock, mock-electroporated AML cells; AML EP R-848, AML cells electroporated with R-848; AML PP R-848, AML cells passively pulsed with R-848; CD4⁺CD45RA⁺ T cells, naïve CD4⁺ T cells. * P < 0.05

Fig. 4

Effect of R-848 addition to cocultures of PBMC and AML cells. The concentration of IFN-γ detected in supernatant after 3 days of coculturing AML cells (AML), allogeneic PBMC, and/or R-848. The mean ± SD shown is of 3 independent experiments with AML cells from AML P21 (a) or NB-4 cells (b). −TW, without transwell; +TW, with transwell. * P < 0.05

Fig. 5

Determination of IFN-γ-producing cells after coculturing PBMC, AML cells, and R-848. Intracellular staining of IFN-γ production in NK cells, NKT cells, and T cells after coculturing PBMC, AML cells, and/or R-848. Dot plots shows the percentage of IFN-γ-positive cells within the NK cell population (above), NKT cell population (middle), and T cell population (below). a Representative result of cocultures of allogeneic PBMC with primary AML cells from AML P3 (n = 3). The results are representative for experiments done with AML cells from 4 different patients. b Representative result of cocultures of allogeneic PBMC with NB-4 cells (n = 4)

Fig. 6

Effect of R-848 on killing of primary AML cells by allogeneic PBMC or NK cells. Flow cytometric detection of AML cell viability following coculture of primary AML cells and allogeneic PBMC (black bars) or NK cells (striped bars) in the presence of R-848. PKH67-positive cells that were positive for annexin V and/or PI were defined as non-viable AML cells. (a) Coculture of effector cells with primary AML cells from AML P17. (b) Coculture of effector cells with primary AML cells from AML P16. Results are representative of experiments done with two different effector cell donors.+ R-848, addition of R-848; + Ab, addition of neutralizing antibodies directed against IL-6 and TNF-α; + R-848 + Ab, addition of both R-848 and neutralizing antibodies against IL-6 and TNF-α