Blood dendritic cells suppress NK cell function and increase the risk of leukemia relapse after hematopoietic cell transplantation

Abstract

NK cells play an important role in hematopoietic stem cell transplantation (HCT) and in cross talk with dendritic cells (DCs) to induce primary T cell response against infection. Therefore, we hypothesized that blood DCs should augment NK cell function and reduce the risk of leukemia relapse after HCT. To test this hypothesis, we conducted laboratory and clinical studies in parallel. We found that although, phenotypically, NK cells could induce DC maturation and DCs could in turn increase activating marker expression on NK cells, paradoxically, both BDCA1(+) myeloid DCs and BDCA4(+) plasmacytoid DCs suppressed the function of NK cells. Patients who received an HLA-haploidentical graft containing a larger number of BDCA1(+) DCs or BDCA4(+) DCs had a higher risk of leukemia relapse and poorer survival. Further experiments indicated that the potent inhibition on NK cell cytokine production and cytotoxicity was mediated in part through the secretion of IL-10 by BDCA1(+) DCs and IL-6 by BDCA4(+) DCs. These results have significant implications for future HCT strategies.

Figure 1. TLR signaling in purified NK cells

Phosphorylation of (A) ZAP70/SYK and (B) SLP76 in purified NK cells after incubation with 10 µg/ml of a-CpG (blue) or 100 ng/ml of LPS (pink), with resultant increased in (C) IFN-γ production. Data are expressed as means ± SEM of three different experiments.

Fig 2. Effects of TLR ligands and blood DC subsets on IFN-γ secretion and natural cytotoxicity of NK cells

(A) IFN-γ secretion by unstimulated NK cells (NK), NK cells stimulated overnight with 100 ng/ml of LPS (NK+LPS) or 10 µg/ml of a-CpG (NK+CpG), and NK cells stimulated overnight with one of these two TLR ligands in the presence of BDCA1+ myeloid DCs (NK+LPS+BDCA1) or BDCA4+ pDCs (NK+CpG+BDCA4) in a starting 2:1 cell ratio (no change in cell number or ratio thereafter was observed overnight). Results are expressed as means ± SEM from three independent experiments. (B) Cytotoxicity against K562 cells (NK:K562 cell ratio were always 8:1) by unstimulated NK cells (NK), NK cells stimulated overnight with 100 ng/ml of LPS (NK+LPS) or 10 µg/ml of a-CpG (NK+CpG), and NK cells stimulated overnight with one of these two TLR ligands in the presence of BDCA1+ myeloid DCs (NK+LPS+BDCA1) or BDCA4+ pDCs (NK+CpG+BDCA4) in a NK:DC cell ratio of 2:1. After cocultured, NK cells were not further purified. Results are expressed as means ± SEM from three independent experiments. * P < 0.05.

Figure 3. Suppressive effect of BDCA4+ cells on the antitumor response of NK cells

Quantitative ventral photon counting analysis on Day 7, 14, and 21 after the initiation of NK cell therapy in a metastatic neuroblastoma mouse model. Animals either received no treatment (Untreated), or received treatment with NK cells stimulated overnight with 10 µg/ml of a-CpG (NK+CpG), or NK cells stimulated with a-CpG in the presence of BDCA4+ pDCs (NK+CpG+BDCA4) in a 2:1 cell ratio. Results are representative of and summarized from three independent experiments (means ± SD) at various time points after receiving NK cellular therapy.

Figure 4. Cumulative risk of leukemia relapse after HCT

The risk of leukemia relapse after transplant was evaluated based on the graft’s DC content in tertiles: (A) BDCA1+ cells (<0.5, 0.5–0.9, and >1.0×10⁸) or (B) BDCA4+ cells (<1.4, 1.5–2.5, and >2.5×10⁸). Those patients who had received a hematopoietic graft containing (C) the upper tertile of BDCA1+ myeloid DCs or (D) the upper two tertiles of BDCA4+ pDCs (high number) had a higher risk of relapse when compared with those who received less DCs (low number), P<0.05.

Figure 5. Role of IL-6 and IL-10

(A) IL-10 and IL-6 levels in the supernatant of cell cultures including unstimulated NK cells (NK), NK cells stimulated overnight with 100 ng/ml of LPS (NK+LPS) or 10 µg/ml of a-CpG (NK+CpG), and NK cells stimulated with these two TLR ligands in the presence of BDCA1+ myeloid DCs (NK+LPS+BDCA1) or BDCA4+ pDCs (NK+CpG+BDCA4) in a 2:1 cell ratio. Results are expressed as means ± SEM from three independent experiments. * P < 0.05 (B) Suppressive effect of IL-6 and IL-10 on natural cytotoxicity of NK cells. Incubation of purified NK cells with soluble IL-10 or IL-6 (with no DCs or TLR ligands) for 48 hours resulted in reduction of natural cytotoxicity against K562 cells (E:T; 2:1) in a dose-dependent manner (P<0.05 for the two highest concentrations of IL-6 and IL-10 when compared with no cytokines, and for dose-response as analyzed by Wilcoxon rank-sum test and trend test).

Figure 6. Two-signal model in HCT for leukemia

In the presence of leukemia cells and TLR ligands (1^st signal), various immune cells may provide NK cells a different 2^nd signal (A) towards “effector” and “helper” phenotype, or (B) towards “indolent” and “tolerant” phenotype. The NK cell effects on leukemia may be direct (solid arrows), especially in T-cell depleted HCT setting as described herein, or mediated through T-cell priming (dotted arrows).