Characterization of the defective interaction between a subset of natural killer cells and dendritic cells in HIV-1 infection

Abstract

In this study, we demonstrate that the in vitro interactions between a CD56(neg)/CD16(pos) (CD56(neg)) subset of natural killer (NK) cells and autologous dendritic cells (DCs) from HIV-1-infected viremic but not aviremic individuals are markedly impaired and likely interfere with the development of an effective immune response. Among the defective interactions are abnormalities in the process of reciprocal NK-DC activation and maturation as well as a defect in the NK cell-mediated editing or elimination of immature DCs (iDCs). Notably, the lysis of mature DCs (mDCs) by autologous NK cells was highly impaired even after the complete masking of major histocompatibility complex I molecules, suggesting that the defective elimination of autologous iDCs is at the level of activating NK cell receptors. In this regard, the markedly impaired expression/secretion and function of NKp30 and TNF-related apoptosis-inducing ligand, particularly among the CD56(neg) NK cell subset, largely accounts for the highly defective NK cell-mediated lysis of autologous iDCs. Moreover, mDCs generated from HIV-1 viremic but not aviremic patients are substantially impaired in their ability to secrete interleukin (IL)-10 and -12 and to prime the proliferation of neighboring autologous NK cells, which, in turn, fail to secrete adequate amounts of interferon-gamma.

Figure 1.

mDC functional profiles and ability to prime autologous NK cells. (A and B) Levels of IL-12p70 (A) and IL-10 (B) secreted by mDCs generated from healthy donors (HD; yellow bars), HIV-1–infected aviremic (green bars) patients, and viremic (red bars) HIV-1–infected patients. (C) Levels of IFN-γ secreted by freshly purified NK cells cocultured with autologous mDCs (yellow bars) for 24 h. mDCs alone (blue bars) and NK cells alone (red bars) served as negative controls. The mDC/NK cell ratio was 1:10. (D) mDC-mediated priming of NK cells; fresh NK cells (responders) purified from HIV-1–infected viremic (red triangles) and aviremic (green squares) individuals and from uninfected subjects (yellow circles) were cocultured with autologous mDCs (stimulators) at different ratios. Cells were harvested after 4 d of coincubation, and the proliferation of NK cells was measured by ³[H]thymidine incorporation. All data are presented as the median (± SD [error bars] for A–C) of experiments conducted on 15 healthy donors and 15 HIV-1 viremic and aviremic infected patients.

Figure 2.

mDC priming of heterologous NK cells. Fresh NK cells (responders) purified from HIV-1–infected viremic (red triangles) and aviremic (green squares) individuals and from healthy donors (HD; yellow circles) were cocultured with heterologous mDCs (stimulators) generated from healthy donors (A) as well as aviremic (B) and viremic (C) HIV-1–infected individuals at different ratios. Cells were harvested after 4 d of coincubation, and the proliferation of NK cells was measured by ³[H]thymidine incorporation. Data are presented as the median of experiments conducted on 15 healthy donors and 15 HIV-1 viremic and aviremic infected patients.

Figure 3.

NK cell–mediated killing of heterologous iDCs and autologous MHC-I masked mDCs. (A) Cytolysis exerted by unfractionated rIL-2–activated NK cells purified from a healthy donor, an aviremic, and a viremic HIV-1–infected individual against heterologous iDCs generated from a healthy donor (HD; yellow bars), an aviremic (green bars), and a viremic (red bars) HIV-1–infected patient. (B) Autologous mDC cytolysis exerted by unfractionated rIL-2–activated NK cells purified from a healthy donor (yellow bars), an aviremic (green bars), and a viremic (red bars) HIV-1– infected individual. (C) Comparison of autologous mDC killing exerted by rIL-2–activated CD56^pos- (dark blue bars) and CD56^neg (light blue bars)-derived NK cell subsets purified from the same HIV-1–infected viremic subject shown in B. NK cells were incubated either in the absence (baseline lysis) or presence of specific mAbs masking classic HLA-A/-B/-C and nonclassic HLA-E. Data were obtained from single experiments performed in triplicate (± SD [error bars]) and are representative of results obtained using cells isolated from 15 viremic and 15 aviremic HIV-1–infected individuals and from 15 healthy donors. The NK cell/iDC ratio in all experiments was 10:1.

Figure 4.

NK cell–mediated killing of autologous iDCs: masking of NKp30. (A) Autologous iDC cytolysis exerted by unfractionated rIL-2–activated NK cells purified from a healthy donor (HD; yellow bars), an aviremic (green bars), and a viremic (red bars) HIV-1–infected individual. (B) Comparison of autologous iDC killing exerted by rIL-2–activated CD56^pos- (dark blue bars) and CD56^neg (light blue bars)-derived NK cell subsets purified from the same HIV-1–infected viremic subject shown in A. NK cells were incubated either in the absence (baseline lysis) or presence of a specific mAb masking NKp30. Data were obtained from single experiments performed in triplicate (± SD [error bars]) and are representative of results obtained using cells isolated from 15 viremic and 15 aviremic HIV-1–infected individuals and from 15 healthy donors. The NK cell/iDC ratio in all experiments was 10:1.

Figure 5.

TRAIL expression and s-TRAIL secretion by rIL-2–activated NK cells. (A) Surface expression of TRAIL by NK cells in representative examples for a healthy donor, an aviremic, and a viremic HIV-1–infected individual and on CD56^pos and CD56^neg NK cell subsets purified from the same HIV-1–infected viremic subject. Histogram graphs show the percentage and geometric mean fluorescence intensity (MFI) of freshly purified (blue lines) and rIL-2–activated (red lines) cells. Gray shaded histograms represent the negative controls stained with the second reagent alone. (B and C) Summary graphs of statistical dot plots with medians (horizontal bars) showing the percentages of unfractionated rIL-2 NK TRAIL^pos cells purified from healthy donors (yellow circles), aviremic (green circles), and viremic (red circles) HIV-1–infected patients and from rIL-2–activated CD56^pos (dark blue circles) and the CD56^neg (light blue circles)-derived NK cell subsets purified from viremic HIV-1–infected individuals. (D and E) Levels of sTRAIL secreted by unfractionated rIL-2–activated NK cells purified from healthy donors (yellow bar), HIV-1–infected aviremic (green bar), and viremic subjects (red bar) and by rIL-2–activated CD56^pos- (dark blue bar) and CD56^neg (light blue bar)-derived NK cell subsets purified from viremic HIV-1–infected individuals. Data are presented as the median ± SD (error bars) of experiments conducted on 15 healthy donors and 15 HIV-1 viremic and aviremic infected patients.

Figure 6.

NK cell–mediated killing of autologous iDCs: masking of TRAIL and NKp30. (A) Autologous iDC cytolysis exerted by unfractionated rIL-2–activated NK cells purified from a healthy donor (HD), an aviremic, and a viremic HIV-1–infected individual. NK cells were incubated either in the absence (baseline lysis; red bars) or presence of a mAb-neutralizing TRAIL (pink bars). Experiments were also performed masking the effectors with an anti-NKp30–specific mAb either in the absence (blue bars) or presence (purple bars) of a specific anti-TRAIL mAb. (B) Comparison of autologous iDC killing exerted by rIL-2–activated CD56^pos- and CD56^neg-derived NK cell subsets purified from the same HIV-1–infected viremic subject shown in A. Cells were incubated either in the absence (baseline lysis; green bars) or presence of a mAb-neutralizing TRAIL (light blue bars). Experiments were also performed masking the effectors with an anti-NKp30–specific mAb either in the absence (black bars) or presence (gray bars) of a specific anti-TRAIL mAb. (C) Autologous cytolysis of iDCs exerted by unfractionated rIL-2–activated NK cells from a representative healthy donor, an HIV-1–infected aviremic and viremic individual (bars on the left side of the arrow), and by CD56^pos- and CD56^neg-derived NK cell subsets purified from the same viremic patient (bars on the right side of the arrow). NK cells were incubated either in the absence (baseline lysis; red bars) or presence of a specific mAb-neutralizing DNAM-1 (purple bars). Experiments were also performed masking the effectors with anti-NKp30, anti-TRAIL, and DNAM-1 mAbs together (yellow bars). Data were obtained from single experiments performed in triplicate (± SD [error bars]) and are representative of results obtained using cells isolated from 15 viremic and 15 aviremic HIV-1–infected individuals and from 15 healthy donors. The NK cell/iDC ratio in all experiments was 10:1.